Genomic Library_Hongming Lam

7/31/2019 Genomic Library_Hongming Lam

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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Construction and Screening ofGene Libraries

Further Readings:

Genome II by TA Brown, Ch. 4;

Current Protocols in MolecularBiology by Ausubel et al., Ch. 5 and 6;

PNAS88:1731-35


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Tissue/Cell

mRNA

cDNA

(methylation; addition oflinkers, etc.)

DNA

Partial or

CompleteRestriction

Size Fractionation

Vectors

(cDNA: plasmids,

phageGenomic: phage, cosmid,BAC, PAC, YAC, TAC)

Restriction

Ligation

Transformation, in vitropackaging, etc

Screening for

desired clones

Amplify for long-

term storage


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Genomic Libraries from genomic DNA

frequency of hitsindependent of geneexpression levels

may contain promoters andintrons

cannot express inheterologous system

useful for genome analysis,map-based cloning,

promoter studies, etc

reverse transcription of mRNA

dependent

no promoters or introns

expression is feasible if linkedto a suitable promoter

useful for analysis of codingregions and gene functions

cDNA Libraries


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Representation and RandomnessQ = (1 1/n)N

P = 1 Q

P = 1 (1 1/n)N

(1 1/n)N = 1 P

ln (1 1/n)N = ln (1 P)

N = ln (1-P) / ln (1-1/n)

Q: probability of a clone not in arandom library

P: probability of including any DNAsequence in a random library

N: independent recombinant clones

to be screened

n: total number of clones needed tocover the total genome if the

clones are not overlapping (totalsize of genome divided by theaverage size of a single clonedfragment)


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Representation and RandomnessN = ln (1 P) / ln (1 1/n)

P: probability of includingany DNA sequence in arandom library

N: independent recombinantclones to be screened

n: total number of clonesneeded to cover the total

genome if the clones arenot overlapping (total sizeof genome divided by the

average size of a singlecloned fragment)

N = ln (1 P) / ln (1 m/T)

P: probability that each mRNAwill be represented once

N: independent recombinantclones to be screened

m: number of molecules of therarest mRNA in a cell

T: total number of mRNAmolecules in a cell

Genomic Libraries cDNA Libraries


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Representation and Randomness

N = ln (1P) / ln (11/n)

To have 99% chance ofgetting a desiredsequence, screen 4.6

times the total number ofbase pairs

N = ln (1P) / ln (1m/T)

Since it is hard to estimatedthe total number of mRNAmolecules and the number of

rarest mRNA molecules, it isdifficult to predict the exactnumber of clones to bescreened. In general, to haveat least one copy of everymRNA, 500,000 to 1,000,000independent cDNA clones

should be screened

Genomic Libraries cDNA Libraries


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Vectors for Genomic Libraries

Propagate in Saccharomycescerevisiae;

Three major elements:centromere for nuclear division;telomeres for marking the end ofthe chromosome; origins ofreplication for initiation of newDNA synthesis when the

chromosome divides Previously an important tool to

map complex genomes;

Problems: chimera, instability

(rearrangement)

230 - 1700 kb(length ofnatural yeast

chromosome)Average: 400-700 kb

YAC (yeastartificialchromosome)

RemarksInsert SizeVector

More details can be found here:

http://batzerlab.lsu.edu/Hum_Mol_Gen_Lectures/Hum%20Mol%20Genet%20Lecture%203%20Feburary%203,%202004.ppt


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A combination of BAC and P1 featuresSimilar to

BAC

PAC (P1-

derived artificialchromosome)

Plasmid vector containing the F factor replicon;

One copy per bacterial cell

Up to 300 kb

Average:

100 kb

BAC (bacterialartificial

chromosome)

With P1 plasmid replicon (single copy in E. coli) andRi plasmid replicon (single copy in Agrobacterium

tumefaciens) With T-DNA border and can transform plant directly

Similar to P1TAC(Transformable

artificialchromosome)

Deletion version of a natural phage genome

P1 phage genome is about 110 kb

Efficient packaging system

paccleavage site for recognition

P1 plamsid replicon and inducible P1 lytic replicon

loxPsite for Cre action

Maximumabout 100 kb

BacteriophageP1



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Sources: www.genetics.wisc.edu/courses/spring04/466/files/bacteria2.pdf


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Sources: www.genetics.wisc.edu/courses/spring04/466/files/bacteria2.pdf


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pAD10SacBII

(30kb)

sacB

Ad2

pBR322 ori

P1 plasmidreplicon

pac

loxP

loxP

kan

P1 lyticreplicon

sacB

Sp6 T7

BamH 1Sfi 1 Not 1c 1 repressorbinding site

E.colipromoter

Sca 1

kan loxPloxP B BS S

pac

kan loxPloxP B BS S

pac

P1 vector for the construction ofP1 vector for the construction of

recombinants by P1 packagingrecombinants by P1 packaging

Pierce et al, Proc. Natl. Acad. Sci.U.S.A. (1992) 89, 2056-2060

Sources: http://batzerlab.lsu.edu/Hum_Mol_Gen_Lectures/Hum%20Mol%20Genet%20Lecture%203%20February%201 ,%202005.ppt


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Constructing P1 recombinants usingConstructing P1 recombinants usingpackaging extractspackaging extracts

S

pac

kan loxPB Skan loxPB Skan loxPB SloxP BloxP B

packaging

loxP B kan loxPB

insert of 80-90 kb

site-specific recombination

loxP



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Electroporation Based System Large insert size

Low copy number origin for propagation

High copy origin for DNA production

Negative selection against non-recombinants

Very stable inserts

P1 Derived ArtificialP1 Derived ArtificialChromosomes (PACs)Chromosomes (PACs)



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Genome size of phages is about 47 kb;

Packaging system is efficient and can

handle a total size of 78-105% of the genome;

Replacement vector system is usuallyemployed;

Pre-digested arms are commerciallyavailable for library constructions;

Useful for study of individual genes

Up to20-30kb

Phages

RemarksInsert

Size

Vector


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Contain F plasmid origin of

replication and cossite;

Low copy number and hence morestable

Similar tocosmid

Fosmids

Plasmid contain the cossite of phage and hence can use phagepackaging system;

Propagate in E. colias plasmids; Useful for subcloning of DNA inserts

from YAC, BAC, PAC, etc.

35-45 kbCosmid

RemarksInsertSize

Vector


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Vectors for cDNA Libraries

Maximum size for mRNA is about 8 kb, hence thecapacity of DNA insert is not a major concernhere;

Insertion vector system is usually employed; Useful for study of individual genes and their

putative functions Efficient packaging system, easy for gene transfer

into E. colicells, more representative thanplasmid libraries, subcloning and subsequentDNA manipulation processes are less convenientthan plasmid systems

Up to 20-30kb (for

replacementvectors) and10-15 kb (forinsertion

vectors)

Phages

Relatively easy to transform E. colicells although

may not be as efficient as the phage system forlarge scale gene transfer;

Less representative than phage libraries,subcloning and subsequent DNA manipulationprocesses are more convenient than the phagesystems

Up to 10-15kb

Bacterialplasmids



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Combining the Advantage of the

Phage and Plasmid Systems

Embed a plasmid vector in a vector In vitropackage, transfect, propagate, and

screen as phages

Excise the plasmid for the vector andpropagate and manipulate as plasmidssubsequently, e.g.

Excise by filamentous helper phages (e.g.Strategene)

Excise by the Cre-loxsystem (Clontech)


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ZAP Library (Stategene)


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AAAAAAAA

mRNA

TTTTTTTTGAGCTC

+ Linker-primerReverse transcriptase (no RNase activities)

5-methyl dCTP, dATP, dGTP, dTTP

First Strand Synthesis

1st Strand

cDNACH3 CH3 CH3

linker-primer: reverse transcription, restriction site (XhoI)

5-methyl dCTP: protect internal sites


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TTTTTTTTGAGCTC

+ RNase H and DNA polymerase I; dNTPs

CH3 CH3 CH3

2nd StrandcDNA

AAAAAAAACTCGAT

RNase H: remove mRNA in DNA-RNA hybrid

DNA Polymerase I: synthesis 2nd strand

Both enzymes added simultaneously

When RNase H starts to degrade the mRNA, residual RNAfragments may act as primers for initiation of DNA synthesis

DNA Polymerase 1 fills the gaps by nick translation

Second Strand Synthesis


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TTTTTTTTGAGCTC

CH3 CH3 CH3

AAAAAAAACTCGAT

EcoRIAdapter and XhoI linker: directional cloning

Addition of Adapter

G...

AATTC...

... CTTAA

... G

+ EcoRI adapter; ligase

TTTTTTTTGAGCT

CH3 CH3 CH3

AAAAAAAAC

G...

AATTC...

+ XhoI


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Primary Library Synthesis Size fractionation Ligation to arms

In vitropackage (packaging extract should be McrA-,McrB-, and Mrr- to prevent digestion ofhemimethylated DNA)

Host for phage infection, e.g. XL1-Blue MRF:Restriction deficient (mcrA)183, (mcrBC-hsdSMR-mrr)173;

supEto allow propagation of helper filamentous phages(with amber mutation); recA- to prevent recombination

F plasmid:

M15lacZfor blue-white screening

lacIq to prevent uncontrolled expression of fusion protein

F pili to allow infection of helper filamentous phages


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ZAP Library (Stategene)


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Excision of pBluescript Plasmid

First host XL1-Blue MRF: Fplasmid provides F pilifor f1 filamentous phage attachment; it is asuppressing strain so that f1 helper phages whichcarry an amber mutation can pack DNA

The ZAP: contains initiation and termination signalsequences for f1 packing flanking pBluescriptsequence

Second host: SOLR, a non-suppressing strain tostop helper phage propagation; a phage resistantstrain to prevent phage propagation


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F

XL1-Blue MRF


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F

Co-transfection of ZAP and fl help phage

XL1-Blue MRF


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F

The ZAP phage clone replicates in the host cell

The fl helper phage protein nicks the fl replication initiation

site I on ZAP and replication continues until reaching the flreplication termination site T

XL1-Blue MRF

T I

BIO L M i l P d b D H Mi L


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F

fl phage coats indiscriminately pack DNA moleculescontaining the fl replication origin; both fl genome

and pBluescript fragments are packed

XL1-Blue MRF

BIO4320 L t M t i l P d b D H Mi L


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F

fl phages are secreted to thegrowth medium

XL1-Blue MRF

BIO4320 Lecture Materials Prepared by Dr Hon Ming Lam


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F

SOLR

fl phages infect a new host SOLR (R

, suppressor free)

F

BIO4320 Lecture Materials Prepared by Dr Hon Ming Lam


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F

SOLR

fl phages infect a new host SOLR (R

, suppressor free)

F

BIO4320 Lecture Materials Prepared by Dr Hon-Ming Lam


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F

SOLR

fl genome (contains an amber mutation) cannot propagate inthe suppressor free SOLR and pBluescript containing cells

can be selected by the ampicillin resistance marker

BIO4320 Lecture Materials Prepared by Dr Hon-Ming Lam


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BIO4320 Lecture Materials, Prepared by Dr. Hon Ming Lam

Screening for the Right Clones

By PCR or hybridization if DNA sequenceinformation of the target gene or homologousgenes, or the amino acid sequence of thetarget gene product is available

By hybridization if DNA fragments of thetarget gene or homologous genes areavailable

By functional assays, e.g.Yeast functional complementation

Microinjection



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BIO4320 Lecture Materials, Prepared by Dr. Hon Ming Lam

Screening by Hybridization Plaque/colony lifting

Making labeled probes

HybridizationPre-hybridization

Hybridization

Washing Detection

Radioactivity

LuminescenceFlorescence

Chemiluminescence

Rescuing clones and further analysis

Pl /C l Lif i


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Plaque/Colony Lifting

Nylon membrane

Pl /C l Lifti


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Master plate

Replica membrane

Hybridization

Labelednucleic acidprobes(radioactive or

non-radioactive)

Plaque/Colony Lifting


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Hybridizationbottle

32P

Labeled probes hybridize toDNA bound on membrane

Wash offunboundprobe

Place Bio-Max film ontohybridized membrane


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Hybridizationbottle

Develop film

Original master plate



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The Result of 2

nd

Round Screening



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Making Probes

Methods of DetectionRadioactive (e.g. P32, P33, S35)

Non-radioactive (e.g. biotinylation,horseradish peroxidase, Digoxigenin)



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Example of Non-Radioactive Probes

(from Boehringer Mannheim)

DIG (Digoxigenin)



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Example of Non-Radioactive Probes

(from Boehringer Mannheim)



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Making Probes Methods of Synthesis

DNA

Nick translation (E. colipolymerase I)

Random priming (Klenow)

Random labeling by PCR 5-end labeling (DNA kinase)

3-end labeling (T4 DNA polymerase,

terminal transferase, modified T7 DNApolymerase, Klenow, reversetranscriptase)



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Nick Translation



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Nick Translation

Nicks created by DNaseI



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Nick Translation

Gaps created by E. coliDNApolymerase I (5 to 3 exonuclease

activities)



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Nick Translation

Gaps filled by E. coliDNA polymerase Iin the presence of dNTPs and labeled

deoxynucleotides (5 to 3 polymeraseactivities, proofreading by 3 to 5

exonuclease activities)



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Nick Translation

Reaction repeats to give uniform labeling



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Random Priming



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Random Priming

Heat denaturing

+ Random primers (hexamers);+ Klenow, dNTPs, and labeleddeoxynucleotides



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Making Probes Methods of Synthesis

RNA

Random labeling by in vitrotranscription(DNA dependent RNA polymerase, e.g.Sp6, T3, T7 RNA polymerase)

3-end labeling (DNA independent RNApolymerase + ATP by polyA tail; RNAligase)



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Making Riboprobes

Promoter

Clone gene of interest under the control

of a strong, specific, and induciblepromoter (e.g. Sp6, T3, T7)

Gene of Interest



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Making Riboprobes

Cut with a restriction enzyme to generate

a blunt end or 5 overhang at the 3 end ofthe gene of interest

Promoter Gene of Interest



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Making Riboprobes

+ DNA-dependent RNA polymerase

corresponding to the promoter used, inthe presence of NTPs and labelednucleotides, to generate randomly labeled

cRNA probes

Promoter Gene of Interest


H b idi ti d W hi


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Hybridization and Washing Prehybridization

for reduction of noise to signal ratio

Hybridization

usually carried out in solutions of high ionicstrength to maximize annealing rate

20-25C below Tm

smaller volume the better: fast reassociation andless probes

to minimize background, hybridize for the shortesttime with minimum probes

Washing to remove non-specific binding

can control the stringency by altering temperatureor [Na+]



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Conditions for Hybridization and Washing

For cloning of a specific gene, use highstringency conditions for hybridization (hightemperature, with formamide) and washing(high temperature, low NaCl)

For cloning of homologous genes (ormembers of a gene family), use low

stringency conditions for hybridization (lowtemperature, without formamide) andwashing (low temperature, high NaCl)



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Factors Affecting Hybrid Stability

Negligible effect with probes >500 bpDuplex length

Tm is reduced by 1oC for each 1% ofmismatching

Mismatchedbase pairs

Each 1% of formamide reduces the Tm byabout 0.6oC for a DNA-DNA hybrid

DestabilizingAgents

AT base pairs are less stable than GC basepairs in aqueous solutions containing NaCl

BaseComposition

Tm increases 16.6oC each 10-fold increase inmonovalent cations (Na+) between 0.01 to 0.40

M NaCl

Ionic Strength



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Tm: Transition Mid-Point;

Melting Temperature

Temperature at which 50% of thenucleotides of DNA-DNA, DNA-

RNA or RNA-RNA duplex arehybridized.



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For Hybrids > 100 Nucleotides

DNA-DNA

Tm=81.5oC + 16.6log10[Na+] + 0.41(%G+C) -0.63(% formamide) - (600/l)

DNA-RNA

Tm=79.8oC + 18.5log10[Na+] + 0.58(%G+C) +

11.8(%G+C)2 - 0.5(% formamide) - (820/l)

RNA-RNATm=79.8oC + 18.5log10[Na

+] + 0.58(%G+C) +11.8(%G+C)2 - 0.35(% formamide) - (820/l)



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For Hybrids > 100 Nucleotides

l = the length of the hybrid in base pairs [Na+] between 0.01 M to 0.4 M

%G+C between 30% to 75%

Stability: RNA-RNA > RNA-DNA > DNA-DNA Tm of a double-stranded DNA decreases by 1-

1.5oC with every 1% decrease in homology

pH between 5-9; when outside this range,stability of DNA decreases drastically due toprotonation or deprotonation of the bases



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For Oligos 14-70 nucleotides

Tm = 81.5oC + 16.6(log10[Na+])

+ 0.41(%G+C) - (600/N)

N=number of nucleotides



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For Oligos < 18 nucleotides

Tm = (#A+T x 2) + (#G+C x 4)


Factors Affecting Hybridization Rate


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g y

Repetitive sequences increase the rateProbecomplexity

Each 10% of mismatching reduces rate by a factor of twoMismatchedbase pairs

Directly proportional to duplex lengthDuplex length

50% formamide: no effect; other concentrations: reducedDestabilizingAgents

Optimal rate at 1.5 M Na+

IonicStrength

Little effectBaseComposition

Increase rate of membrane hybridization; 10% dextransulfate increases rate by factor of tenViscosity

Maximum rate: 20-25oC below Tm for DNA-DNA hybrids, 10-15oC below Tm for DNA-RNA hybrids

Temperature

Little effect between pH 5.0 to 9.0pH



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Screening for the Right Clones

By PCR or hybridization if DNA sequenceinformation of the target gene or homologousgenes, or the amino acid sequence of thetarget gene product is available

By hybridization if DNA fragments of thetarget gene or homologous genes areavailable

By functional assays, e.g.Yeast functional complementation

Microinjection


Screening by Yeast Functional


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g y

Complementation

Saccharomyces cerevisiaeis a simpleeukaryotic system

Relatively easy to grow and perform

genetic manipulation

Mutants, expression vector,

transformation protocol, etc. areavailable


Cloning by Yeast Functional Complementation


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A yeast mutant which is defective

in a function of your interest

A library containing cDNAs of theorganism of interest is inserted in

a yeast vector;

Yeastexpressionpromoter

cDNAs express under an

inducible yeast expression promoter




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Yeast mutant is complementedwhen the transgene expression is induced




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Designing a Suitable Vector

E. coli

replicationorigin

Yeast

replicationorigin

E. coli

selection

marker

Yeastselection marker

Multiple

cloning site

Yeast expressionpromoter


A Case Study: Cloning of Potassium Channels


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A yeast mutant which cannot survive

on K+

-limiting medium

A plant cDNA library constructed ina yeast vector;

Yeastexpressionpromoter

Plant cDNAs express under an

inducible yeast expression promoter




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Survives in K+-limiting mediumwhen the transgene expression is induced





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Mutant Transformant

Both survive on K+

-rich medium

Only the transformant surviveson K+-limiting medium

Transgene not

induced

Anderson et al., 1992


Genomic Library_Hongming Lam

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