Chapter 2shodhganga.inflibnet.ac.in/bitstream/10603/13612/14/14_chapter 2.pdf · host chromosome....

Chapter 2

233

41 Introduction

In recent years a number of compounds previously considered non-

degradable are also being degraded by microorganisms suggesting that

under selective pressure of environmental pollution microbes develop the

ability to degrade recalcitrant xenobiotics However the fact that many

pollutants are still persistent in the environment reflects the inadequacy of the

current microbial catabolic capacity to deal with such pollutants That is the

kinetics of process may be much slower than desired This has stimulated

the development of bioremediation technologies which may be applied to

many different environmental situations One of the new developing

technologies involves the genetic engineering of natural microorganisms with

enhanced or new degradative capabilities for bioaugmentation of selected

contaminated environments Molecular biology offers the tools to optimize

the biodegradative capacities of microorganisms accelerate the evolution of

new activities and construct totally new pathways through the assemblage

of catabolic segments from different microbes Although the number of

genetically engineered microbes (GEMs) for potential use in biodegradation is

not large these recombinant microbes function in microcosms according to

their design The survival and fate of recombinant microbes in different

ecological niches under laboratory conditions is similar to what has been

observed for the unmodified parental strains rDNA both on plasmids and on

the host chromosome is usually stably inherited by GEMs The potential

lateral transfer of rDNA from the GEMs to other microbes is significantly

diminished though not totally inhibited when rDNA is incorporated on the

host chromosome The behaviour and fate of GEMs can be predicted more

accurately through the coupling of regulatory circuits that control the

expression of catabolic pathways to killing genes so that the GEMs survive in

polluted environments but die when the target chemical is eliminated (Ramos

et al 1994)

The formation of DDD from DDT is also a common reaction among soil

microorganisms (Guenzi and Beard 1968) Chacko et al (1966) isolated

numerous actinomycetes (Nocardia sp Streptomyces aureofaciens

Streptomyces cinnamoneus Streptomyces viridochromogenes) from soil

Chapter 2

234

which readily degraded DDT to DDD Wedemeyer (1967) studied DDT

metabolic pathway by incubation of proposed intermediates with organisms

and examining the products formed The metabolism of DDT in Aerobacter

aerogenes goes in order DDT DDD DDMU DDNU DDOH DDA

DBP and direct conversion of DDT to DDE (Wedemeyer 1967) Reports

on the involvement of enzymes in the degradation of DDT indicate the

presence of enzymes like dioxygenases (Nadeau et al 1994)

dehydrogenases (Bourquin 1977) oxygenases (Ahmed et al 1991) Only a

few enzymes have been described in DDT degradative pathway (Singh et al

1999) The conversion of DDT to DDD involves a dehydrochlorination step

In our laboratory an attempt was made to clone sequence and express the

gene responsible for this step

42 Materials

Luria- Bertoni broth and ampicillin were procured from Hi media

Mumbai Agarose was procured from Sisco Research Laboratories Pvt Ltd

Taq polymerase dNTPs and restriction enzymes were procured from Genei

Bangalore PCR purification kit was purchased from GE Healthcare UK

Ethidium bromide nitrocellulose membrane and nylon membrane were

procured from Sigma Aldrich Chemical Company MO US The designed

primers were synthesized by Sigma MO US Cloning kit was procured from

Fermentas Lifesciences EU DIG kit was obtained from Roche Company

Germany EDTA was procured from Qualigens Fine Chemicals All other

chemicals used in the study were procured from standard chemical

companies

43 Reagents and buffers

431 Reagents for plasmid isolation

Solution 1 50 mM Glucose 25 mM Tris- Cl (pH-80) 10 mM EDTA (pH-80)

Solution 2 02 N NaOH (freshly prepared from 10 N NaOH) 1 SDS

(Prepared freshly before use)

Chapter 2

235

Solution 3

50 M Potassium acetate 600mL

Glacial acetic acid 115mL

Distilled water 285mL

432 Ampicillin stock solution (100 mg mL)

Ampicillin was dissolved in distilled water filter sterilized and stored at

-20 0C

433 RNAse stock solution

10 mg of RNAse was dissolved in 1mL of distilled water and boiled for

15 min and used

434 CaCl2 solution

60 mM CaCl2 in MOPrsquos buffer (pH 65)

435 Luria Bertani broth

Tryptone peptone 100 g L

Yeast extract 50 g L

Sodium chloride 100 g L

The medium was autoclaved at 15 lbs 121 0C for 15 min

436 Tris- EDTA buffer (pH 80)

10 mM Tris Hcl (pH 80)

1 mM EDTA (pH 80)

pH was adjusted to 80 autoclaved and stored

437 Tris- acetate EDTA buffer (50X)

Tris buffer 242 g

05 M EDTA 10 mL

Glacial acetic acid 57 mL

Distilled water 84 mL


Chapter 2

236

438 DNA loading buffer

Bromophenol blue 025

Xylene Cyanol FF 025

Glycerol 30

This was prepared in double distilled water and stored in aliquots at -

20oC

439 Alkaline phosphatase buffer

Tris- HCl (pH 95) 100 mM

NaCl 100 mM

MgCl2 50 mM

1 M Tris (pH 95) 5 M NaCl and 1 M MgCl2 stocks were prepared in

double distilled water autoclaved and stored at RT Alkaline phosphatase

buffer was prepared by adding appropriate amounts of stock solutions

volume made up with double distilled water and stored at RT

4310 Maleic acid buffer

Maleic acid 100 mM

NaCl 150 mM

Maleic acid was dissolved in double distilled water containing NaCl

pH was adjusted to 7 with NaOH autoclaved and stored at RT

4311 Blocking solution

10 (wv) BSA was dissolved in maleic acid buffer by stirring and

heating autoclaved and stored at RT

4312 Colour development buffer

BCIP (50 mg mL) 70 μL

NBT (50 mg mL) 70 μL

Alkaline Phosphatase buffer 10 mL

50 mg mL NBT in 70 DMF and 50 mg mL BCIP in DMF were

prepared and stored at 4 0C protected from light BCIP may precipitate during

storage and should be warmed at RT to dissolve

Chapter 2

237

4313 Church hybridisation buffer

SDS 7 (wv)

BSA 1 (wv)

EDTA 1 mM

Na-PO4 (pH 74) 025 M

1M NaH2PO4 and 774mL of 1M Na2HPO4 were prepared and mixed to

produce 1M Na-PO4 pH 74 stock For long term storage this was

autoclaved and stored at 4 0C

The hybridization solution was prepared by dissolving 5 g BSA in

~100mL double distilled water 125 mL of 1M NaPO4 175 mL of 20 SDS

and 1 mL of 05 M EDTA were added The volume was made up to 500 mL

and stored at RT SDS precipitates out at cool temperature When this

happens the hybridization buffer was pre- warmed before use to redissolve

the SDS

4314 20X SSC

NaCl 3 M

Sodium citrate 03 M

1753 g of NaCl and 882 g of Sodium citrate were dissolved in ~700

mL of double distilled water The pH was adjusted to 70 with HCl and the

volume was made up to 1 L The solution was autoclaved and stored at RT

44 Methods

441 Microorganisms and culture conditions

All the members of the consortium were grown individually in Luria

Bertoni broth for 18 h under shaking conditions (180 rpm) Cells were

harvested by centrifuging at 10 000 rpm at 4 0C for 15 min The supernatant

was discarded and pellet was washed with minimal medium (331) induced

with 10 ppm DDT and used for the isolation of genomic and plasmid DNAs

442 Isolation of genomic DNA from the bacterial isolates

Genomic DNA was isolated using the method described below

Suspended a 30 h old induced culture (after centrifugation) in 500L

Chapter 2

238

lysozyme solution (containing 2mgmL lysozyme and 50mgmL heat treated

RNase) and incubated at 37 0C for around 30 min or till the cells became

translucent To this was added 250 L of 2 SDS and vortexed gently to mix

until the viscosity of the solution decreased noticeably 250 L of neutral

chloroform solution was added to this and vortexed for 30 seconds spun for 2

min in micro centrifuge and the supernatant was removed leaving the white

interface behind This was repeated twice or till no or very little interface was

seen To this 01 volume of 3M sodium acetate (pH 48) was added and

mixed This was followed by the addition of 1 volume of isopropanol and

mixing Contents were incubated for 24 h at -20 0C and spun for 5 min in the

micro centrifuge Supernatant was poured off The pellet was redissolved in

500 L of TE buffer An agarose gel electrophoresis was carried out to check

the genomic DNA

443 Isolation of plasmid DNA from the bacterial isolates

Plasmid isolation was done according to Maniatis et al (1982)

Single colonies of appropriate strain grown for 24 h inoculated to 2 mL Luria

Bertoni and harvested cells were induced with 10 ppm DDT The cells were

then harvested by centrifuging at 8000 rpm for 5 min and lysed with solution

1 200 L of freshly prepared solution 2 was added and mixed 300 L of

solution 3 was added and centrifuged for 15 min The supernatant was

transferred to a fresh tube and equal volume of phenol- chloroform was

added and vortexed thoroughly The upper aqueous phase was transferred

to a fresh tube equal volume of chloroform was added and centrifuged at 10

000 rpm for 10 min to remove traces of phenol To the upper aqueous layer

2 volumes of absolute ethanol was added and kept at -20 0C for precipitation

The tube was centrifuged at 10000 rpm for 10 min supernatant was

discarded carefully The pellet was air- dried and dissolved in 20 L TE

buffer An agarose gel electrophoresis was carried out to check the plasmid

DNA To isolate cloned plasmids ampicillin was added during inoculation

The cells were not induced with DDT

Chapter 2

239

444 Designing of the oligonucleotide primers

DDT dehydrochlorinase sequences available in the databank were

aligned using Dialign 20 software program and primers were designed using

Primer 30 program and the primer sequence is given in Table 41

Table 41 Nucleotide sequences of the primers used for

screening DDT- dhl1 producing bacterial spp

Primer Primer name Primer sequence

Forward primer dhl1F CTCGAGGCGATTGGCCGCGGCATAAC

Reverse primer dhl1R AAGCTTAAATTTCGGTTTCGGCACGCT

445 PCR amplification of DDT- dhl1 gene from Pseudomonas putida

The PCR amplification was carried out with genomic DNA and plasmid

DNA with Takara thermocycler unit The PCR mixture consisted of

Sterile deionised water 1898 L

Taq Buffer 25 L

dNTP mix 05 L

Taq Polymerase 03 L

Forward Primer 10 L

Reverse Primer 10 L

Template DNA plasmid DNA 10 L

PCR conditions used are as follows

4451 Gradient PCR

A gradient PCR was done with different temperatures from 50- 70 0C

720C 720C940C 940C

500 045

045

50- 700C

100 800

40C

25 Cycles

Chapter 2

240

4452 Touchdown PCR

A touchdown PCR was done as programmed below

4453 PCR with optimised conditions

The amplified PCR products were analysed by agarose gel

electrophoresis

446 Analysis of the PCR products

The gel casting tray was set up with the appropriate comb Agarose

(1) was dissolved in 1X TAE buffer by heating It was cooled to 50 to 60 0C

and was poured into the gel casting unit The comb was removed carefully

from the solidified gel The agarose gel was taken in the electrophoresis tank

containing 1X electrophoresis buffer The wells were loaded with the DNA

samples mixed with gel loading buffer Markers (100 bp markers) were run

along with samples unless otherwise stated After 3- 4 h run at 40 V the gels

were stained with 1 ethidium bromide solution for 10 min and then

destained in double distilled water The DNA was visualised as bands under

U V transilluminator and documented

720C 72 0C 72 0C 4 0C

infin

045

100 030

940C 940C 600C 940C 500C

500 030

10 cycles 30 cycles

045

100 15

72 0C 94 0 C 94 0 C

500 045

115

55 0C

100 800

4 0 C

30 Cycles

72 0C

Chapter 2

241

447 Purification of amplicons of DDT- dhl 1 gene of Pseudomonas

putida T5

The obtained PCR product was purified by using GE gel purification kit

The bands were excised from the agarose gel by cutting with a clean blade

under UV light and transferred into a 20 mL microcentrifuge tube The

following steps were involved in purification of the DNA bands 10 microL capture

buffer type 3 was added for each 10 mg of agarose gel slice The contents of

the tubes were mixed by inverting the tubes and kept in water bath at 60 0C

till agarose completely dissolved GFXtm microspin column was placed in 20

mL collection tube provided by the kit For binding the DNA the sample was

passed though the column and centrifuged at 8 000 rpm for 60 sec The flow

through liquid was discarded and the GFXtm microspin column was placed in

new 20mL collection tube The same procedure was repeated until the entire

sample was poured Now 500 microL of wash buffer type1was added and

centrifuged at 8 000 rpm for 30 sec The collection tube was discarded and

GFXtm microspin column was transferred to clean 15 mL DNase free

microcentrifuge tube 10- 50microL of elution buffer type 6 (nuclease free water)

was added and kept at RT for 60 sec Then it was centrifuged at 8 000 rpm

for 60 sec to elute the DNA The obtained purified DNA sample was stored at

-200C and checked by agarose gel electrophoresis

448 Cloning of DDT- dhl1 gene of Pseudomonas putida T5

4481 Ligation of the purified PCR amplicon into pTZ57RT vector

Ligation of the purified PCR amplicon was done by using Fermentas

kit To a 20 mL microcentrifuge tube 3 microL vector pTZ57RT (018ρ mol

ends) (Fig41) 6 microL 5X ligation buffer 4 microL PCR product (054ρ mol ends)

16 microL nuclease free water and 1 microL T4 DNA ligase were added and incubated

at 4 0C overnight

Chapter 2

242

Fig41 Vector map of pTZ57RT

4482 Transformation of E coli DH5α

44821 Preparation of competent cells

A single colony of E coli DH5α was inoculated to 50 mL of Luria

Bertoni broth and incubated overnight at 37 0C in a shaker The cells were

transferred aseptically to sterile tubes and harvested at 10 000 rpm 10 min

4 0C The above cells were then treated with 10 mL of 60 mM CaCl2 in MOPrsquos

buffer at a pH 65 and vortexed for few minutes The tubes were centrifuged

at 10 000 rpm 4 0C for 10 min The supernatant was discarded and the

pellet was resuspended in the 4 mL solution containing 01 M CaCl2 The

tubes were kept at 4 0C overnight

Chapter 2

243

44822 Transformation of competent cells

Transformation of competent cells into E coli DH5α was done by using

Fermentas transformation kit 150 microL of overnight stored competent cells

were taken and 15 mL of C- media was added The tubes were incubated at

37 0C for 20 min The tubes were centrifuged at 8 000 rpm 4 0C for 1 min

and supernatant was discarded The pellet was suspended in 300 μL of T-

solution The tubes were placed on ice for 5 min Again the tubes were

centrifuged at 8 000 rpm 4 0C for 1 min and supernatant was discarded

The pellet was resuspended in 120 μL of T- solution and kept in ice for 5 min

To 50 μL of above solution 25 μL of ligation mixture containing 14 ng vector

DNA was added and kept in ice for 5 min The solution was poured to pre-

warmed Luria Bertoni media and incubated at 37 0C overnight

449 Selection of transformants

100 μL of transformation mix was plated onto Luria Bertoni agar plates

containing 100 μL mL ampicillin The plates were incubated at 37 0C

overnight for the colonies to grow

4410 Analysis of the transformants

The plasmid profile of the transformed colonies was checked by gel

electrophoresis of plasmids with and without insert

4411 Isolation of plasmid DNA from transformed colonies

The transformed cells in Luria Bertoni broth were taken in 2 mL micro

centrifuge tube and plasmid isolation was done as described under section

443 An agarose gel electrophoresis was carried out to check the

transformation

4412 Sequencing of the cloned genestransformed plasmids

The transformed plasmid pTZ57RT containing the DDT-dhl 1 insert

was sent for sequencing to Bio-serve Biotechnologies Pvt Ltd Hyderabad

Chapter 2

244

4413 Sub- cloning of DDT- dhl1 gene of Pseudomonas putida T5


Plasmid DNA from the transformed colonies was isolated by alkali lysis

as described under section 4411

44132 Restriction digestion of recombinant plasmids for insert release

The cloned vector was double digested with EcoR1 and Hind III for the

release of cloned insert The restriction digestion was carried out as per

suppliersrsquo instructions After the addition of restriction enzyme along with its

buffer the tubes were incubated at 37 0C for 1 h The insert release was

cross- checked on agarose gel electrophoresis

44133 Sub- cloning the DDT- dhl1 insert into E coli DH5α

The insert DNA was ligated to pUC18 (Fig42) vector and also pET28a

as per the procedure described under 4413 The cloned plasmid pUC 18

was transformed into E coli DH5α and pET28a (Fig43) to E coli BL21

according to 44822 The transformants were selected on Luria Bertoni

agar ampicillin plates The E coli BL21 transformed with pET28a vector were

selected with IPTG- X- Gal selection

Fig42 Vector map of pUC18

Chapter 2

245

Fig43 Vector map of pET28a

44134 Isolation of DDT- dhl1 enzyme

The cells of E coli were grown on Luria Bertoni broth containing

ampicillin for 6 h and then were induced with IPTG (04 mM) The cells were

harvested after 16 h by centrifugation at 10 000 rpm 4 0C for 10 min Cells

were washed with phosphate buffer The crude extracts of recombinant E

coli were prepared by sonication The supernatant was purified by using

HIS- tag purification kit The eluent was subjected to electrophoresis on a

12 (wv) SDS- PAGE The amount of protein was determined by

determining O D at 280 nm The enzyme was estimated for activity in micro

titre plate The assay was based on decrease in the pH of a weakly buffered

medium containing phenol red as an indicator dye The colour change from

reddish orange to yellow which occur when Cl- is released because of

enzyme action on DDT was identified as the enzyme activity The culture

showing dehydrohalogenase activity in micro titre plate was tested for DDT

Chapter 2

246

clearance assay on minimal- DDT- agar plate to conform the activity Spray

plates were prepared with 15 agar in minimal medium on Petri dishes

The cloned isolate was streaked on to the plate The surface of the plate was

sprayed with 01 DDT as acetone solution The plates were incubated at

37 0C for 4- 5 days The formation of clearance surrounding the colonies was

used as an indicator of DDT-dehydrohalogenase activity

4414 Labelling DDT- dhl1 gene by using DIG kit (digoxigenin- dUTP

alkali labile labelling)

The labelling of the DDT- dhl1 gene was done by using DIG kit The

components of the kit are given in Table 42 For the labelling of the DDT-

dhl1 gene of Pseudomonas putida T5 PCR was carried out as per the

procedure 4453 and the obtained PCR product was purified by the

procedure given in 447

The O D of the purified product was determined by using

spectrophotometer (Schimadzu Japan) at 260 280 nm

10 ng- 3 μg of DNA was taken and sterile double distilled water was

added and the final volume was made up to 15 μL The DNA was denatured

by heating in a boiling bath for 10 min and quickly chilled in ice for 10 min

To this 2 μL of hexanucleotide mix (vial 5) 2 μL of dNTP labelling mix (vial 6)

and 1μl of Klenow enzyme (vial 7) were added mixed and centrifuged briefly

The reaction vials were incubated at 37 0C for 20 h After 20 h the reaction

was stopped by adding 2 μL of 02 M EDTA (pH 80) The labelled probe was

stored at -20 0C

4415 Restriction digestion of genomic DNA

For the restriction digestion of genomic DNA of Pseudomonas putida

T5 the restriction enzymes used are given in Table 43

Chapter 2

247

Table 42 Components of DIG kit

Bottlecap

Label Content (including function)

1 Unlabelled

control DNA

20 μL unlabelled control DNA1 [100 μg mL]

10 mM Tris- HCl1 mM EDTA pH 80

Mixture of pBR328 DNA digested separately with

Bam H1 Bgl 1and Hinf 1 The separate digests

are combined in a ratio of 2 3 3

Sizes of 16 pBR328 fragments 490 2176 1766

1230 1033 653 394 298(2X) 234 (2X) 220 and

154 (2X) bp

Clear solution

Controls target in a southern blot

2 Unlabelled

controlled DNA

20 μL unlabelled control DNA2

[200 μg mL]

pBR328 DNA that has been linearized with Bam H1

Clear solution

To practice labelling and to check labelling

efficiency

3 DNA dilution

buffer

2 x 1mL DNA dilution buffer

[50 μg mL herring sperm DNA in 10 mM Tris-HCl

1 mM EDTA pH 80 (20 0C)]

Clear solution

For the dilution steps in the semi- quantitative

determination of labelling efficiency

4 Labelled control

DNA

50μl labelled control DNA

Linearized pBR328 DNA labelled with digoxigenin

according to the standard protocol

1μg template DNA and aprox 250ng digoxigenin-

labelled DNA

Clear solution

Chapter 2

248

Table 43 Restriction enzymes used for digestion

Sl No Type of restriction enzyme

1 Sau 3A

2 Bgl ΙΙ

3 Sal І

4 Bam H І

5 Xho І

6 Hind Ш

7 Pvu ΙΙ

Estimation of the yield of DIG- labelled DNA

5 Hexa-

nucleotide mix

80 μL of 10X conc Hexanucleotide mix

[625 A260 unitsmL] random hexanucleotides

500 mM Tris-HCl 100 mM MgCl2 1 mM

Dithioerythritol [DTE] 2 mg mL BSA pH72

Clear solution

Component of the labelling reaction

6 dNTP Labelling

Mix

80 μL of 10X conc dNTP Labelling mix

1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM

dTTP 035 mM DIG- 11- dUTP alkaline- labile

pH75 ( 20 0C)]

Clear solution


7 Klenow Enzyme

Labelling Grade

40 μL Klenow Enzyme Labelling Grade

[2 units μL DNA Polymerase 1 (Klenow Enzyme

large fragment)]

Clear solution

Synthesis of DIG- labelled DNA

Chapter 2

249

To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme

buffer and enzyme were added respectively The vials were kept in the water

bath at 37 0C for 24 h

Agarose gel electrophoresis was carried out for the restriction digested

genomic DNA of Pseudomonas putida T5 For the restriction digested

genomic DNA southern blot analysis was carried out

4416 Southern blot analysis of DDT- dhl1 gene

The conventional transfer method introduced by Southern relies on a

gel-sandwich setup The southern blot was carried out as follows the gel

was placed over a buffer-soaked wick and overlaid with a piece of transfer

membrane A stack of dry paper towels was then placed on the top of the gel

sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick

through the gel and the membrane and up towards the dry paper towels

Fractioned DNA fragments in the gel will be carried upward by capillary buffer

flow and retained on the membrane thus generating an imprint that is

identical to the electrophoresis pattern of the gel After 18 h of transfer the

DNA was then permanently fixed on the membrane by using U V cross-

linking Capillary action was usually conducted with a neutral high salt 20X

SSC buffer The membrane was removed and kept in the hybridization bottle

containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the

probe was taken in a fresh Eppendorf tube and placed in boiling water for 10

min and immediately chilled on ice The denatured probe was added to 15

mL fresh hybridization solution and the hybridization was carried out overnight

at 65 0C The hybridization solution was discarded and the membrane was

thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed

membrane was transferred into a clean dish containing maleic acid buffer and

the membrane was washed for 2- 5 min with gentle shaking at RT The

membrane was then replaced in the freshly prepared blocking solution and

incubated with shaking for 60 min The blocking solution was discarded and

diluted anti-DIG- AP antibody was added and the membrane was incubated

at RT for 40- 60 min with gentle shaking on the rocker The membrane was

then washed with maleic acid buffer for 20 min with shaking and this step

Chapter 2

250

was repeated once by using the fresh washing tray The membrane was

equilibrated in the Alkaline Phosphatase buffer with gentle shacking The

colour development solution was added to the membrane and incubated in

the dark at RT for 30 min The colour development was monitored with clear

zones

4417 Production and isolation of antibody

Twenty week old Single comb white leg horn poultry (layers) were

immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos

complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)

was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to

poultry intramuscularly (at breast muscle) Subsequent immunizations were

done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every

15 days for 6 months

Egg yolk antibody (IgY) was isolated by PEG method Yolk was

separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH

72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform

yolk was added and stirring was continued for another 30 min at RT The

precipitate formed was removed by centrifugation To the supernatant 14

(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The

solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus

obtained (which contains IgY) was dissolved in a known quantity of

phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water

for 24h at 4 0C to obtain purified antibodies

4418 Western blot analysis of DDT- dhl1 gene

44181 SDS- PAGE was done according to Laemmli (1970)

44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose

membrane

Whatman No3 filter papers were cut according to the gel size The

nitrocellulose membrane was cut to the same size of SDS-PAGE gel The

Chapter 2

251

filter papers containing 8 sheets were wetted in transfer solution Glycine 15

Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the

transfer unit Then nitrocellulose membrane was placed on the filter papers

and then gel was placed over it 8 sheets of Whatman No 3 were placed over

the gel The current passed through at the rate of 08mA cm2 for 75 min A

part of membrane was stained with amido black (002 in distilled water) and

rest was taken for western blot

44183 Western blot

SDS-PAGE transferred Nitrocellulose membrane was blocked with 2

gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes

The membrane was incubated with the primary antibody (IgY 15000 in

TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10

min with three changes in TBST buffer Then the membrane was incubated

with the secondary antibody Goat anti chicken IgY conjugated to alkaline

phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by

washing with TBST for 10 min with three changes

44184 Colour development

To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM

MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70

dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl

formamide) were added as substrates The colour development reaction was

stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or

simply by replacing the buffer by water

45 Results

451 Screening for DDT- dhl 1 producers

Genomic and plasmid DNA from all the ten bacterial isolates were

screened by gradient PCR and touchdown PCR for the presence of DDT-

dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in

Table 41 All the isolates gave positive amplification with genomic DNA

(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose

Chapter 2

252

gel Distinct PCR products of around 750- 800 bp were obtained In our

studies genomic DNA of Pseudomonas putida T5 was chosen for further

work

Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA

Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10

Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA

Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10

To optimise the PCR conditions gradient PCR between 50- 70 0C was

carried out But in all the temperatures the amplification was not much

(Fig46) The touchdown PCR conditions also did not give better

amplification (Fig46) The PCR done with optimised conditions of

1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253

denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and

extension at 72 0C for 45 sec gave very good amplification (Fig47)

Fig46 Amplification of the gene at different temperatures

Fig47 Amplification of the gene by optimised PCR conditions

Lane 1 PCR amplification at 50 0C





Lane 6 PCR amplification using

touchdown PCR

Lane 1 100 bp marker

Lane 2 3 4 PCR products

Chapter 2

254

452 Large batch amplification and purification of the PCR product

Large batch trails were done to get good quantity of amplified product

The PCR conditions were maintained the same as described under section

4453 A PCR product was run in preparatory agarose gel The amplified

PCR product band was cut and separated from the gel and was purified using

PCR product purification kit (GE health care)


The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as

per instructions provided along with the kit E coli DH5α grown overnight in

Luria Bertoni broth was treated with CaCl2 to give competent cells The

ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene

was transformed into competent cells of E coli DH5α

The transformants were plated on Luria Bertoni agar containing

ampicillin (100 microg mL) (Fig48) The transformation efficiency was around

50 A total of about 50 transformant colonies that grew on Luria Bertoni-

ampicillin plates were replica plated on to minimal media- DDT- agar

ampicillin plates by toothpick method to select DDT dehydrohalogenase

clones 20 of these colonies grew on minimal media- DDT- agar ampicillin

plates These colonies showed good growth in minimal media- DDT- agar

ampicillin plates

Fig48 Transformant cells on Luria Bertoni- ampicillin agar

Chapter 2

255

454 Selection of the transformants

The colonies that showed good growth in minimal media- DDT- agar

ampicillin plates were then picked up and inoculated to Luria Bertoni broth

containing ampicillin (100 microg mL) The transformants showing maximum

growth in the medium were selected for further studies The transformants

from the medium were harvested and the plasmids were isolated from the

transformants by alkali lysis method (443)

The agarose gel electrophoresis of the transformed plasmids run along

with untransformed pTZ57RT plasmid showed that one of the colonies (E

coli DH- pTZ15) showed an increase in molecular weight indicating the

presence of the complete insert (lane 5 Fig49) The insert in the

transformed clone (E coli DH- pTZ12) was sequenced

Fig49 Gel showing the transformed plasmids

The sequence analysis of Pseudomonas putida T5 indicated that the

protein had 238 residues This amounted to a reverse translation product of

714 base sequences of most likely codons The molecular weight of the

reverse translated protein has been given as 277893 with a theoretical pI of

921

The ExPASSy blast results for finding out the homology of DDT- dhl 1

gene with Pseudomonas spp genome indicated that the sequence had 24-

1 2 3 4 5 6 7

Chapter 2

256

26 identity with Pseudomonas putida genome The DDT-dhl 1 gene

showed homology with acetolactate synthase gene sequence The southern

blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1

probe was done The genomic DNA of Pseudomonas putida yielded positive

hybridisation More than 1 hybridising bands were observed for few

restriction enzymes digested EcoR1 yielded around 5 bands whereas others

yielded only 2 bands The cloned insert was sub- cloned into pUC18 and

pET28a vectors The expressed protein was analysed for enzyme activity

product of enzyme action and western blot analysis The colorimetric assay

is shown in Fig410 The enzyme exhibited colour change of the dye

because of chloride release The DDT- dehalogenase positive colonies also

exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)

DDT precipitation disappeared in 5 days because of the degradation of the

compound The western blot analysis gave positive colour with the cloned

protein (Fig412) The product of analysis of the enzyme action indicated

formation of DDD as identified by GC- MS analysis (Fig413)

Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme

Red colour Control Yellow colour DDT- dehydrohalogenase positive

Chapter 2

257

Fig411 DDT-dehydrohalogenase positive plates

Fig412 Western blot hybridization of DDT-dhl1 protein of

Pseudomonas putida probed with antibody raised

against DDT-dehydrohalogenase enzyme

Chapter 2

258

Fig413 GC- MS pattern

A DDT

B DDD

The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15

was recovered by restriction digestion and was ligated to restriction digested

pUC18 cloning vector The transformants were selected as given above

The insert was recovered from the cloned vector by restriction digestion and

this was transformed into pET28a vector This transformed plasmid vector

was cloned into E coli BL 21

The purified PCR product of Pseudomonas putida T5 was labelled

using non radio active nucleic acid labelling by DIG- labelling kit system

according to users guide as described above The genomic DNA of

Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I

Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers

and incubating for 16hrs at 37 0C The restricted fragments were separated

on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL

Chapter 2

259

probes were allowed to hybridize with the denatured DNA on the nylon

membrane The southern hybridization of genomic DNA from the strain

Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-

DHL probes tested More than one hybridizing band was observed for few

restriction enzymes used for digestion (Fig414) These results indicated that

presence of more than one band may be due to the presence of more than

one copy of the gene

Fig414 Southern blot hybridization of genomic DNA

of Pseudomonas putida T5 probed with DIG

labelled T5 probe

46 Discussion

The recalcitrance of many synthetic chemicals to biodegradation is

mainly due to the lack of enzymes that can carry out critical steps in the

catabolic pathway This especially holds good for low- molecular weight

halogenated compounds These xenobiotic chemicals are less water soluble

and less bio- available Theoretically these could be converted by short

metabolic routes to intermediates that support cellular growth under aerobic

Chapter 2

260

conditions Yet no organisms have been found that oxidatively degrade and

use these important environmental chemicals as a carbon source Attempts

to obtain enrichments or pure cultures that aerobically grow on these

chemicals have met no success However some other halogenated

chemicals are easily biodegradable and cultures that utilize chloroacetate 2-

chloropropionate and 1-chlorobutane can be readily enriched from almost any

soil sample (Leisinger 1996 van Agteren et al 1998) For still other

compounds degradative organisms have been isolated but only after

prolonged adaptation due to pre- exposure to halogenated chemicals in the

environment In our studies also a DDT-degrading microbial consortium was

isolated after a long enrichment of the DDT- contaminated soil All the

individual isolates ie ten bacterial strains that constituted the consortium

were found to be necessary for the complete degradation of DDT These

individual isolates were found to act synergistically during degradation of

DDT

With halogenated compounds an obvious critical step in a potential

biodegradation pathway is dehalogenation Biochemical research with

organisms that grow on halogenated compounds has shown that a broad

range of dehalogenases exists both for aliphatic and aromatic compounds

The first enzyme that is responsible in the dehalogenation of DDT to DDD has

been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are

hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)

enzyme and genes encoding the enzyme An attempt was made in our

laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-

dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by

gradient PCR and touchdown PCR The PCR amplified gene product was

cloned and sequenced The gene showed sequence similarity of 93 to

Acetolactate synthase of Serratia spp There are many xenobiotic

degrading enzymes which have shown such sequence similarities to other

family of enzymes An interesting type of aliphatic dehalogenase is the

enzyme (LinA) that is responsible for the first step in the bacterial degradation

of lindane (-hexachlorocyclohexane) where HCl was eliminated converting

the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et

Chapter 2

261

al 2001) The structure has not been solved but a mechanism was

predicted on the basis of the stereochemistry of the reaction and low but

significant sequence similarity to scytalone dehydratase Similarly hydrolytic

dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has

been shown to belong to the enoyl hydratase superfamily A specific

hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of

atrazine has been shown to be related to melamine deaminase (TriA)

(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing

the conversion of dichloromethane to formaldehyde in a glutathione-

dependent reaction and another group of dehalogenating proteins

chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that

degrade the nematocide 13-dichloropropene have been found to belong to

the tautomerase superfamily of proteins Halohydrin dehalogenases belong

to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de

Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of

proteins that also includes non- haem integral membrane desaturases

epoxidases acetylenases conjugases ketolases decarbonylases and

methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the

amidohydrolase superfamily Other members of the amidohydrolase

superfamily are triazine deaminase hydantoinase melamine deaminase

cytosine deaminase and phosphotriesterase Rhodococcus haloalkane

dehalogenase (DhaA) the dehalogenase gene was preceded by the same

invertase gene sequence and a regulatory gene and on the downstream side

an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene

(Poelarends et al 2000b)

Analysis of the genetic organization of biodegradation pathways

provides insight into the genetic processes that led to their evolution It

appears that catabolic genes for xenobiotic compounds are often associated

with transposable elements and insertion sequences They are also

frequently located on transmissible plasmids One striking example of a

mobile element that has assisted catabolic genes in their dissemination is IS

1071 This insertion element flanks the haloacetate dehalogenase gene

dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)

Chapter 2

262

the haloalkane dehalogenase gene dhaA on the chromosome in P

pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes

atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett

2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas

putida UCC22 (Fukumori and Saint 1997) and presumably also the p-

sulfobenzoate degradative genes on plasmids pTSA and pPSB in

Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and

Cook 1997) These observations clearly indicate that gene mobilization

between and within replicons is an important process during genetic

adaptation It also suggests that genes that are involved in biodegradation of

xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration

transposition homologous recombination and mobilization Association of

dehalogenase sequences with mobile genetic elements has also been

observed in other cases viz with haloacetate dehalogenases (Slater et al

1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane

dehalogenase (Schmid-Appert et al 1997) and with -

hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies

the native plasmid did not show amplification of the dehalogenase sequence

indicating that the DDT-dehydrohalogenase gene is a genomic character but

not a plasmid borne character

It has become clear that most dehalogenases belong to protein

superfamilies that harbour both dehalogenases and proteins that carry out

completely different reactions (de Jong and Dijkstra 2003) Hisano et al

(1996) and Ridder et al (1999) defined the haloalkane dehalogenases

belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase

fold main domain the α β- hydrolase structural fold is shown also to be found

in lipases acetylcholinesterases esterases lactonases epoxide hydrolases

and others showing that the haloalkane dehalogenases belong to a protein

superfamily of which the members carry out diverse reactions mostly with

non-halogenated compounds

Less is known about the origin of the structural genes that encode

critical enzymes such as dehalogenases and the degree of divergence that

occurred during evolution of the current sequences Theoretically it is

Chapter 2

263

possible that a current gene for a specific critical (dehalogenase) reaction was

already present in the pre-industrial gene pool Alternatively there could be a

short evolutionary pathway that led from an unknown pre- existing gene to the

gene as we currently find it in a biodegradation pathway It has even been

suggested that a new sequence for an enzyme acting on a synthetic

compound could evolve through the activation of an unused alternative open

reading frame of a pre-existing internal repetitious coding sequence (Ohno

1984) Here it should be noted that the similarity of a dehalogenase to

members of an enzyme superfamily that catalyse other reactions generally

does not provide information about the process of adaptation to xenobiotic

compounds The level of sequence similarity that exists between a

dehalogenase and other proteins in a phylogenetic family is usually less than

50 Therefore the time of divergence should be much earlier than a

century ago and the process of divergence thus cannot be related to the

introduction of industrial chemicals into the environment If the

dehalogenases and other critical enzymes that occur in catabolic pathways

have undergone recent mutations there should be closely related sequences

in nature that differ from the current enzymes by only a few mutations No

such primitive dehalogenase has yet been detected with the notable

exception of TriA the enzyme that dehalogenates the herbicide atrazine

Another issue is the function of the pre-industrial dehalogenase or

dehalogenase-like sequences from which the current catabolic systems with

their activated and mobilized genes originate The original genes may have

been involved in the dehalogenation of naturally occurring halogenated

compounds of which there are many (Gribble 1998) Such proteins may

fortuitously also have been active with a xenobiotic halogenated substrate

just because of their lack of substrate specificity Alternatively the

evolutionary precursor of a dehalogenase may have catalysed a different

reaction that has some mechanistic similarity to dehalogenation in which

case the original enzyme may or may not have shown some dehalogenation

activity due to catalytic promiscuity Finally the precursor genes for

dehalogenases may have been silent or cryptic genes with no clear function

for the pre- industrial host (Hall et al 1983) In all cases the gene could

Chapter 2

264

have become functional in a dehalogenation pathway as a result of the

fortuitous ability to catalyse dehalogenation of a xenobiotic compound

possibly after acquisition of some mutations

One way to obtain information about the evolutionary origin of

dehalogenase genes is to compare the dehalogenase sequences that have

been detected in different bacterial cultures If closely related sequences are

present this would make it possible to identify sequence differences and to

determine the effect of the mutations on substrate selectivity Currently the

whole sequence of more than 300 bacterial genomes is available If we find

closely related sequences in these databases they could define evolutionary

ancestors of the current dehalogenases

When searching for putative alkane hydroxylase genes in

environmental DNA (Venter et al 2004) a large number of alkB and alkM

homologues were again detected including two sequences that were 82

similar to alkB

Even though the same dehalogenase sequences are detected in

organisms that are isolated in different geographical areas and in some

cases even on different substrates they are not the most abundant

dehalogenase sequences identified in whole genome sequencing projects

and massive random sequencing Thus it appears that enrichment

techniques explore a different segment of sequence space than massive

sequencing of environmental DNA The presence of large numbers of

unexplored functional sequences in genomic databases suggests that the

biotransformation scope of microbial systems has an enormous potential for

further growth

47 Conclusions

Bacterial dehalogenases catalyse the cleavage of carbon-halogen

bonds which is a key step in aerobic mineralization pathways of many

halogenated compounds that occur as environmental pollutants DDT-

dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with

the removal one HCl The gene responsible for the dehalogenation was

cloned and sequenced The gene sequence showed similarity to acetolactate

Chapter 2

265

synthase gene There is a broad range of dehalogenases which can be

classified in different protein superfamilies and have fundamentally different

catalytic mechanisms Identical dehalogenases have repeatedly been

detected in organisms that were isolated at different geographical locations

indicating that only a restricted number of sequences are used for a certain

dehalogenation reaction in organohalogen-utilizing organisms

One of the striking observations concerning the distribution and

evolution of key catabolic genes is that identical sequences have repeatedly

been detected in organisms that are enriched on xenobiotic halogenated

substrates as a carbon source Probably the number of solutions that nature

has found to degrade these compounds is very small and horizontal

distribution occurs faster than generation of new pathways Indeed the

dehalogenase genes are often associated with integrase genes invertase

genes or insertion elements and they are usually localized on mobile

plasmids

It is likely that ongoing genetic adaptation with the recruitment of silent

sequences into functional catabolic routes and evolution of substrate range by

mutations in structural genes will further enhance the catabolic potential of

bacteria toward synthetic organohalogens and ultimately contribute to

cleansing the environment of these toxic and recalcitrant chemicals

References

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Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden

HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A

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Bourquin AW 1977 Degradation of malathion by salt- marsh

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Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides

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de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial

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de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB

Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol

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Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization

of lin genes and IS6100 among different strains of

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evidence for horizontal gene transfer Journal of Bacteriology 186

2225- 2235

Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis

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putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408

Gribble GW 1998 Naturally occurring organohalogen compounds Acc

Chemical Research 31 141- 152

Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and

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Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial

evolution Molecular Biology and Evolution 1 109- 124

Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal

structure of L-2-haloacid dehalogenase from Pseudomonas spp YL

An alpha beta hydrolase structure that is different from the alpha beta

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Junker F Cook AM 1997 Conjugative plasmids and the degradation of

arylsulfonates in Comamonas testosterone Applied Environmental

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Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology

between two haloacetate dehalogenase genes encoded on a plasmid

from Moraxella sp strain B Journal of General Microbiology 138

1317- 1323

Laemmli UK 1970 Cleavage of structural proteins during the assembly of the

head of bacteriophage T4 Nature (London) 227 680- 685

Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds

Current Opinions in Biotechnology 7 295-300

Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory

manual Cold Spring Harbour Laboratory New York

Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of

111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes

eutrophus A5 Applied and Environmental Microbiology 60 51- 55

Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of

protein fold and catalytic residues of gamma-

hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-

477

Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of

the preexisted internally repetitious coding sequence Proceedings of

National Academy of Sciences USA 81 2421- 2425

Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen

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evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative

pathways Journal of Bacteriology 182 2191- 2199

Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR

2000b Haloalkane utilizing Rhodococcus Strains isolated from

geographically distinct locations possess a highly conserved gene

cluster encoding haloalkane catabolism Journal of Bacteriology 182

2725- 2731

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Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis

KN 1994 The behavior of bacteria designed for biodegradation

Biotechnology 12 1349- 1356

Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of

intermediates in the dehalogenation of haloalkanoates by L-2-haloacid

dehalogenase Journal of Biological Chemistry 274 30672- 30678

Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997

Association of newly discovered IS elements with the dichloromethane

utilization genes of methylotrophic bacteria Microbiology 143 2557-

2567

Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine

deaminase and atrazine chlorohydrolase 98 percent identical but

functionally different Journal of Bacteriology 183 2405- 2410

Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and

molecular basis of pesticide degradation by microorganisms Critical

Reviews in Biotechnology 19(3)197- 225

Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of

Pseudomonas putida PP3 on chromosomally located transposable

elements Molecular Biology and Evolution 2 557- 567

Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI

from Pseudomonas putida PP3 is carried on an unusual mobile genetic

element designated DEH Journal of Bacteriology 174 1932- 1940

Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V

Damborsky J 2001 Reaction mechanism and stereochemistry of

gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of


van Agteren MH Keuning S Janssen DB 1998 Handbook on

Biodegradation and Biological Treatment of Hazardous Organic

Compounds Dordrecht the Netherlands Kluwer Academic Publishers

van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of

Xanthobacter autotrophicus GJ10 to bromoacetate due to activation

and mobilization of the haloacetate dehalogenase gene by insertion

element IS1247 Journal of Bacteriology 177 1348- 1356

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269

Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA

2004 Environmental genome shotgun sequencing of the Sargasso

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Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical

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41259- 41262

Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-

chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology

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270

4A1 Introduction

The biological destruction of toxic and hazardous chemicals is an

important contribution of microbial metabolism Microorganisms convert

complex hazardous organic compounds via their central metabolic routes to

CO2 or other simple organic compounds Organisms that bring about their

degradation have been isolated from their natural environments During

aerobic degradation of chlorinated compounds by microorganisms molecular

oxygen serves as the electron acceptor Several chloroaliphatic compounds

have been shown to be degraded aerobically Microbes play an essential role

in the bioconversion and total breakdown of pesticides Among the microbial

communities bacteria and fungi are the major degraders of pesticides

Yeasts microalgae and protozoa are less frequently encountered in the

degradation process

Several microorganisms are known to degrade DDT anaerobically

(Wedemeyer 1967) The primary metabolic mechanism that was studied was

the reductive dechlorination of DDT with the formation of DDD (11-dichloro-

22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman

and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the

first product of DDT metabolism has been found to be DDD by

dehydrochlorination DDD was further degraded through dechlorination

dehydrochlorination and decarboxylation to DBP or to a more reduced form

DDM

In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5

was shown to be converted to DDD The enzyme protein was purified and

characterized An attempt was made to study the binding pattern of

substrates and inhibitors of DDT-dehydrohalogenase using a computational

method Threendashdimensional models were constructed and substrate

specificity binding site of enzymes and the activity of the selected substrates

were compared using the molecular docking A comparison of theoretical and

experimental results of enzyme activity was also investigated

Chapter 2

271

4A2 Materials and methods

4A21 Softwares

The following softwares were used in molecular modelling of DDT-

dehydrohalogenase

4A211 BLAST computer programme

Similarity to the DDT-dehydrohalogenase nucleotide sequence was

obtained by using BLAST computer programme

4A212 Clustal W

Multiple sequence alignment was done using Clustal W

4A213 COMPUTE PI

Primary structure analysis tools such as COMPUTE PI was used for

isoeletric point and molecular weight

4A214 PROTPARAM

This was used for deciphering computation of various physical and

chemical parameters for a given protein sequence

4A215 Rapid automatic Detection and alignment of repeats (Radar)

Rapid automatic Detection and alignment of repeats in protein

sequences programme is used for alignment of repeats in protein sequences

4A216 MOTIFS

MOTIFS is to identify the number of motifs in a sequence

4A217 CONSERVED DOMAIN DATABASE

This is used to identify the conserved domain present in a protein

sequence

4A218 PROSITE

This is to scan a protein sequence for the occurrence of patterns and

profiles stored in the prosite data

Chapter 2

272

4A129 PEPINFO

This is used to plot simple amino acids properties in parallel and Plot

of hydrophobicity

4A2110 PEPWINDOW

This will display protein hydropathy

4A2111 PEPSTATS

This was used to report information on Molecular Weight Number of

residues Average residue weight Charge Isoeletric point for each physic

chemical class of aminoacids and Molar extinction coefficient at 1mgmL

4A2112 Peptide Cutter

This predicts potential cleavage sites cleaved by proteases or

chemicals in a given Protein sequence

4A2113 Reverse Translate

Reverse Translate accepts a protein sequence as input and uses a

codon usage table to generate a DNA sequence representing the most likely

non-degenerate coding sequence

4A2114 Neural Network

Neural network was used to predict the secondary structure

4A2115 3DPSSM OR PHYRE

3DPSSM OR PHYRE was used to predict the Tertiary structure

4A2117 Discovery studio 20

Docking was processed by Discovery studio 20

Homology modelling

Structural modelling of the sequence of DDT-dehydrohalogenase

obtained from the above sequencing process (Uniprot

tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the

Phyre2 remote homology modelling server since the conventional BLAST

program could not find homologues with satisfying sequence similarity and

Chapter 2

273

structure Phyre protocols can be used to achieve high accuracy models at

very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)

The query sequence was submitted to the Phyre2 server The Phyre2 server

uses a library of known protein structures taken from the Structural

Classification of Proteins (SCOP) database (Alexey et al 1995) The

sequence of each of these structures is scanned against a non-redundant

sequence database and a profile constructed and deposited in the lsquofold

libraryrsquo The query sequence is scanned against the non-redundant

sequence database and a profile is constructed Five iterations of PSI-Blast

are used to gather both close and remote sequence homologs and are

combined into a single alignment with the query sequence as the master

Following profile construction the query secondary structure is predicted

Three independent secondary structure prediction programs are used in

Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet

(Cuff etal 1999) These programs provide a confidence value at each

position of the query for each of the three secondary structure states These

confidence values are averaged and a final consensus prediction is

calculated In addition the program Disopred (Ward et al 2004) is run to

calculate a two-state prediction of which regions of the query are most likely

to be structurally ordered (o) and which disordered (d) This profile and

secondary structure is then scanned against the fold library using a profilendash

profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This

alignment process returns a score on which the alignments are ranked An e-

value is calculated by fitting the scores to an extreme value distribution The

top ten highest scoring alignments are then used to construct full 3D models

of the query Where possible missing or inserted regions caused by

insertions and deletions in the alignment are repaired using a loop library and

reconstruction procedure Finally side- chains are placed on the model using

a fast graph-based algorithm and side chain rotamer library Phyre2 also

incorporates ab initio folding simulation called Poing2 to model regions of the

proteins with no detectable homology to known structures The model

generated by Phyre2 is then retrieved and the quality of the model was

Chapter 2

274

evaluated Profiles- 3D and assessed using Ramachandran map using

Accelrys Discovery studio 25 (DS25)

4A2118 CDOCKER

CDOCKER is an implementation of a CHARMm based docking tool

using a rigid receptor

The following steps are included in the CDOCKER protocol

A set of ligand conformations were generated using high-temperature

molecular dynamics with different random seeds

Random orientations of the conformations were produced by

translating the center of the ligand to a specified location within the receptor

active site and performing a series of random rotations A softened energy

was calculated and the orientation was kept if the energy was less than a

specified threshold This process was continued until either the desired

number of low-energy orientations were found or the maximum number of

bad orientations was tried

Each orientation was subjected to simulated annealing molecular

dynamics The temperature was heated up to a high temperature then cooled

to the target temperature

A final minimization of the ligand in the rigid receptor using non-

softened potential was performed

For each final pose the CHARMm energy (interaction energy plus

ligand strain) and the interaction energy alone were calculated The poses

were sorted by CHARMm energy and the top scoring (most negative thus

favorable to binding) poses were retained

4A3 Results and Discussion

4A31 Sequence analysis

The sequence of DDT-dehydrohalogenase along with amino acid

sequence is given in the fig4A31

Chapter 2

275

Fig4A31 Sequence analysis of DDT-dehydrohalogenase

The enzyme was found to have 238 amino acid residues (Table

4A31)

Chapter 2

276

4A32 Sequence similarity by BLAST searching

DDT-dehydrohalogenase was found to have substantial sequence

similarity to that of acetolactate synthase of few bacterial strains The N-

terminal and C-terminal homology is given in fig4A32a and fig4A32b

Db AC Description Score E-value

tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029

tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038


tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21

tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21

tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35

Fig4a31a Graphical overview of the alignments

Chapter 2

277

Alignments

tr Q9HVA0 Q9HVA0_PSEAE

SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]

574 AA

Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566

Chapter 2

278

Fig4A32a Multiple sequence alignment of N terminal

region of homologous

Chapter 2

279

Fig4A32b Multiple sequence alignment of C terminal


4A33 Composition of the enzyme

Number of amino acids 238

Molecular weight 277893

Theoretical pI 921

Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)

Total number of positively charged residues (Arg + Lys) 44

Grand average of hydropathicity (GRAVY) -053

The amino acid with highest composition is lysine and the protein was found

to be stable


Chapter 2

280

Table 4A31 Amino acid composition of DDT-dehydrohalogenase

Amino acid Amino acid Amino acid

Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21

Lysine was 113 followed by alanine (8) Histidine and tryptophan were

low in concentration (21)

4A34 Atomic composition

Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23

Formula C1234H1927N345O341S23

Total number of atoms 3870

4A35 Extinction coefficients

Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water

Ext coefficient 44765

Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines

The estimated half-life is 30 hours

4A36 Instability index

The instability index (II) is computed to be 3624 this classifies the protein as

stable

Chapter 2

281

4A37 Aliphatic index

Aliphatic index is 6315

4A38 Formula

C1234H1927N345O341S23 and total number of amino acid in the given

sequence is 238

4A39 RADAR RESULTS

RADAR did not find any repeats in the sequence

4A310 MOTIF RESULTS

Number of found motifs 112

No motif was found in prosite pattern

No motif was found in prosite profile

4A311 CD DOMAIN RESULTS

Fig4A33a CD DOMAIN RESULTS

10 20 30 40 50 60 70

80

||||||||

MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG

GEKHLCDKFAKILR 43

ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-

AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121

cd03183 3

ARVDEYLAWQHTNLRLGCAKY-

FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--

PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282

GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-

MTAWKARVRAKICNCKYQAHKRL 165

cd03183 79

SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII

123

Fig4A33b Hphob Kyte amp Doolittle Plot

Property Residues Number Mole

Tiny (A+C+G+S+T) 63 26471

Small (A+B+C+D+G+N+P+S+T+V) 107 44958

Aliphatic (I+L+V) 37 15546

Aromatic (F+H+W+Y) 33 13866

Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361

Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639

Charged (B+D+E+H+K+R+Z) 78 32773

Basic (H+K+R) 49 20588

Acidic (B+D+E+Z) 29 12185

Chapter 2

283

4A312 Secondary Structure Analysis

Fig4A34 Secondary Structure Prediction

The protein structure belongs to a β fold The structure has 12 β-

sheets 9 long and 3 short (Fig4A34 amp Fig4A35)

Chapter 2

284

Fig4A35 Predicted secondary structure and the

disorder prediction of the protein

Homology modelling

BLAST search against the structure database could find only two

models with sequence identity of only around 27 Homology modelling of

the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot

tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling

server Phyre2 BLAST search against the sequence database the predicted

GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-

Theta subfamily in the region of residues 83-161 The GST fold contains an

Chapter 2

285

N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain

with an active site located in a cleft between the two domains GSH binds to

the N-terminal domain while the hydrophobic substrate occupies a pocket in

the C-terminal domain Class Theta subfamily comprises of eukaryotic class

Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-

Bauer A et al 2011) Fig4A34 shows the secondary structure and the

disorder prediction of the protein as predicted by Phyre2 The protein

structure belongs to β fold DDT-dehydrohalogenase is composed of 8

helices and has 4 β-sheets inter-attached by random coils Phyre2 server

predicted the model by aligning regions of domains of PDB structures

c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA

c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A

c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with

100 confidence and the percentage of identity were between 12-24

PSIPRED graphical out put from prediction of DDT-

dehydrohalogenase produced by PSIPRED view

PSIPRED ndashprotein structure prediction server incorporates three

recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for

predicting structural information about a protein from its amino acid sequence

alone PSIPRED (Jones 1999) carried out reliable secondary structure

prediction on a protein The DDT-dehydrohalogenase is composed of 4

helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38

show the 3D modelling of the enzyme

Chapter 2

286

Fig4A36 Hydrophobic index of DDT-dehydrohalogenase

+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287

4A313 3D structure analysis

Fig4A37 3D structure viewed in Accelrys DS Visualizer Software

Stereo view of Cartoon representation of DDT-

dehydrohalogenase enzyme

A B

C D

A is Wire model B is Ribbon model showing β sheets

C is Barallel model D is Ribbon model showing α helices

Chapter 2

288

Fig4A38 Ribbon model of the modelled protein generated by Phyre2

Chapter 2

289

Fig4A39 Ramachandran plot

Ramachandran plot developed by Gopalasamudram Narayana

Ramachandran is a way to visualize dihedral angles φ against ψ of amino

Chapter 2

290

acid residues in protein structure It shows the possible conformations of φ

and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha

and C alpha-C bonds relatively are free to rotate These rotations are

represented by the torsion angles phi and psi respectively Computer

models were used to systematically vary phi and psi with the objective of

finding stable conformations For each conformation the structure was

examined for close contacts between atoms Atoms were treated as hard

spheres with dimensions corresponding to their van der Waals radii

Therefore phi and psi angles which caused spheres to collide correspond to

sterically disallowed conformations of the polypeptide backbone In the

diagram given above the white areas correspond to conformations where

atoms in the polypeptide come closer than the sum of their van der Waals

radii These regions are sterically disallowed for all amino acids except

glycine which is unique in that it lacks a side chain The red regions

correspond to conformations where there are no steric clashes ie these are

the allowed regions namely the alpha-helical and beta-sheet conformations

The yellow areas show the allowed regions if slightly shorter van der Waals

radii are used in the calculation ie the atoms are allowed to come a little

closer together This brings out an additional region which corresponds to the

left-handed alpha-helix L-amino acids cannot form extended regions of left-

handed helix but occasionally individual residues adopt this conformation

These residues are usually glycine but can also be asparagine or aspartate

where the side chain forms a hydrogen bond with the main chain and

therefore stabilises this otherwise unfavourable conformation The 3(10) helix

occurs close to the upper right of the alpha-helical region and is on the edge

of allowed region indicating lower stability Disallowed regions generally

involve steric hindrance between the side chain C-beta methylene group and

main chain atoms Glycine has no side chain and therefore can adopt phi

and psi angles in all four quadrants of the Ramachandran plot Hence it

frequently occurs in turn regions of proteins where any other residue would be

sterically hindered

Chapter 2

291

Fig4A310 Ramachandran map of the modeled protein

The plot combines the four separate Ramachandran maps (as shown

to the right) using shapes to distinguish the membership to a particular class

(General Proline Glycine and Pre-Proline) Within this scheme Glycines are

shown as diamonds Prolines as triangles residues preceding Prolines (Pre-

Proline) have a rectangular shape otherwise (General case) they are drawn

as small squares

Ramachandran map of the model calculated using DS25 is shown in

fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed

region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier

region

Before energy minimization in the figure above the white areas

corresponds to conformations where atoms in the polypeptide come closer

than the sum of their van der Waals radii These regions are sterically

disallowed for all amino acids except glycine which is unique in that it lacks a

Chapter 2

292

side chain The red regions correspond to conformations where there are no

steric clashes ie these are the allowed regions namely the alpha-helical

and beta-sheet conformations The yellow areas show the allowed regions if

slightly shorter van der Waals radii are used in the calculation ie the atoms

are allowed to come a little closer together This brings out an additional

region which corresponds to the left-handed alpha-helix L-amino acids

cannot form extended regions of left-handed helix but occasionally individual

residues adopt this conformation These residues are usually glycine but can

also be asparagine or aspartate where the side chain forms a hydrogen bond

with the main chain and therefore stabilises this otherwise unfavourable

conformation The 3(10) helix occurs close to the upper right of the alpha-

helical region and is on the edge of allowed region indicating lower stability

Disallowed regions generally involve steric hindrance between the side chain

C-beta methylene group and main chain atoms Glycine has no side chain

and therefore can adopt phi and psi angles in all four quadrants of the

Ramachandran plot Hence it frequently occurs in turn regions of proteins

where any other residue would be sterically hindered

After energy minimization the colour scheme encodes the affiliation to

a certain region within the respective Ramachandran Map Residues in the

core region are rendered in green or yellow if they are found to be in the

allowed region and red if in the outlier region Outliers are always marked as

crosses independent of their class

The colour option allows for viewing the plot in either black or white

background colour When White background is selected the above

mentioned colouring scheme for assignment to core allowed or outlier region

within the map is not used instead filled or non-filled shapes are used to

make class

Chapter 2

293

4A313b Molecular surface analysis

Fig4A311 Surface analysis of DDT-dehydrohalogenase

Using molecular surface analysis active site could be predicted

Hydrophobic region is in dark green colour rose colour indicated hydrogen

bonding region and Blue colour indicated the mid polar region The green

shade is the predicted active site It indicated a hydrophobic core It favours

hydrophobic ligands (Fig4A311)

Chapter 2

294

4A314 Rotamer strain energy plot

The Plot depicts the rotamer strain calculated energy (kcalmol) The

X-Axis shows the residues in sequential order within MOE Residues with

strain energies larger than 50 kcalmol (marked by the dotted horizontal line

in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a

closer inspection The strain energy threshold of the result set may be

adjusted with the E-Threshold slider reducing the results to entries with strain

energies above the designated cut off

Fig4A312 Rotamer profile of DDT-dehydrohalogenase

4A315 Prediction of active site

Active site was predicted using binding site molecule of discovery studio

software which predicted 6 possible ligand binding sites (active site) Since

the DDT was reported to be ligand for this protein Based on the chemical

Chapter 2

295

properties of this ligand or DDT we have chosen site no1 as the probable

active site which comprises of

Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu

104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified

as substrate binding site

4A316 Molecular docking

4A316a Molecular docking of DDT into active site

Fig4A313 Molecular docking of ligand DDT into the active site

Chapter 2

296

Fig4A314 Interaction of the ligands with the active site

4A316b Docking of ligand DDT along with glutathione into the active Site

The GST active site is composed of a GSH binding site (G-site)

common to all GSTs and a xenobiotic binding site (H-site) which varies

between different classes and isotypes Residues from the N-terminal TRX-

fold domain form the G-site while the H-site is comprised mainly of residues

from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)

In order to comprehend the molecular level interaction of ligands

Glutathione and DDT docking of the co-factor Glutathione was carried out first

followed by docking of DDT to the modelled Glutathione DDT dehalogenase

complex The predicted glutathione binding site was similar to GST- N family

Class Theta subfamily composed of eukaryotic class Theta GSTs and

bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was

Chapter 2

297

successfully docked into the predicted binding site which comprised of

residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41

Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of

glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance

from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)

Glutathione makes three hydrogen bond contact with the active site residues

The Sulphur atom of Glutathione is making a hydrogen bond contact with

Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a

bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also

making a hydrogen bond contact with O4 atom of Glutathione (h-bond

distance 315 Aring)

GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on

nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur

atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate

DDT was docked into the active site cleft located between the two domains

TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand

DDT is making a number of van der walls contact with the active site G-site

which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31

Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218

and to the docked Glutathione Fig4A313 shows the interaction of the

ligands to the active site residues and the Fig4A314 shows the molecular

surface representation of the docked complex Calculated free energy of

binding was -4062 and -2645 kcalmol for GSH and DDT respectively

The structures of reduced form of Glutathione and DDT were built

using the Build fragment module of DS25 and energy minimised using

standard energy minimisation protocols of DS25 Conserved residues of the

binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57

(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were

rendered flexible during the docking simulation of Glutathione (Fig4A315)

(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to

both the protein and ligand and Monmany Rone force field was used for the

calculation of partial charges Glutathione was flexibly docked into the

Chapter 2

298

identified glutathione binding site based on the standard flexible docking

protocols implemented in DS25 in which docking refinement was carried out

using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of

cooling Free energy of binding was calculated with the Calculate Free

Energy Binding module with a distance depended di-electric constant as

implicit solvent model and a di-electric constant of 78 The final docked

complex was selected based on orientation of the ligand in respect to other

structural homologs and estimated Free Energy of binding

The Substrate DDT was similarly docked in the identified substrate

binding site of the structure of Glutathione DDT dehalogenase complex DDT

is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride

(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond

distance 229200 A and 5th chloride of DDT is attached by hydrogen bond

with amino acids GLN130 HE21 Bond distance 248 A In this picture

orange colour indicates DDT and yellow colour indicate co factors such as

glutathione (Fig4A316)

Name Neighbour Monitor1

Distance 5000000

Show distances No

Show intermolecular only Yes

Number of Neighbours 418

Chapter 2

299

Fig4A315 Docking of glutathione as a cofactor into the active site

Fig4A316 Molecular surface representation of the docked complex

ligand DDT on the left and GSH on the right represented as stick

Chapter 2

300

4A316c Docking of ligand DDT along with glutathione into the active

Site

Glutathione which enhanced the activity of DDT-dehydrohalogenase to

312 folds was docked along with DDT

The structure of a glutathione was built with minimized energy using

builder module of discovery studio This was then docked into the active site

of the modelled structure using c-docker Green dotted line indicates

hydrogen bond with aminoacids in the activesite

Fig4A317 Ligand DDT contact with Wanderwall force

In the Fig4A317 green colour shows wanderwall interaction towards

ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5

The vander waals radii were completely attached with DDT DDT

dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig

4A318)

Chapter 2

301

Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase

Chapter 2

302

4A4 Discussion

The PCR amplified gene product was cloned and sequenced The

gene showed sequence similarity of 93 to Acetolactate synthase of Serratia

spp There are many xenobiotic degrading enzymes which have shown such

sequence similarities to other family of enzymes An interesting type of

aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first

step in the bacterial degradation of lindane (-hexachlorocyclohexane) where

HCl was eliminated converting the substrate to pentachlorocyclohexene

(Nagata et al 2001 Trantirek et al 2001) The structure has not been

solved but a mechanism was predicted on the basis of the stereochemistry of

the reaction and low but significant sequence similarity to scytalone

dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate

(CbzA) (Benning et al 1998) has been shown to belong to the enoyl

hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in

the bacterial degradation of atrazine has been shown to be related to

melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane

dehalogenase (DcmA) catalysing the conversion of dichloromethane to

formaldehyde in a glutathione- dependent reaction and another group of

dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are

present in bacteria that degrade the nematocide 13-dichloropropene have

been found to belong to the tautomerase superfamily of proteins Halohydrin

dehalogenases belong to short- chain dehydrogenase reductase (SDR)

superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)

belongs to a large superfamily of proteins that also includes non- haem

integral membrane desaturases epoxidases acetylenases conjugases

ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase

(AtzA) belongs to the amidohydrolase superfamily Other members of the

amidohydrolase superfamily are triazine deaminase hydantoinase melamine

deaminase cytosine deaminase and phosphotriesterase Rhodococcus

haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by

the same invertase gene sequence and a regulatory gene and on the

downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase

encoding gene (Poelarends et al 2000)

Chapter 2

303











The nucleic acid sequence of Pseudomonas putida T5 was used for

bioinformatics work Based on BLAST search against sequence database

putative conserved domain have been detected The predicted domain is

GST-C-family superfamily It is in Class Theta subfamily composed of

eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)

dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain

and a C-terminal alpha helical domain with an active site located in a cleft

between the two domains GSH binds to the N-terminal domain while the

hydrophobic substrate occupies a pocket in the C-terminal domain Using

compute PI and protparam molecular weight and physical and chemical

properties have been detected using RADAR and prosite repeats and blocks

pfam was detected The identified domains were then designated according

to their pore forming residues The secondary structure was predicted by

nnpredict

For construction of 3D model Pseudomonas putida T5 sequence was

submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The

comparative model failed to find a suitable template for the Pseudomonas

putida T5 sequence Therefore threading method was employed to find an

appropriate template using 3D Position Specific Scorings Matrix server

(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has

predicted many structures similar to Pseudomonas putida T5 sequence From

these c1v2ab was selected based on their sequence identity and some

logical ideas The sequence identity is 18 and length is 210cm Its family

Chapter 2

304

and superfamily are Glutathione transferase gst1-6 and glutathione-s-

transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model

was constructed using 3dpssm or phyre The aligned sequence of ppt5 and

c1v2ab was adjusted manually (minimization) to get better structural quality of

the model corresponding to the template structure using DISCOVERY

STUDIO 20 The side chain conformations of the constructed model were

refined to minimize the error using the Side Chain with Rotamer Library

The constructed model of the Pseudomonas putida T5 protein was

evaluated for its backbone conformation (structural quality) using

Ramachandran plot The Ramachandran plot shows the residue backbone

conformations for the modelled protein as well as the template The plot

combines the four separate Ramachandran maps using shapes to distinguish

the membership to a particular class (General Proline Glycine and Pre-

Proline) The Auto Deposition Input Tool (ADIT) available online

(httpdepositpdborgvalidate) was used to check the favourable and

unfavourable properties of the model structure before it was deposited in the

Protein Data Bank Presence of active sites and pockets in the protein

Pseudomonas putida T5 was predicted using binding site molecule of

discovery studio software which predicted six possible ligand binding

The three-dimensional model was constructed for Pseudomonas

putida T5 with the receptor protein and DDT analogues as flexible ligands

The DDT analogues having more than 90 structural similarity with DDT

were retrieved from the PubChem database

(httppubchemncbinlmnihgov) Since DDT was reported to be ligand

for this protein based on the chemical properties of this ligand we have

chosen site no1 as the probable active site which comprises of ASP18

MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD

domain predicted the protein to have GST-C-SUPERFAMILY which generally

takes part in cellular detoxification by catalyzing the conjugations (GSTs) with

a wide range of endogenous and xenobiotic alkylating agents The predicted

active site shows big cavity In that big cavity we docked two things one

cofactor Glutathione and other ligand DDT Glutathione was chosen because

we observed slight enhancement of enzyme activity with the addition of

Chapter 2

305

glutathione Glutathione cofactor was docked first followed by DDT ligand

The potential energy of the overall reaction was -324278kcalmol

In general blast search of the enzyme showed sequence homology to

that of bacterial acetolactate synthase With specific blast search with the

reported sequence of DDT resistance gene of Aedes aegypti the sequence

showed great similarity with GST of Aedes aegypti However its identity was

observed to be only 59 Then second similarity was shown with GST of

Culex quinquefasciatus The blast search revealed that the sequence identity

between the VGSC of An gambiae and VGSC of Homo sapiens was 312

The DDT-dehydrohalogenase protein showed a molecular weight of

2778936 and isoelectric point of 921 Using protaparam the physical and

chemical properties of DDT-dehydrohalogenase protein was found out

Number of amino acids found was 238 with a formula is

C1234H1927N345O341S23 Total number of atoms is 3870

Radar tool showed no repeats in DDT dehydrohalogenase protein

sequence because protein sequence had no repeats Motif indicated

structural part of protein Number of Motif found in DDT dehydrohalogenase

was 125 and block showing an ungapped aligned motif consisting of

sequence segments that are clustered to reduce multiple contribution from

groups of highly similar or identical sequences Here CD domain predicted

that glutathione-s-transferase is the domain of DDT dehydrohalogenase

protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in

DDT dehydrohalogenase protein sequence because the protein sequence

doesnrsquot contain pattern and profile The average residue weight was 116762

and charge was 238 Pepinfo tool showed the plot of hydropathy value and

hydropathy plot of residues There were large number hydrophobic

aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are

isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)

and lysine This is very important in protein structure Hydrophobic

aminoacids tend to be internal while hydrophilic amino acids are more

commonly found towards the protein sequence Pep window tool shows kyte-

doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)

small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)

Chapter 2

306

aromatic residues (F+H+W+Y) non-polar residues

(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z

charged residues (B+D+E+H+K+R+Z) in the protein DDT

dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-

Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-

specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase

sequences

Secondary structure prediction of FAP52 based on its primary

structure indicated that itrsquos NH2-terminus has a long stretch with an -helical

arrangement with a high-degree of propensity to coiled-coil arrangements

This could be fulfilled either by intra- or inter molecular interactions In order

to distinguish between these possibilities the capacity of FAP52 to self-

associate were analysed The PAIRCOIL program predicted that FAP52

included three regions comprising the residues 146ndash179 185ndash219 and 248ndash

280 with the probability of 53 100 and 9 respectively to form coiled-

coils While another program MULTICOIL further discriminated the

propensity for dimeric and higher oligomeric arrangements giving the

likelihoods of dimer formation of 3 74 and 0 and of trimer formation of

4 10 and 05 for the same regions respectively Here the PAIRCOIL

program predicted that DDT dehyrohalogenase had no coiled coils in the

residue position and MULTICOIL predicted that the probability cut off for the

coiled-coil locater was 050 The scoring distances for dimeric table was 2 3

4 for trimeric table it was 1 2 3

For construction of 3D model of VGSC protein of An gambiae the

retrieved sequence was submitted to PSI-BLAST The comparative model

failed to find a suitable template for the VGSC sequence Therefore

threading method was employed to find an appropriate template using 3D

Position Specific Scorings Matrix server (3DPSSM)

(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated

potassium channel (VGPC) of A pernix as a template for the domain II of

VGSC For the other regions of VGSC no template with the required

sequence identity of gt20 was found Therefore a 3D model was

constructed only for domain II of VGSC using MODELLER programmes

Chapter 2

307

httpsalilaborgmodellerdownload_installationhtml) For construction of 3d

model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved

sequence was submitted to PSI-BLAST The comparative model failed to find

a suitable template for the DDT-dehalogenase sequence Therefore threading

method was employed to find an appropriate template using 3DPSSM or

PHYRE online software Many structures were predicted by phyre software

Among these structures one structure was selected based on its sequence

identity and e value

Ramachandran plot of VGSC Domain II of each of the 20 amino

acids that included glycine and proline the ADIT validation indicated that the

VGSC model had 6 amino acids in unfavourable region where as in the

template the unfavourable region contained a total of 19 amino acids Thus

the analysis (in the absence or in the presence of glycine and proline) clearly

suggested that the backbone conformations of the model have suitable

structural quality when compared with the template Ramachandran plot of

Pseudomonas putida T5 of each 20 aminoacids that included glycine proline

and aparagine (the ADIT validation) indicated that the asparagine or

aspartate in the side chain formed a hydrogen bond with the main chain and

therefore stabilised the enzyme which was otherwise an unfavourable

conformation Disallowed regions generally involved steric hindrance between

the side chain C-beta methylene group and main chain atoms The active

sites of the VGSC domain II model were predicted by the PASS programme

In the modelled protein the ASTp program illustrated the presence of a total

of 30 pockets meant for ligands interaction Here the active site of DDT-

dehydrohalogenase of Psueodomonas putida T5 was predicted by binding

site molecule of discovery studio 20 docking software which predicted 6

possible active sites Based on the chemical properties of the ligand site no1

was chosen as the probable active site which comprised of SP18 MET20

SER21 CYS42 TRP 129 AG117

The side chain conformations of the constructed model of

Pseuedomonas putida T5 domain were refined to minimize the error DDT

dehydrochlorinase docking result had been reported previously in A gambaie

GSTs but this is the first report of docking DDT-dehydrohalogenase in

Chapter 2

308

Pseudomonas putida T5 Here the predicted domain is GST_C family Class

Theta subfamily composed of eukaryotic class Theta GSTs and bacterial

dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins

involved in cellular detoxification by catalyzing the conjugation of glutathione

(GSH) with a wide range of endogenous and xenobiotic alkylating agents

including carcinogens therapeutic drugs environmental toxins and products

of oxidative stress and here the cofactor glutathione is defined as tripeptide

with many roles in cells It conjugates to drugs to make them more soluble for

excretion is a cofactor for some enzymes is involved in protein disulfide

bond rearrangement and reduces peroxides

The report of A gambiae GSTs are of particular interest because of

their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-

chlorophenyl) ethane] which is an important malaria vector In A gambiae

an increased rate of DDT dehydrochlorination in the resistant strain was

associated with quantitative increase in multiple GST enzymes The report of

Aedes aegypti found no evidence for increased levels of this GST protein in

DDTpyrethroid-resistant populations from Thailand In A gambiaea also

gluthathione was cofactor Glutathione in Drosophilla indicated detoxification

and is of interest for two reasons First the ecological niche of Drosophila

species is largely defined by the larval feeding substrate and therefore the

genes enabling Drosophila to identify detoxify and utilize these substrates as

nutritional resources are obvious candidates for genes that have been the

targets of natural selection Glutathione S-transferases encoded in the

D melanogaster genome are divided into two nonhomologous families the

microsomal GSTs for which there are three genes and the canonical GSTs

that are cytosolic (36 genes)

Docking result of VGC DOMAIN was done with this altered model

the ligand DDT interacting with a different group of residues such as glutamic

acid methionine cysteine leucine tyrosine and phenylalanine with a binding

energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was

done with ligand DDT with a differing group of residues such as arg lys leu

tyr leu with a binding energy is -324278 kcalmol DDT was docked within

predicted catalytic sites by using the DOCK function of MOE in case of

Chapter 2

309

Agambaie Here DDT was docked within the predicted active sites by using

DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In

Pseudomonas putida T5 and Agambaie Visual inspection indicated that

DDT dock best in the presence of glutathione with the trichloro group of DDT

orientied toward the sulphur atom of glutathione This conformation agreed

that with GSH depended dehydrochlorinase activity proposed by Matsumura

(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted

to be distant enough from DDT to be free of van der Waals clashes But in

Pseudomonas putida T5 the vander waals radii were completely attached with

DDT

4A5 Conclusion

DDT- dehydrohalogenase is an enzyme that is involved in the removal

of one chlorine from DDT molecule The gene responsible was isolated from

Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21

using pET 28a plasmid vector

In the computational study the enzyme sequence deciphered by gene

sequence was used for the construction of models employing homology

modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT

substrate was subsequently studied Docking was also done along with GSH

a co-factor like substrate These two were found to have different binding

pockets regarding the size and the key amino acids involved in binding

Predicted binding modes of these two with DDT ndash degrading enzyme was

compared The calculated docking interaction energy of DDT showed high

affinity suggesting specificity of the enzyme

Chapter 2

310

References

Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995

SCOP A structural classification of proteins database for the

investigation of sequences and structures Journal of Molecular

Biology 247(4)536ndash540

Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse

tissues Canadian J Zool 42324ndash325

Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring

the extremes of sequencestructure space with ensemble fold

recognition in the program Phyre Proteins 70 611-625





ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein

structure and structural feature prediction server Nucleic Acids Res

33 web server issue W72ndashW76

Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple

Sequence Methods for Protein Secondary Structure

Prediction PROTEINS Structure Function and Genetics 34508-519













Chapter 2

311

Jones DT 1999 Protein secondary structure prediction based on position-

specific scoring matrices J Mol Biol 292(2)195-202

Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by

yeast Science 141(3585)1050-1

Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web

a case study using the Phyre server Nature Protocols 4 363 ndash 371

Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005

Nomenclature for mammalian soluble glutathione transferases

Methods Enzymol 4011-8

Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved

Domain Database for the functional annotation of proteins Nucleic

Acids Res 39(D) 225-9

Matsumura F 1985 Toxicology of Insecticides Plenum Press New

York

McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The

Genomic Threading Database Bioinformatics applications note

20(1)131ndash132 (DOI 101093bioinformaticsbtg387)


protein fold and catalytic residues of gamma- hexachlorocyclohexane

dehydrochlorinase LinA Proteins 45 471- 477

Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR




2725- 2731

Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase

from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution

Acta Cryst D551273 ndash 1290




Supariya S 2009 prediction of molecular structures of DDT

dehydrohalogenase and docking

Chapter 2

312





Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004

The DISOPRED server for the prediction of protein disorder

Bioinformatics applications note 20(13) 2138ndash2139

(doi101093bioinformaticsbth195)


chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology

15569- 574

Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone

metabolites of naphthalene covalently bound to sulfur nucleophiles of

proteins of murine Clara cells after exposure to naphthalene Chem

Res Toxicol 10 1008 ndash1014

Chapter 2

234

which readily degraded DDT to DDD Wedemeyer (1967) studied DDT

metabolic pathway by incubation of proposed intermediates with organisms

and examining the products formed The metabolism of DDT in Aerobacter

aerogenes goes in order DDT DDD DDMU DDNU DDOH DDA

DBP and direct conversion of DDT to DDE (Wedemeyer 1967) Reports

on the involvement of enzymes in the degradation of DDT indicate the

presence of enzymes like dioxygenases (Nadeau et al 1994)

dehydrogenases (Bourquin 1977) oxygenases (Ahmed et al 1991) Only a

few enzymes have been described in DDT degradative pathway (Singh et al

1999) The conversion of DDT to DDD involves a dehydrochlorination step

In our laboratory an attempt was made to clone sequence and express the

gene responsible for this step

42 Materials

Luria- Bertoni broth and ampicillin were procured from Hi media

Mumbai Agarose was procured from Sisco Research Laboratories Pvt Ltd

Taq polymerase dNTPs and restriction enzymes were procured from Genei

Bangalore PCR purification kit was purchased from GE Healthcare UK

Ethidium bromide nitrocellulose membrane and nylon membrane were

procured from Sigma Aldrich Chemical Company MO US The designed

primers were synthesized by Sigma MO US Cloning kit was procured from

Fermentas Lifesciences EU DIG kit was obtained from Roche Company

Germany EDTA was procured from Qualigens Fine Chemicals All other

chemicals used in the study were procured from standard chemical

companies

43 Reagents and buffers

431 Reagents for plasmid isolation

Solution 1 50 mM Glucose 25 mM Tris- Cl (pH-80) 10 mM EDTA (pH-80)

Solution 2 02 N NaOH (freshly prepared from 10 N NaOH) 1 SDS

(Prepared freshly before use)

Chapter 2

235

Solution 3






-20 0C



15 min and used

434 CaCl2 solution









1 mM EDTA (pH 80)



Tris buffer 242 g

05 M EDTA 10 mL




Chapter 2

236




Glycerol 30


20oC



NaCl 100 mM

MgCl2 50 mM






Maleic acid 100 mM

NaCl 150 mM













Chapter 2

237


SDS 7 (wv)

BSA 1 (wv)

EDTA 1 mM

Na-PO4 (pH 74) 025 M









the SDS

4314 20X SSC

NaCl 3 M

Sodium citrate 03 M




44 Methods










Chapter 2

238













the genomic DNA


















Chapter 2

239














Taq Buffer 25 L

dNTP mix 05 L

Taq Polymerase 03 L

Forward Primer 10 L

Reverse Primer 10 L



4451 Gradient PCR


720C 720C940C 940C

500 045

045

50- 700C

100 800

40C

25 Cycles

Chapter 2

240

4452 Touchdown PCR




electrophoresis












720C 72 0C 72 0C 4 0C

infin

045

100 030

940C 940C 600C 940C 500C

500 030

10 cycles 30 cycles

045

100 15

72 0C 94 0 C 94 0 C

500 045

115

55 0C

100 800

4 0 C

30 Cycles

72 0C

Chapter 2

241


putida T5

























at 4 0C overnight

Chapter 2

242












Chapter 2

243
























transformation




Chapter 2

244



















Chapter 2

245
















Chapter 2

246























stored at -20 0C




Chapter 2

247


Bottlecap


1 Unlabelled

control DNA







1230 1033 653 394 298(2X) 234 (2X) 220 and

154 (2X) bp

Clear solution


2 Unlabelled

controlled DNA


[200 μg mL]


Clear solution


efficiency

3 DNA dilution

buffer



1 mM EDTA pH 80 (20 0C)]

Clear solution



4 Labelled control

DNA





labelled DNA

Clear solution

Chapter 2

248



1 Sau 3A

2 Bgl ΙΙ

3 Sal І

4 Bam H І

5 Xho І

6 Hind Ш

7 Pvu ΙΙ


5 Hexa-

nucleotide mix





Clear solution


6 dNTP Labelling

Mix




pH75 ( 20 0C)]

Clear solution


7 Klenow Enzyme

Labelling Grade



large fragment)]

Clear solution


Chapter 2

249

































Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

235

Solution 3






-20 0C



15 min and used

434 CaCl2 solution









1 mM EDTA (pH 80)



Tris buffer 242 g

05 M EDTA 10 mL




Chapter 2

236




Glycerol 30


20oC



NaCl 100 mM

MgCl2 50 mM






Maleic acid 100 mM

NaCl 150 mM













Chapter 2

237


SDS 7 (wv)

BSA 1 (wv)

EDTA 1 mM

Na-PO4 (pH 74) 025 M









the SDS

4314 20X SSC

NaCl 3 M

Sodium citrate 03 M




44 Methods










Chapter 2

238













the genomic DNA


















Chapter 2

239














Taq Buffer 25 L

dNTP mix 05 L

Taq Polymerase 03 L

Forward Primer 10 L

Reverse Primer 10 L



4451 Gradient PCR


720C 720C940C 940C

500 045

045

50- 700C

100 800

40C

25 Cycles

Chapter 2

240

4452 Touchdown PCR




electrophoresis












720C 72 0C 72 0C 4 0C

infin

045

100 030

940C 940C 600C 940C 500C

500 030

10 cycles 30 cycles

045

100 15

72 0C 94 0 C 94 0 C

500 045

115

55 0C

100 800

4 0 C

30 Cycles

72 0C

Chapter 2

241


putida T5

























at 4 0C overnight

Chapter 2

242












Chapter 2

243
























transformation




Chapter 2

244



















Chapter 2

245
















Chapter 2

246























stored at -20 0C




Chapter 2

247


Bottlecap


1 Unlabelled

control DNA







1230 1033 653 394 298(2X) 234 (2X) 220 and

154 (2X) bp

Clear solution


2 Unlabelled

controlled DNA


[200 μg mL]


Clear solution


efficiency

3 DNA dilution

buffer



1 mM EDTA pH 80 (20 0C)]

Clear solution



4 Labelled control

DNA





labelled DNA

Clear solution

Chapter 2

248



1 Sau 3A

2 Bgl ΙΙ

3 Sal І

4 Bam H І

5 Xho І

6 Hind Ш

7 Pvu ΙΙ


5 Hexa-

nucleotide mix





Clear solution


6 dNTP Labelling

Mix




pH75 ( 20 0C)]

Clear solution


7 Klenow Enzyme

Labelling Grade



large fragment)]

Clear solution


Chapter 2

249

































Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

236




Glycerol 30


20oC



NaCl 100 mM

MgCl2 50 mM






Maleic acid 100 mM

NaCl 150 mM













Chapter 2

237


SDS 7 (wv)

BSA 1 (wv)

EDTA 1 mM

Na-PO4 (pH 74) 025 M









the SDS

4314 20X SSC

NaCl 3 M

Sodium citrate 03 M




44 Methods










Chapter 2

238













the genomic DNA


















Chapter 2

239














Taq Buffer 25 L

dNTP mix 05 L

Taq Polymerase 03 L

Forward Primer 10 L

Reverse Primer 10 L



4451 Gradient PCR


720C 720C940C 940C

500 045

045

50- 700C

100 800

40C

25 Cycles

Chapter 2

240

4452 Touchdown PCR




electrophoresis












720C 72 0C 72 0C 4 0C

infin

045

100 030

940C 940C 600C 940C 500C

500 030

10 cycles 30 cycles

045

100 15

72 0C 94 0 C 94 0 C

500 045

115

55 0C

100 800

4 0 C

30 Cycles

72 0C

Chapter 2

241


putida T5

























at 4 0C overnight

Chapter 2

242












Chapter 2

243
























transformation




Chapter 2

244



















Chapter 2

245
















Chapter 2

246























stored at -20 0C




Chapter 2

247


Bottlecap


1 Unlabelled

control DNA







1230 1033 653 394 298(2X) 234 (2X) 220 and

154 (2X) bp

Clear solution


2 Unlabelled

controlled DNA


[200 μg mL]


Clear solution


efficiency

3 DNA dilution

buffer



1 mM EDTA pH 80 (20 0C)]

Clear solution



4 Labelled control

DNA





labelled DNA

Clear solution

Chapter 2

248



1 Sau 3A

2 Bgl ΙΙ

3 Sal І

4 Bam H І

5 Xho І

6 Hind Ш

7 Pvu ΙΙ


5 Hexa-

nucleotide mix





Clear solution


6 dNTP Labelling

Mix




pH75 ( 20 0C)]

Clear solution


7 Klenow Enzyme

Labelling Grade



large fragment)]

Clear solution


Chapter 2

249

































Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

237


SDS 7 (wv)

BSA 1 (wv)

EDTA 1 mM

Na-PO4 (pH 74) 025 M









the SDS

4314 20X SSC

NaCl 3 M

Sodium citrate 03 M




44 Methods










Chapter 2

238













the genomic DNA


















Chapter 2

239














Taq Buffer 25 L

dNTP mix 05 L

Taq Polymerase 03 L

Forward Primer 10 L

Reverse Primer 10 L



4451 Gradient PCR


720C 720C940C 940C

500 045

045

50- 700C

100 800

40C

25 Cycles

Chapter 2

240

4452 Touchdown PCR




electrophoresis












720C 72 0C 72 0C 4 0C

infin

045

100 030

940C 940C 600C 940C 500C

500 030

10 cycles 30 cycles

045

100 15

72 0C 94 0 C 94 0 C

500 045

115

55 0C

100 800

4 0 C

30 Cycles

72 0C

Chapter 2

241


putida T5

























at 4 0C overnight

Chapter 2

242












Chapter 2

243
























transformation




Chapter 2

244



















Chapter 2

245
















Chapter 2

246























stored at -20 0C




Chapter 2

247


Bottlecap


1 Unlabelled

control DNA







1230 1033 653 394 298(2X) 234 (2X) 220 and

154 (2X) bp

Clear solution


2 Unlabelled

controlled DNA


[200 μg mL]


Clear solution


efficiency

3 DNA dilution

buffer



1 mM EDTA pH 80 (20 0C)]

Clear solution



4 Labelled control

DNA





labelled DNA

Clear solution

Chapter 2

248



1 Sau 3A

2 Bgl ΙΙ

3 Sal І

4 Bam H І

5 Xho І

6 Hind Ш

7 Pvu ΙΙ


5 Hexa-

nucleotide mix





Clear solution


6 dNTP Labelling

Mix




pH75 ( 20 0C)]

Clear solution


7 Klenow Enzyme

Labelling Grade



large fragment)]

Clear solution


Chapter 2

249

































Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

238













the genomic DNA


















Chapter 2

239














Taq Buffer 25 L

dNTP mix 05 L

Taq Polymerase 03 L

Forward Primer 10 L

Reverse Primer 10 L



4451 Gradient PCR


720C 720C940C 940C

500 045

045

50- 700C

100 800

40C

25 Cycles

Chapter 2

240

4452 Touchdown PCR




electrophoresis












720C 72 0C 72 0C 4 0C

infin

045

100 030

940C 940C 600C 940C 500C

500 030

10 cycles 30 cycles

045

100 15

72 0C 94 0 C 94 0 C

500 045

115

55 0C

100 800

4 0 C

30 Cycles

72 0C

Chapter 2

241


putida T5

























at 4 0C overnight

Chapter 2

242












Chapter 2

243
























transformation




Chapter 2

244



















Chapter 2

245
















Chapter 2

246























stored at -20 0C




Chapter 2

247


Bottlecap


1 Unlabelled

control DNA







1230 1033 653 394 298(2X) 234 (2X) 220 and

154 (2X) bp

Clear solution


2 Unlabelled

controlled DNA


[200 μg mL]


Clear solution


efficiency

3 DNA dilution

buffer



1 mM EDTA pH 80 (20 0C)]

Clear solution



4 Labelled control

DNA





labelled DNA

Clear solution

Chapter 2

248



1 Sau 3A

2 Bgl ΙΙ

3 Sal І

4 Bam H І

5 Xho І

6 Hind Ш

7 Pvu ΙΙ


5 Hexa-

nucleotide mix





Clear solution


6 dNTP Labelling

Mix




pH75 ( 20 0C)]

Clear solution


7 Klenow Enzyme

Labelling Grade



large fragment)]

Clear solution


Chapter 2

249

































Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

239














Taq Buffer 25 L

dNTP mix 05 L

Taq Polymerase 03 L

Forward Primer 10 L

Reverse Primer 10 L



4451 Gradient PCR


720C 720C940C 940C

500 045

045

50- 700C

100 800

40C

25 Cycles

Chapter 2

240

4452 Touchdown PCR




electrophoresis












720C 72 0C 72 0C 4 0C

infin

045

100 030

940C 940C 600C 940C 500C

500 030

10 cycles 30 cycles

045

100 15

72 0C 94 0 C 94 0 C

500 045

115

55 0C

100 800

4 0 C

30 Cycles

72 0C

Chapter 2

241


putida T5

























at 4 0C overnight

Chapter 2

242












Chapter 2

243
























transformation




Chapter 2

244



















Chapter 2

245
















Chapter 2

246























stored at -20 0C




Chapter 2

247


Bottlecap


1 Unlabelled

control DNA







1230 1033 653 394 298(2X) 234 (2X) 220 and

154 (2X) bp

Clear solution


2 Unlabelled

controlled DNA


[200 μg mL]


Clear solution


efficiency

3 DNA dilution

buffer



1 mM EDTA pH 80 (20 0C)]

Clear solution



4 Labelled control

DNA





labelled DNA

Clear solution

Chapter 2

248



1 Sau 3A

2 Bgl ΙΙ

3 Sal І

4 Bam H І

5 Xho І

6 Hind Ш

7 Pvu ΙΙ


5 Hexa-

nucleotide mix





Clear solution


6 dNTP Labelling

Mix




pH75 ( 20 0C)]

Clear solution


7 Klenow Enzyme

Labelling Grade



large fragment)]

Clear solution


Chapter 2

249

































Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

240

4452 Touchdown PCR




electrophoresis












720C 72 0C 72 0C 4 0C

infin

045

100 030

940C 940C 600C 940C 500C

500 030

10 cycles 30 cycles

045

100 15

72 0C 94 0 C 94 0 C

500 045

115

55 0C

100 800

4 0 C

30 Cycles

72 0C

Chapter 2

241


putida T5

























at 4 0C overnight

Chapter 2

242












Chapter 2

243
























transformation




Chapter 2

244



















Chapter 2

245
















Chapter 2

246























stored at -20 0C




Chapter 2

247


Bottlecap


1 Unlabelled

control DNA







1230 1033 653 394 298(2X) 234 (2X) 220 and

154 (2X) bp

Clear solution


2 Unlabelled

controlled DNA


[200 μg mL]


Clear solution


efficiency

3 DNA dilution

buffer



1 mM EDTA pH 80 (20 0C)]

Clear solution



4 Labelled control

DNA





labelled DNA

Clear solution

Chapter 2

248



1 Sau 3A

2 Bgl ΙΙ

3 Sal І

4 Bam H І

5 Xho І

6 Hind Ш

7 Pvu ΙΙ


5 Hexa-

nucleotide mix





Clear solution


6 dNTP Labelling

Mix




pH75 ( 20 0C)]

Clear solution


7 Klenow Enzyme

Labelling Grade



large fragment)]

Clear solution


Chapter 2

249

































Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

241


putida T5

























at 4 0C overnight

Chapter 2

242












Chapter 2

243
























transformation




Chapter 2

244



















Chapter 2

245
















Chapter 2

246























stored at -20 0C




Chapter 2

247


Bottlecap


1 Unlabelled

control DNA







1230 1033 653 394 298(2X) 234 (2X) 220 and

154 (2X) bp

Clear solution


2 Unlabelled

controlled DNA


[200 μg mL]


Clear solution


efficiency

3 DNA dilution

buffer



1 mM EDTA pH 80 (20 0C)]

Clear solution



4 Labelled control

DNA





labelled DNA

Clear solution

Chapter 2

248



1 Sau 3A

2 Bgl ΙΙ

3 Sal І

4 Bam H І

5 Xho І

6 Hind Ш

7 Pvu ΙΙ


5 Hexa-

nucleotide mix





Clear solution


6 dNTP Labelling

Mix




pH75 ( 20 0C)]

Clear solution


7 Klenow Enzyme

Labelling Grade



large fragment)]

Clear solution


Chapter 2

249

































Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

242












Chapter 2

243
























transformation




Chapter 2

244



















Chapter 2

245
















Chapter 2

246























stored at -20 0C




Chapter 2

247


Bottlecap


1 Unlabelled

control DNA







1230 1033 653 394 298(2X) 234 (2X) 220 and

154 (2X) bp

Clear solution


2 Unlabelled

controlled DNA


[200 μg mL]


Clear solution


efficiency

3 DNA dilution

buffer



1 mM EDTA pH 80 (20 0C)]

Clear solution



4 Labelled control

DNA





labelled DNA

Clear solution

Chapter 2

248



1 Sau 3A

2 Bgl ΙΙ

3 Sal І

4 Bam H І

5 Xho І

6 Hind Ш

7 Pvu ΙΙ


5 Hexa-

nucleotide mix





Clear solution


6 dNTP Labelling

Mix




pH75 ( 20 0C)]

Clear solution


7 Klenow Enzyme

Labelling Grade



large fragment)]

Clear solution


Chapter 2

249

































Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

243
























transformation




Chapter 2

244



















Chapter 2

245
















Chapter 2

246























stored at -20 0C




Chapter 2

247


Bottlecap


1 Unlabelled

control DNA







1230 1033 653 394 298(2X) 234 (2X) 220 and

154 (2X) bp

Clear solution


2 Unlabelled

controlled DNA


[200 μg mL]


Clear solution


efficiency

3 DNA dilution

buffer



1 mM EDTA pH 80 (20 0C)]

Clear solution



4 Labelled control

DNA





labelled DNA

Clear solution

Chapter 2

248



1 Sau 3A

2 Bgl ΙΙ

3 Sal І

4 Bam H І

5 Xho І

6 Hind Ш

7 Pvu ΙΙ


5 Hexa-

nucleotide mix





Clear solution


6 dNTP Labelling

Mix




pH75 ( 20 0C)]

Clear solution


7 Klenow Enzyme

Labelling Grade



large fragment)]

Clear solution


Chapter 2

249

































Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

244



















Chapter 2

245
















Chapter 2

246























stored at -20 0C




Chapter 2

247


Bottlecap


1 Unlabelled

control DNA







1230 1033 653 394 298(2X) 234 (2X) 220 and

154 (2X) bp

Clear solution


2 Unlabelled

controlled DNA


[200 μg mL]


Clear solution


efficiency

3 DNA dilution

buffer



1 mM EDTA pH 80 (20 0C)]

Clear solution



4 Labelled control

DNA





labelled DNA

Clear solution

Chapter 2

248



1 Sau 3A

2 Bgl ΙΙ

3 Sal І

4 Bam H І

5 Xho І

6 Hind Ш

7 Pvu ΙΙ


5 Hexa-

nucleotide mix





Clear solution


6 dNTP Labelling

Mix




pH75 ( 20 0C)]

Clear solution


7 Klenow Enzyme

Labelling Grade



large fragment)]

Clear solution


Chapter 2

249

































Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

245
















Chapter 2

246























stored at -20 0C




Chapter 2

247


Bottlecap


1 Unlabelled

control DNA







1230 1033 653 394 298(2X) 234 (2X) 220 and

154 (2X) bp

Clear solution


2 Unlabelled

controlled DNA


[200 μg mL]


Clear solution


efficiency

3 DNA dilution

buffer



1 mM EDTA pH 80 (20 0C)]

Clear solution



4 Labelled control

DNA





labelled DNA

Clear solution

Chapter 2

248



1 Sau 3A

2 Bgl ΙΙ

3 Sal І

4 Bam H І

5 Xho І

6 Hind Ш

7 Pvu ΙΙ


5 Hexa-

nucleotide mix





Clear solution


6 dNTP Labelling

Mix




pH75 ( 20 0C)]

Clear solution


7 Klenow Enzyme

Labelling Grade



large fragment)]

Clear solution


Chapter 2

249

































Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

246























stored at -20 0C




Chapter 2

247


Bottlecap


1 Unlabelled

control DNA







1230 1033 653 394 298(2X) 234 (2X) 220 and

154 (2X) bp

Clear solution


2 Unlabelled

controlled DNA


[200 μg mL]


Clear solution


efficiency

3 DNA dilution

buffer



1 mM EDTA pH 80 (20 0C)]

Clear solution



4 Labelled control

DNA





labelled DNA

Clear solution

Chapter 2

248



1 Sau 3A

2 Bgl ΙΙ

3 Sal І

4 Bam H І

5 Xho І

6 Hind Ш

7 Pvu ΙΙ


5 Hexa-

nucleotide mix





Clear solution


6 dNTP Labelling

Mix




pH75 ( 20 0C)]

Clear solution


7 Klenow Enzyme

Labelling Grade



large fragment)]

Clear solution


Chapter 2

249

































Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

247


Bottlecap


1 Unlabelled

control DNA







1230 1033 653 394 298(2X) 234 (2X) 220 and

154 (2X) bp

Clear solution


2 Unlabelled

controlled DNA


[200 μg mL]


Clear solution


efficiency

3 DNA dilution

buffer



1 mM EDTA pH 80 (20 0C)]

Clear solution



4 Labelled control

DNA





labelled DNA

Clear solution

Chapter 2

248



1 Sau 3A

2 Bgl ΙΙ

3 Sal І

4 Bam H І

5 Xho І

6 Hind Ш

7 Pvu ΙΙ


5 Hexa-

nucleotide mix





Clear solution


6 dNTP Labelling

Mix




pH75 ( 20 0C)]

Clear solution


7 Klenow Enzyme

Labelling Grade



large fragment)]

Clear solution


Chapter 2

249

































Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

248



1 Sau 3A

2 Bgl ΙΙ

3 Sal І

4 Bam H І

5 Xho І

6 Hind Ш

7 Pvu ΙΙ


5 Hexa-

nucleotide mix





Clear solution


6 dNTP Labelling

Mix




pH75 ( 20 0C)]

Clear solution


7 Klenow Enzyme

Labelling Grade



large fragment)]

Clear solution


Chapter 2

249

































Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

249

































Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

250





zones






















membrane



Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

251








44183 Western blot
















45 Results







Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

252



work









1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

253











touchdown PCR



Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

254




















ampicillin plates


Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

255


















921



1 2 3 4 5 6 7

Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

256



















Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

257





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

258


A DDT

B DDD














Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

259










labelled T5 probe

46 Discussion







Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

260















DDT



















Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

261


































Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

262


































Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

263


































Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

264















similar to alkB










further growth

47 Conclusions







Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

265















plasmids






References





2887





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

266



362















2225- 2235















Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

267







1317- 1323













477












2725- 2731

Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

268










2567
























Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

269






41259- 41262



15 569- 574

Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

270

4A1 Introduction












degradation process









DDM









Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

271


4A21 Softwares


dehydrohalogenase




4A212 Clustal W


4A213 COMPUTE PI



4A214 PROTPARAM






4A216 MOTIFS




sequence

4A218 PROSITE



Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

272

4A129 PEPINFO


of hydrophobicity

4A2110 PEPWINDOW


4A2111 PEPSTATS

















Homology modelling






Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

273

































Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

274



4A2118 CDOCKER


























Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

275



4A31)

Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

276













Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

277

Alignments



574 AA


Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

278



Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

279






Theoretical pI 921





to be stable


Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

280



Ala (A) 19 80

80 Ile (I) 10 42 Tyr (Y) 11 46

Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55

Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00

Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00

Cys (C) 14 59 Phe (F) 12 50 (B) 0 00

Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00

Glu (E) 14 59 Ser (S) 11 46 (X) 0 00

Gly (G) 11 46 Thr (T) 8 34

His (H) 5 21 Trp (W) 5 21




Carbon C 1234

Hydrogen H 1927

Nitrogen N 345

Oxygen O 341

Sulfur S 23










stable

Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

281



4A38 Formula


sequence is 238

4A39 RADAR RESULTS


4A310 MOTIF RESULTS






10 20 30 40 50 60 70

80

||||||||


GEKHLCDKFAKILR 43



cd03183 3



PFLAGDEI-SIADL 78

90 100 110 120

||||


Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

282



cd03183 79


123



Tiny (A+C+G+S+T) 63 26471







Basic (H+K+R) 49 20588


Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

283





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

284



Homology modelling








Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

285
























Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

286


+0531

-0103

1 200

Hydrophobicity

indices at

ph 34

determined

by HPLC

+0827

-082

0

1 200

Hydropathicity

Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

287





A B

C D



Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

288


Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

289




Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

290































sterically hindered

Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

291







as small squares




region





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

292



























make class

Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

293








Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

294














Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

295









Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

296














Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

297











distance 315 Aring)






















Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

298


















Distance 5000000

Show distances No



Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

299




Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

300


Site












4A318)

Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

301


Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

302

4A4 Discussion

































Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

303
























nnpredict










Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

304


































Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

305


































Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

306







sequences



























Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

307


































Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

308


































Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

309










DDT

4A5 Conclusion














Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

310

References
































Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

311














York











2725- 2731









Chapter 2

312











15569- 574





Chapter 2

312











15569- 574





Chapter 2shodhganga.inflibnet.ac.in/bitstream/10603/13612/14/14_chapter 2.pdf · host chromosome....

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Transcript of Chapter 2shodhganga.inflibnet.ac.in/bitstream/10603/13612/14/14_chapter 2.pdf · host chromosome....