Genomes Slides

8/3/2019 Genomes Slides

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Microbial Genomes

1) Methods for Studying Microbial Genomes

2) Analysis and Interpretation of Whole Genome Sequences


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Why study microbial genomes?

until whole genome analysis became viable, life sciences have been

based on a reductionist principle dissecting cell and systems into

fundamental components for further study

studies on whole genomes and whole genome sequences in particulargive us a complete genomic blueprint for an organism

we can now begin to examine how all of these parts operate

cooperatively to influence the activities and behavior of an entire

organism a complete understanding of the biology of an organism

microbes provide an excellent starting point for studies of this type asthey have a relatively simple genomic structure compared to higher,

multicellular organisms

studies on microbial genomes may provide crucial starting points for

the understanding of the genomics of higher organisms


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Why study microbial genomes?

analysis of whole microbial genomes also provides insight into

microbial evolution and diversity beyond single protein or gene

phylogenies

in practical terms analysis of whole microbial genomes is also apowerful tool in identifying new applications in for biotechnology and

new approaches to the treatment and control of pathogenic organisms


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H

istory of microbial genome sequencing

1977 - first complete genome to be sequenced was bacteriophage

JX174 - 5386 bp

first genome to be sequenced using random DNA fragments -

Bacteriophage P - 48502 bp

1986 - mitochondrial (187 kb) and chloroplast (121 kb) genomes of

Marchantia polymorpha sequenced

early 90s - cytomegalovirus (229 kb) and Vaccinia (192 kb) genomes

sequenced

1995 - first complete genome sequence from a free living organism -

Haemophilus influenzae (1.83 Mb)

late 1990s - many additional microbial genomes sequenced including

Archaea (Methanococcus jannaschii - 1996) and Eukaryotes

(Saccharomyces cerevisiae - 1996)


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Microbial genomes sequenced to date

currently there are 32 complete, published microbial genomes 25

domain Bacteria, 5 Domain Archaea, 1 domain Eukarya

(www.tigr.org)

around 130 additional microbial genome and chromosome sequencingprojects underway


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L

aboratory tools for studying whole genomes

conventional techniques for analysing DNA are designed for the

analysis of small regions of whole genomes such as individual genes or

operons

many of the techniques used to study whole genomes are conventionalmolecular biology techniques adapted to operate effectively with DNA

in a much larger size range


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Pulsed Field Gel Electrophoresis

agarose gel electrophoresis is a fundamental technique in molecular

biology but is generally unable to resolve fragments greater than 20

kilobases in size (whole microbial genomes are usually greater than

1000 kilobases in size) PFGE (pulsed field gel electrophoresis) is a adaptation of conventional

agarose gel electrophoresis that allows extremely large DNA

fragments to be resolved (up to megabase size fragments)

essential technique for estimating the sizes of whole

genomes/chromosomes prior to sequencing and is necessary forpreparing large DNA fragments for large insert DNA cloning and

analysis of subsequent clones

also a commonly used and extremely powerful tool for genotyping and

epidemiology studies for pathogenic microorganisms


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Principle ofPFGE

two factors influence DNA migration rates through conventional gels

- charge differences between DNA fragments

- molecular sieve effect of DNA pores

DNA fragments normally travel through agarose pores as spherical

coils, fragments greater than 20 kb in size form extended coils and

therefore are not subjected to the molecular sieve effect

the charge effect is countered by the proportionally increased friction

applied to the molecules and therefore fragments greater than 20 kb do

not resolve

PFGE works by periodically altering the electric field orientation

the large extended coil DNA fragments are forced to change

orientation and size dependent separation is re-established because the

time taken for the DNA to reorient is size dependent


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Principle ofPFGE


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Principle ofPFGE

the most important factor in PFGE resolution is switching time, longer

switching times generally lead to increased size of DNA fragments

which can be resolved

switching times are optimised for the expected size of the DNA beingrun on the PFGE gel

switch time ramping increases the region of the gel in which DNA

separation is linear with respect to size

a number of different apparatus have been developed in order to

generate this switching in electric fields however most commonly usedin modern laboratories are FIGE (Field Inversion Gel Electrophoresis)

and CHEF (Contour-Clamped Homogenous Electrophoresis)


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Electric Field 1 Electric Field 2

Switch Time

CHEF


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Preparation of DNA forPFGE

ideally a genomic DNA preparation that contains a high proportion of

completely or almost completely intact genome copies would be

suitable forPFGE

conventional means of DNA preparation are unsuitable forPFGE asmechanical shearing and low-level nuclease activity will result in

fragmented DNA with an average size much smaller than an entire

microbial genome (usually less than 200 kb in size)

the solution to this is to prepare genomic DNA from whole cells in a

semisolid matrix (ie. agarose) that eliminates mechanical shearing a very high concentration of EDTA is also used at all times in order to

eliminate all nuclease activity


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Procedure

1) intact cells are mixed with molten LMT agarose and set in a mold

forming agarose plugs

2) enzymes and detergents diffuse into the plugs and lyse cells

3) proteinase K diffuses into plugs and digests proteins

4) if necessary restriction digests are performed in plugs (extensive

washing orPMSF treatment is required to remove proteinase K

activity)

5) plugs are loaded directly onto PFGE and run


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for restriction digests, conventional enzymes are unsuitable as they cut

frequently on an entire genome sequence producing DNA fragments

that are far too small

rare cutter restriction endonucleases cut genomic DNA with far lessfrequency than conventional restriction enzymes such as HindIII,

BamHI etc.

many rare cutter REs have 6-bp (or longer) recognition sites eg. NotI

GCGGCCGC

in many cases the frequency of cutting is highly species dependent eg.BamHI will cut far less frequently on a low GC% genome when

compared to a intermediate or high GC content genome

suitable rare cutter enzymes therefore have to be determined

experimentally for each new species being studied


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Large insert cloning vectors BACs and

PACs

DNA cloning is another technique fundamental to molecular biology

that requires adaptation in order to be useful in studying DNA at a

whole genome scale

conventional plasmid derived cloning vectors are only able to reliablymaintain inserts less than 20 kb in size

there are a number of approaches to generating clones with inserts in

an intermediate size range (20 80 kB) such as cosmids, etc.

the most commonly used vectors for cloning extremely large DNA

inserts are BACs (Bacterial Artificial Chromosomes) and PACs (P1-derived Artificial Chromosomes)

both BAC and PAC vectors are plasmid derived vectors distinguished

from conventional vectors by extremely tightly controlled low copy

numbers


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Large insert cloning vectors BACs and

PACs

these very low copy numbers help to limit the strain on host cellular

resources generated by very large DNA inserts thus eliminating the

rejection of large insert clones

low copy numbers also help to limit recombination events with hostgenomic DNA

BAC and PAC vectors both utilise E. coli as the host organism

BAC vectors are based on the E. coli single copy F-factor plasmid

the F-factor origin of replication is very tightly controlled

PAC vectors are based on an identical principle but instead use a singlecopy origin of replication derived from P1 phage

PAC vectors also contain a pUC19 cassette for improved vector

purification


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Approaches to whole microbial genome

sequencing

aim of microbial genome sequencing projects is to construct, from 500

800 bp sequencing reads containing about 1% mistakes, a genome

sequence of several megabases with an error rate lower than 1 per

10000 nucleotides with improving software, decreasing computation costs and

advancements in automated DNA sequencing, an entire microbial

genome project can be completed in a small laboratory in 1-2 years

there are two main approaches to sequencing microbial genomes the

ordered clone approach and direct shotgun sequencing both require both large and small insert genomic DNA libraries in

order to be effective


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Approaches to whole microbial genome

sequencing


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Ordered Clone Approach

essentially this technique involves constructing a map of overlapping

large insert clones covering the whole genome and then completely

sequencing the minimum subset of these ordered clones

there are a number of methods used to order clones including

restriction fingerprinting and hybridisation mapping

once an ordered large insert clone set is identified, a whole genome

sequence is determined by either shotgun or partial primer walk

sequencing of each insert

the ordered clone approach to DNA sequencing requires a large

amount of characterisation prior to actual DNA sequencing and istherefore a relatively time consuming approach, however, it may be

cheaper than shotgun sequencing an entire genome as less redundant

sequencing is required

with rapid decreases in costs for computing power and sequencing this

method is no longer considered viable for small (< 5 Mb) genomes


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Whole Genome

Large

DNA

fragmentDigest and

subclone

Randomly

sequence

fragments

Fill gaps

Repeat for entire genome map


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Random sequencing (shotgun) approach

this is the currently the most commonly used strategy for microbial

whole genome sequencing

sequences from both ends of a large number of small and large insert

clones are generated and overlapping sequences joined together to

form a contig of the whole genome sequence (whole inserts notsequenced)

although this requires enormous amounts of DNA sequencing (often

up to 10x genome coverage) and computational power for sequence

assembly, it is a relatively rapid approach to whole genome sequencing

the first 90 95% of the genome sequence is relatively easy togenerate by shotgun sequencing resulting in several hundred discrete

contigs

filling the gaps to produce a single contig is the most difficult and time

consuming phase of this process


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Whole Genome

Shear and

subclone

Randomly

sequence

fragments

Fill gaps


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Random sequencing (shotgun) approach

There are a number of steps in the process -

1) Random large and small insert library construction

2) High throughput DNA sequencing

3) Sequence assembly

4) Ordering of contigs

5) Primer walking to complete sequence

6) Annotation


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Library construction

Both conventional and large insert genomic DNA libraries should be

constructed

the small insert library will be used for the bulk of the sequencing in

order to generate suitable coverage of the complete genome

the large insert library (BAC, PAC, cosmid etc.) will be used as a

scaffold during the sequence closure phase

it is crucial to ensure that both libraries are as random as possible -

mechanical shearing is often used to generate small DNA fragments

it is also important that each clone contains only one DNA fragment

and as such specialised methods for library construction must be used


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DNA Sequencing

DNA sequences are generated using vector primers for both ends of

inserts

at least 6X coverage of the genome is required although 9 to 10X

coverage is often generated


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Sequence assembly and gap closure

4 major steps in sequence assembly and gap closure -

1) random sequences initially interpreted using highly accurate base

calling software and assembled to generate primary contigs usingsoftware such as PHRAPP

2) computational and experimental techniques used to identify linking

clones and order primary contigs

3) primer walk sequencing of linking clones and PCR products to fill

sequence gaps between contigs 4) confirmation of contig order by PCR


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Linking Clones

one of the most effective means of contig ordering and gap filling is

linking clones

linking clones are those whose terminal sequences (from either end of

the insert) belong to different contigs if the orientation of the sequences and the distance to the end of the

contig are compatible with with the size of the insert, the two contigs

are likely to be linked

the larger the insert the more likely a clone will be a linking clone

this is why random sequencing is also performed on large insert clones- they are far more likely to form linking clones


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Contig 1 Contig 2

Random Sequencing Random Sequencing

Gap


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Contig 1 Contig 2

FWD REV

Large Insert

Linking Clone

Once all possible linking clones are identified -

gaps are classified into two categories - those with linking clones

(template available for sequencing) and physical gaps without linking

clones ( no DNA template for the region)

for those gaps with suitable linking clones, the gaps confirmed by

PCR and closed by primer walk sequencing


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Contig 1 Contig 2

FWD REV

Large insert

Linking Clone

Primer Walking


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Contigs separated with physical gaps (no linking clones) are usually

spanned by PCR on genomic DNA using primers from each end of the

contigs

the PCR products can then be sequenced to close the gaps

without linking clones other techniques to order contigs must be used

in order to guide the selection ofPCR products

Physical Gaps


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Supercontig 1

Supercontig 2

Supercontig 3

Linking

clone

For those contigs without

linking clones, how do you fill

the gaps?


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contigs can be ordered by -

peptide linking - contig ends having regions with homology to the

same gene (or operon / gene cluster)

southern hybridisation of labelled contig terminal oligonucleotidesagainst large restriction fragments

Physical Gaps


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Contig 2 Contig 6

FWD REV

Primer Walking

PCRProduct

Linked by Southern Hybridisation


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Gapped Microbial Genomes

considering the cost and difficulty in filling gaps between contigs some

interest has been generated by the analysis of gapped microbial

genomes

each gap is usually very small on average (approximately 75 bp for a3.2x coverage library)

increasing bioinformatic resources available mean that these gaps have

little influence on functional reconstruction

eg. Thiobacillus ferroxidans - all assigned amino acid biosynthesis

genes (140 in total) identified from a gapped genome of 1912 contigs error rates tend to be relatively high compared to genome sequences

with greater coverage


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Example -Haemophilus influenzae

first complete genome sequence of a free living organism (1995)

important pathogen

genome is around 1.83 megabases in size

random sequencing was done for both small insert and large insert

(lambda) libraries

sequencing reactions performed by eight individuals using fourteen

ABI 377 DNA sequencers per day over a three month period

in total around 33000 sequencing reactions were performed on 20000

templates

plasmid extraction performed in a 96 well format

11 mb of sequence was intially used to generate 140 contigs

gaps were closed by lambda linking clones (23), peptide links (2),

Southern analysis (37) and PCR (42)


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Annotation of Genome Sequences

a microbial genome sequence alone is only raw data it needs to be

interpreted in order to be of any scientific significance

the process of predicting the location and function of all possible

coding sequences (genes) in a genome sequence is known as

annotation

although an annotated genome sequence provides a large amount of

important information it is still merely a starting point for completely

characterising an organism


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Identifying ORFs

most genomes will contain genes with very little or no homology to

known genes of other organisms

for this reason all of the possibleORFs need to be identified without

relying totally on homology most efficient means for identifying potential genes in genome

sequences is a three step process

1) submit entire sequence as a 6-frame translation for BLAST analysis

in order to identify some protein coding regions on the basis of high

levels of homology 2) use these initial coding regions to determine the sequence

characteristics (GC content, codon bias etc.) that distinguish coding

and non-coding regions of the genome (training the software)


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Identifying ORFs

3) reanalyse the genome sequence using this data (plus potential

ribosome binding sequences) in order to identify all the potential genes

using this process it has been experimentally shown that around 94%

of genes can be accurately predicted algorithms are also available to identifyORFs without using the

training procedure with only slightly reduced accuracy


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Assigning function to ORFs

in order to assign function, all predicted ORFs are translated to amino

acid sequence and analysed by homology searches against sequence

databases (usually Genbank)

for each ORF there are three possible results - i) clear sequence homology indicating function

ii) blocks of homology to defined functional motifs

- these should be confirmed experimentally

iii) no significant homology or homology to proteins of unknown

function


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ORFs of unidentified function

in most genome sequences many of theORFs identified cannot be

assigned a specific function based on homology

although the figure varies, usually between 40 and 50% ofORFs fall

into this category clearly this represents a significant gap in our knowledge of microbial

metabolism

these ORFs can be further divided into two categories

i) conserved hypothetical proteins ORFs with no homology to

proteins of known function but with significant homology tounidentified ORFs of other species

these ORFs are therefore functionally conserved across numerous

species and may represent important components of central

metabolism that have not yet been identified


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ORFs of unidentified function

the more universal the distribution of theseORFs the more likely they

have a fundamental role in metabolism

ii) ORFs without homologues these are ORFs that have no

homology to any known sequences these may represent genesencoding proteins related to more specific organism adaptations

eg. Deinococcus radiodurans is a radiation resistant organism that

contains many ORFs without homologues many of these are thought

to be involved in specialised processes of DNA repair


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Organism (total

ORFs)

Homologues to

known proteins (%)

Homologues to

conserved

hypothetical

proteins (%)

No homologues (%)

E. coli (4277) 33.3 10.3 56.4

Pyrococcus

horikoshii (2064)

35 33.3 31.7

Haemophilus

influenzae (1709)

58.8 18.2 23

B. subtilis (4099) 58 5 37

Methanococcus

jannaschii (1735)

38.1 40.6 21.3


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Structural genomics

in order to gain a complete understanding of an organism and fully

exploit the potential offered by microbial genome sequencing, it is

essential that these unidentified ORFs are assigned function

in most cases classical molecular biology tools will be necessary for

this task, however, some suggestion of function for theseO

RFs wouldgreatly improve the efficiency of this process

one possibility is structural genomics

this is the process of determining three dimensional structures of all the

gene products encoded in a microbial genome (1000s of structures!!)

function can then be inferred on the basis of 3d structure comparisonsto other proteins

this relies on the principle that structure determines functions and

although two proteins with similar amino acid sequences can be

assumed to have similar structures, two proteins with similar structure

dont necessarily have the same aa sequence


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Microarray hybridisation

a completely annotated microbial genome sequence, whilst a powerful

scientific tool, still doesnt provide all of the information needed to

understand the complete biology of an organism as it essentially a

static picture of the genome

for truly complete characterisation, the dynamic nature of geneexpression within a microbial cell needs to be determined

microarray technology allows whole organism gene expression to be

investigated

PCR products of every gene from a complete genome sequence are

bound in a high density array on a glass slide these arrays are probed with fluorescently labelled cDNA prepared

from whole RNA under specific environmental conditions

the level of cDNA for each ORF is then quantified using high

resolution image scanners


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Microarray hybridisation

example a microarray containing 97% of the predictedORFs from

Mycobacterium tuberculosis was used to investigate the response to

the antituberculosis drug isoniazid (INH)

INH was found to induce several genes related to outer lipid envelopebiosynthesis consistent with the drugs physiological mode of action

a number of additional genes were also induced which may provide

potential drug targets in the future


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INH untreated - green

INH treated - red

Yellow = Red + Green (no

change in expression)

Green = only expressed

without INH treatment

Red = only expressed after

INH treatment

Overlay


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Characteristics of sequenced genomes

the 32 complete genome sequences currently available cover a diverse

range in terms of phylogeny and environments (eg. human pathogens,

plant pathogens, extremophiles etc.)

what conclusions can be made by comparing the genomes of theseorganisms regarding specific adaptations to proliferation in remarkably

different environments?

What conclusions can be made about evolutionary relationships

between these organisms?


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Horizontal gene transfer

before microbial genome sequences became available most of the

focus of microbial evolution was on vertical transmission of genetic

information mutation recombination and rearrangement within the

clonal lineage of a single microbial population genome sequences have demonstrated that horizontal transfer of genes

(between different types of organisms) are widespread and may occur

between phylogentically diverse organisms

generally speaking, essential genes (such as 16S rRNA) are unlikely to

be transferred because the potential host most likely already containsgenes of this type that have co-evolved with the rest of its cellular

machinery and and cannot be displaced

genes encoding non-essential cellular processes of potential benefit to

other organisms are far more likely to be transferred (eg. those

involved in catabolic processes)


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Horizontal gene transfer

clearly, lateral transfer of genomic information has enormous potential

in improving an microorganisms ability to compete effectively - this

may explain why horizontally transferred genes appear so frequently

and ubiquitously in microbial genomes an example of this is horizontally transferred genes between Archaeal

and Bacterial hyperthermophiles -

Thermotoga maritima has 15 clusters of genes (4-20kb) most similar to

equivalent Archaeal hyperthermophile gene regions


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Whole genome phylogenetic analysis

most of the evolutionary relationships between microorganisms are

inferred by comparison of single genes usually 16s rRNA genes

although extremely effective, single gene phylogenetic trees only

provide limited information which can make determining broadrelationships between major groups difficult

phylogenetic relationships can be determined by whole genome

comparisons of the observed absence or presence of protein encoding

gene families

in effect this is similar to using the distribution of morphologicalcharacteristics to determine phylogeny without the problem of

convergent evolution

trees produced using this method are similar to 16s rRNA trees,

however, as more genome sequences become available more detailed

conclusions can be drawn using this method


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Archaeal Genomes

analysis of the 5 complete genome available for members of the

domain Archaea has provided new insights into relationships between

Archaea, Bacteria and Eukaryotes

around 35% of the Archaeal genes form a stable core conservedthroughout the domain

most of these encode proteins involved in transcription, translation and

DNA metabolism and some central metabolic pathways

the remainder of the genome is classified as a variable shell

a relatively high proportion of the variable shell genes are mosthomologous to their bacterial counterparts - this suggests horizontal

gene transfer events

a relatively high proportion of the stable core genes are most similar to

Eukaryotic genes


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A - Stable core B - Variable shell


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Species and strain specific genetic diversity

although genome sequencing and analysis is very useful when

comparing phylogenetically distant taxa, it is also of interest to

examine the genomes of very closely related microorganisms

this allows a more quantitative approach for examining the

relationships between genotype and phenotype

complete genome sequences have been determined for two species of

the genus Chlamydia (pneumoniae and trachomatis)

although the overall genome structure was quite similar, C.pneumoniae

contained an additional 214 genes most of which have an unknown

function two strains of the bacteriumHelicobacter pylori have been completely

sequenced (26695 and J99)

overall the two strains were very similar genetically with only 6% of

genes being specific to each strain


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Case study - Deinococcus radiodurans

D. radiodurans R1 is an extremely radiation resistant bacterium

genome (total of 3.3 megabases) consists of two chromosomes (2.6

and 0.4 mb) a megaplasmid (177 kb) and a small plasmid (44 kb)

considerable genetic redundancy was observed in both thechromosomal and plasmid sequences

numerous systems for DNA repair, DNA damage export were

identified

a significant proportion of theORFs identified had no database

matches - these may be involved in unique cellular adaptations to

radiation and stress response


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Case study - Neisseria meningitits

N. meningititis causes bacterial meningitis and is therefore an

important pathogen

genome is 2.2 megabases in size

2121O

RFs were identified with many having extremely variableG+C% (recently acquired genes)

many of these recently acquired genes are identified as cell surface

proteins

there is a remarkable abundance and diversity of repetitive DNA

sequences

nearly 700 neisserial intergenic mosaic elements (NIMEs) - 50 to 150

bp repeat elements

these repeat elements may be involved in enhancing recombinase

specific horizontal gene transfer


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Case study - Borellia burgdorferi

B. burgdorferi is a spirochaete which causes Lyme disease

it has a 0.91 megabase linear genome and at least 17 linear and circular

plasmids which total 0.53 megabases

853 predictedO

RFs identified - these encode a basic set of proteinsfor DNA replication, transcription, translation and energy metabolism

no genes encoding proteins involved in cellular biosynthetic reactions

were identified - appears to have evolved via gene loss from a more

metabolically competent precursor

there is significant amount of genetic redundancy in the plasmid

sequences although a biological role has not been determined

it is possible the these plasmids undergo frequent homologous

recombination in order to generate antigenic variation in surface

proteins


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Summary

Microbial genome sequencing and analysis is a rapidly expanding and

increasingly important strand of microbiology

important information about the specific adaptations and evolution of

an organism can be determined from genome sequencing

however, genome sequencing merely a strong starting point on road to

completely understanding the biology of microorganisms

further characterisation ofORFs of unknown function, in combination

with gene expression analysis and proteomics is required

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