Molecular Biology. Yeast Transformation. Yeast Plasmids. Gene Disruption, tagging. Cloning by Complementation. Epistasis

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1 Molecular Biology Yeast Transformation Yeast Plasmids Gene Disruption, tagging Cloning by Complementation Epistasis

2 Transformation Transformation: introduction of DNA 1978, ca 1000x less efficient than E. coli Selectable marker Spheroplasts, Li 2+ salts, electroporation Yeast plasmids are shuttle plasmids, i.e. can be propagated in yeast and E. coli, E. coli: ori, β-lactamase Yeast: CEN, URA3

3 Yeast Plasmids Different properties for different uses

4 4 types of yeast vectors

5 Gene-disruption Homologous recombination is very efficient in yeast. Is the knock-out strain viable? What is the phenotype of a strain without the gene of interest? GENE SELECTABLE MARKER SELECTABLE MARKER

6 The Saccharomyces Genome Project has revealed the presence of more than 6000 open reading frames (ORFs) in the S. cerevisiae genome. Construct a set of deletion mutants corresponding to all these ORFs. The method used was a PCR-based gene deletion strategy to generate a start- to stop- codon deletion of each of the ORFs in the yeast genome. Each gene disruption was replaced with a KanM module and uniquely tagged with one or two 20mer sequence(s). The presence of the tags can be detected via hybridization to a high-density oligonucleotide array, enabling growth phenotypes of individual strains to be analyzed in parallel.

7 Adding a Bar Code

8 Heterozygotic Disruption Collection Deletion mutants available in haploid or diploid backgrounds (each ~4 500 strains). This work uses heterozygous knockout collection. Haploid or homozygous knockout typically has clearest phenotype. Why is this library used?

9 Cell Volume 116, January 2004, Pages

10 Gene-tagging In a similar way, a gene can be tagged. For instance, if the cassette is inserted in frame to the end of the ORF it will generate a fusion protein, with lacz, GFP or an immuno-tag for protein detection and purification For instance, there are now sets of strains available in which each yeast has been tagged with GFP or TAP-tag YFG1 GFP URA3

11 Localising proteins in the cell: GFP The green-fluorescent protein is used now systematically to localise proteins within the yeast cells A main advantage of the GFP technology is that it allows watching processes in the living cell! Usually the coding sequence of GFP is fused to the end of the coding region of the gene of interest This can be done on a plasmid but also within the genome The resulting construct is tested for functionality by complementing the corresponding deletion mutant GFP shines green in the fluorescence microscope and the subcellular localisation can be deduced using control staining of different compartments There are now many different versions of GFP with different detection threshold and different emission colours: CFP, RFP, YFP... This allows simultaneous observation of several proteins in the cell and even protein-protein interaction

12 yeastgfp.ucsf.edu YEAST GFP FUSION LOCALIZATION DATABASE >> Advanced Query < Go > >> Quick Search yal001c clr Welcome to yeastgfp.ucsf.edu The database of our global analysis of protein localization studies in the budding yeast, S. cerevisiae. > quick case-insensitive searches of the database may be performed on yeast orf names (yal001c) or gene names (TFC3) > separate multiple orfs/genes with a space (e.g. yal001c zwf1 bud2 etc.) > more advanced searching and downloading can be done in Advanced Query > GFP-tagged strains can be obtained from Invitrogen. > TAP-tagged strains can be obtained from Open Biosystems. > more details available in >> info >> faq >> help This web site supports Huh, et al., Nature 425, (2003). <pdf> The quantitation data presented here is published in Ghaemmaghami, et al., Nature 425, (2003). <pdf> Detailed collection construction methods can be found in Howson et al., Comp Funct Genom 6, 2-16 (2005). <pdf> This research is the work of the laboratories of Erin O'Shea and Jonathan Weissman at the University of California San Francisco. Please direct comments, concerns, and questions to <jan.ihmels@gmail.com> Copyright University of California Regents. All rights reserved.

13 Only a very small percentage of genes are essential (1000 of 6000) 50% of all genes have NO DETECTABLE PHENOTYPE when disrupted. Not all important pathways are essential. Redundant genes may exist. The robustness of yeast is only partly due to redundant genes. The problem in figuring out the function of a non-essential gene is determining what question to ask!

14 How to deal with essential genes? GAL-YFG1, Tet-YFG1 Ts alleles Degron DAmP (decreased expression of essential genes through mrna perturbation) DAmP + Degron

15 Integration of plasmids into the yeast genome Integration occurs by homologous recombination, this means that a plasmid like YIp5 will integrate into the URA3 locus Integration results in the duplication of the target sequence The duplicated DNA flanks the vector Integration can be targetted by linearisation within one of the sequences: cut DNA is highly recombinogenic Integrated plasmids are stably propagated but occasional pop-out by recombination between the duplicated sequences plasmid URA3 ura3 ura3 URA3 genome genome

16 Marker rescue By counterselection agains URA3 on 5-flouro-orotic acid, which is toxic to URA3 cells Or use of a Cre/loxP recombination system Very important for multiple deletions, i.e. 7-fold knockout strains URA3 plasmid YFG1 YFG1 genome URA3

17 The Cre/loxP system Omportant if one wants to re-use the marker in order to make many deletions in one and the same strain (there are strains with more than 20 deletions!) It is also important for industrial yeast strains; when one wants to engineer those at the end no foreign DNA should be left behind (but for hardliners on genetic engineering the intermediate presence of foreign DNA ina yeast is already dangerous ) All these methods use homologous recombination a second time, i.e. to pop-out the integrated DNA again An example for this are the loxp-kanr-loxp cassettes; recombination between the two loxp cassettes is stimulated by the Cre-recombinase (transformed on a separate plasmid); recombination just leaves behind a single loxp site Also used for conditional, time- or tissue specific knockouts in mice

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19 Cloning in yeast by gap repair Gene fragment YFG1 YFG1 gapped plasmid genomic copy is used to repair the gap; the template is duplicated. Allele rescue

20 Plasmid shuffling

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22 Cloning by complementation How do you clone the gene that you have mutated and is now present in your mutant? You clone it by complementing the mutant phenotype by transformation with a genomic library.

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24 Cloning by Complementation: Transform yeast mutant with library of expression plasmids and screen for return to wild type phenotype Recover plasmid, shuttle into E. coli, sequence the clone Celebrate?! Must prove is the gene and not a suppressor

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26 You ve cloned the gene. Now what? Define the pathway by defining interacting genes i.e., suppressors and enhancers of original mutation

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28 Types of suppressors: Allele specific --will only suppress specific point mutations may identify physical interactions Bypass-- will suppress loss of gene may identify downstream factors High copy-- overproduction of one gene product compensates for loss or defects in another often identifies upstream factors

29 Pbs2p and Hog1p are in the same pathway and Hog1p is activated by Pbs2p. Overexpressed Hog1p may confer sufficient activity to mediate the required function even in the absence of Pbs2p. common target Two parallel pathways share one or several common targets. Overexpression and hence higher activity of the parallel pathway may be sufficient to activate the target.

30 Getting further: synthetic lethality Synthetic lethality is a powerful method to identify genes whose products operate (in a pathway) parallel to the one that is affected by the primary mutation Typically, the primary mutant is transformed with a plasmid that carries the corresponding gene; the gene is either expressed through the GAL1 promoter (i.e. on on galactose and off on glucose) or is on a plasmid with URA3 as marker, which can be counter selected with 5- FOA Mutations are then screened that cause the yeast to grow only in the presence of the plasmid (i.e. not on 5-FOA) or only when the gene is expressed (i.e. not on glucose) The principle approach is so powerful that synthetic lethality screens are now done at a genome wide scale using the yeast deletion mutant collection: this means 4,200 x 4,200 crosses, sporulations and tetrad analyses done by robotics

31 Synthetic lethality common target common target The two pathways control some common targets; mutation of PBS2 alone causes only a moderate phenotype. The second mutation in the parallel pathway leads to lethality -> see SGA Analysis

32 Getting further: epistasis I Establishing the order of genes in a pathway Generate a double mutant; the phenotype of the double mutant may reveal if the two gene products work in the same or in parallel pathways and they may reveal the order within a pathway. common target mutation in all these four proteins cause similar phenotypes, such as moderate sensitivity to salt When we combine the hog1 and the pbs2 in a hog1 pbs2 double mutant then we would expect that the double mutant has the same level of sensitivity as each single mutant; we would conclude that they function in the same pathway When we combine the pbs2 and the cba1 mutation in a pbs2 cba1 double mutant we would expect a strongly enhanced sensitivity of the double mutant as compared to the single mutants; we would conclude that Hog1p and Cba1p work in different, though parallel pathways

33 Getting further: epistasis II Let us now assume that deletion of PBS2 (and of HOG1) causes sensitivity to high salt concentrations while deletion of SKO1 causes higher tolerance to salt in the medium Sko1 If those proteins act in the same pathway there are different possibilities for the phenotype of the pbs2 sko1 or hog1 sko1 double mutant If Sko1p were downstream of Pbs2p and Hog1p we would expect that the double mutant is tolerant, i.e. has the same phenotype as the sko1 single mutant: sko1 would be epistatic ( dominant over ) to pbs2 and hog1 (and this is really the case) If Sko1p were upstream of Hog1p and Pbs2p we would expect that the double mutant pbs2 sko1 and hog1 sko1 is sensitive to salt

34 Getting further: epistasis III Also multi copy suppression or activating mutations are useful tools in epistasis analysis Suppression by overexpression can only work for a gene/ protein functioning downstream of the primary lesion, as indicated here for Hog1p; overexpression of PBS2 would not suppress a hog1 mutation In a similar way, an activating mutation of HOG1 can suppress the salt sensitivity of a pbs2 mutant, but not vice versa, and this is indeed exactly how it works The epistasis concept has been used in very many examples to analyse the order of events in signalling pathways and other cellular systems: if the phenotype of the double mutant resembles that of one of the single mutants the latter gene product functions further downstream in the system, i.e. closer to the physiological effect

35 Getting further: suppressor mutations An extragenic suppressor mutation alters a different gene product such that the, or one of the, effects of a certain mutation are overcome Like with multi-copy suppression there are many ways in which this can happen and the outcome of such an approach is often quite surprising but very informative Typical suppressor mutations are those that activate a gene product downstream of the primary lesion in the same pathway; since such mutations cause a gain of function they are usually dominant Other typical suppressor mutations knock out a repressor downstream in the same or in a parallel pathway; since such mutations cause a loss of function they are recessive A suppressor mutation may also activate or inactivate pathways/ systems that affect in some way the same physiological system than the primary lesion If a given protein is part of a multimeric complex and the primary mutation is a point mutation, extragenic suppressor mutations might occur such that protein interactions are restored; hence this is a method to identify interacting proteins

36 Getting further: suppressor mutations Pbs2p and Hog1p are in the same pathway and Hog1p is activated by Pbs2p. A mutation that renders Hog1p active even without activation would suppress the pbs2 mutation and is probably dominant The pathway ultimately inactivates a negative regulator, e.g. the repressor Sko1p; knock out of the repressor could overcome inactivation of the pathway; the mutation is most likely recessive Sko1

37 Interplay between genetics and biochemistry/molecular biology Classical genetics Mutant -> gene -> molecular function -> new mutants in given pathway -> new gene etc. until pathway is resolved Reverse genetics: Gene, i.e., from genome sequencing project -> phenotype -> function (annotation)

38 Mutagenesis in vitro Structure <-> function study of a protein by site directed mutagenesis Generation of ts allele by error prone PCR Plasmid shuffling