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Prof. Fahd M. Nasr

Lebanese universityFaculty of sciences I

Department of Natural Sciences

fnasr@ul.edu.lb

B3206Microbial Genetics

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Eukaryotic M. G.The yeast

Saccharomyces cerevisiaeas a genetic model system

Lectures XVIII and XIX

Cloning by complementation

• To identify the gene clone by complementation of the mutation

• A gene library the entire yeast genome– Digest the entire yeast DNA partially– Clone fragments in appropriately cut plasmids

• Cloned fragments are 5-9kb– 2,000 plasmids genome once– 10,000 plasmids give a more than 90%

probability that all genes are functionally represented

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Use of partial digestion with a restriction enzyme

to produce DNA fragments of appropriate

size for constructing a genomic library

Cloning genes by complementation

Transform in Plasmid Genomic Library

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the cloning of the yeast ARG1 gene

Cloning by complementation• The library is transformed into the yeast mutant

of interest• Transformants are screened or selected for

restoration of the wild type phenotype• Plasmids are isolated from positive clones

amplified into E. coli and further analysed• Seq. information reveals the identity of the clone• Retransformation to confirm complemention

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Complementation

Vector with Genomic Fragment Containing YFG

yfg ts (recessive)

Live at 37°C

Non-complementation (Allelic)

Hybrid Mating with Lines having Known Mutations that have similar phenotype

yfg ts

Dead at 37°CDead at 37°C

Known mutant

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Non-complementation (dominant negative)

Vector with Genomic Fragment Containing YFG

yfg ts (DN)

Dead at 37°C

• Cloning by complementation straightforward approach– Done with recessive mutants

– With dominant mutants a gene library from each mutant and transformed into the wild type strain transformants showing the mutant phenotype are then screened or selected

– Complementation a multi-copy suppressor

Cloning by complementation

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• To demonstrate that the cloned gene is the one that is mutated in the mutant– A deletion mutant has to be constructed by homologous

recombination– Good evidence original and the deletion mutants

have the same phenotype• Final proof cross two mutants

– Diploid has the mutant phenotype– All spores are mutants two genes are the same

• Deletion of genes by homologous recombination is one of the most powerful techniques in yeast

Cloning by complementation

Deleting a yeast geneYFG1 Your favourite gene on a plasmid

URA3Your favourite gene on a plasmid,ORF replaced by marker

Your favourite gene deletedfrom the genome

X X Recombination in yeastURA3

URA3

in vitro

in vivo

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Deleting a yeast gene• Different ways piece of DNA for yeast transformation

– Restriction enzymes and DNA ligation– PCR/restriction/ligation– PCR without any cloning step amplify flanking

regions of YFG1 use as primers to amplify the marker gene

– PCR primers marker is amplified and recombination is mediated by the primer sequences

– The latter two approaches do not even require the gene to be cloned gene deletion project hence may take only a couple of days

Deleting a yeast gene

YFG1 First PCR to amplify the flanking parts of your favourite gene

URA3 Final PCR product ready for transformation

URA3Second PCR to amplify the marker

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Gene deletion in yeast

HIS3

YFG1X X

Chromosomal DNA

DNA cassette~50nc

Chromosomal DNA

HIS3

Smart gene deletion• Smart ways gene deletion/disruption approach• Marker cassette contains in addition the lacZ

reporter gene places the lacZ gene under control of the yeast promoter of YFG1

• Gene deletion in a diploid– Study gene expression by monitoring b-galactosidase

activity in diploid– Study the mutant phenotype in the haploid progeny

• A gene can be tagged insert cassette in frame to the end of the ORF generate a fusion protein, with lacZ, GFP or an immuno-tag for protein detection and purification

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Smart gene deletion

lacZ

YFG1 Diploid cell

URA3

URA3YFG1 GFP

Tagging genes

HA, GST, TAP tags

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Smart gene deletion• Delete a yeast gene without leaving the marker gene• Important to re-use the marker make many deletions in

one and the same strain• Important for industrial yeast strains no foreign DNA

should be left behind• All these methods use homologous recombination a second

time pop-out the integrated DNA again• The loxP-kanR-loxP cassettes recombination between

the two loxP by the Cre-recombinase leaves behind a single loxP site

Selectable Integration Systems

• Integrate selectable marker into target site

• Integration of cloned gene into target site by double recombination eliminates marker gene

• Choose marker gene carefully….

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Removable Selectable Markers

The Cre-loxP strategy• Cre – a 38 kDA integrase family protein

– Encoded by the bacteriophage P1

– Recognizes a 34 bp DNA target on the P1 genome called loxP

• loxP (locus of X-over of P1)

• Cre catalyzes reciprocal DNA recombination between two loxP sites

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The Cre-loxP strategy• loxP sites

– Two inverted DNA segment repeats of 13 bp flanking a spacer (~8 bp)

– Two Cre monomers bind to the loxP site

– Two loxP sites are assembled in anti-parallel fashion by four Cre monomers to form a synaptic structure

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Smart gene deletion• URA3 a very useful marker select for

and against its presence– Selection for URA3 on medium lacking

uracil– Selection against URA3 uses the drug 5-FO

toxic to URA3 cells• Integration of the plasmid creates a

duplication (pop-in)• Recombination a pop-out of the entire

plasmid plus the YFG1 coding region

URA3

URA3plasmid

genomeYFG1

YFG1

Smart gene deletion

Pop-in

Pop-out

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The pyrimidine biosynthetic pathwayURA2 URA2

URA4

URA1

URA5URA10

URA3

5-fluoro-OMP

5-fluoro-UMP

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Gene function• Gene product function

– In what biological processes does the gene product act?

– What is the biochemical function of the gene product?

– Where and when does the gene product function?

Systems Biology• Biological process

– To execute the biological process, what are all the gene products that are necessary?

– How do all the gene products act together in a pathway/network/machine to execute the biological process?

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Network of Networks

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Approaches to function• One fundamental approach to understanding gene

function is to examine the cellular/organismal phenotype following the loss of gene function

• Gene loss-of-function or gene inactivation:– Deletion– Point mutation (nonsense or missense)– RNA mediated interference (RNAi)– Transposable element insertion– Preferable if gene activity is completely eliminated

(null)

Approaches to function• The loss-of-function phenotype allows one to infer wild-

type gene function

• The wild-type function necessary to correct the observed phenotype (opposite of the mutant phenotype)– CDC28 mutant in yeast has a G2 cell cycle arrest phenotype the

wt of the CDC28 gene is to promote G2 cell cycle progression

– ced-3 mutant in C. elegans is defective in apoptosis the wt function of the ced-3 gene is to promote apoptosis

– The greater depth of phenotypic characterization the greater the specificity of the definition of wild-type function

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How to deal with essential genes

• How can we identify and work with essential genes knockout mutation is lethal– For essential gene the deletion is done in a

diploid– Only two spores survive the gene is regarded

as essential– Work with conditional mutations ts mutants

Isolating temperature-sensitive (ts) mutants allows essential genes to be studied

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Testing the viability of null mutants

Phenotype rescue of Drtc1Growth

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Phenotype rescue of Drtc1Growth

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How to deal with essential genes

• Working with mutants in essential genes– The mutant is transformed with plasmid that

expresses the relevant gene conditionally– Put the essential gene under the control of the

promoter of the GAL1 gene• ON on galactose medium• OFF on glucose medium• Shift cells to glucose watch cells dying (yfg1D

pGAL1-YFG1)

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How to deal with essential genes

• To analyze point mutants– The mutant is first transformed with the wild

type gene and then with a mutant gene

– The plasmid with the wt gene carries URA3 forced to be lost on medium with 5-FOA

– The mutant grows on 5-FOA medium the mutant allele is functional (yfg1D pURA3::YFG1 pLEU2::yfg1-1)

Plasmid shuffling

*

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Plasmid Shuffle

Plasmid Shuffle Assay

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Corresponding tools in the two yeast species

The end