Yeast as a Model System II

1. Yeast as a Model System II MBIOS 520/420 October 4, 2005

2. MBios 420/520 Course Announcements • Review Session Time? • Sample Exam • Study guide for exam will be provided next class • Other issues/questions?

3. Yeast Homologous Recombination • Homologous recombination (HR) is exchange or crossover • that occurs between two (nearly) identical DNA sequences • In mammalian cells, this process occurs very rarely • In yeast, HR occurs very frequently, due to small genome and • different recombination machinery • This can be used to target or “tag” a gene with a marker, to • knockout a gene or to replace a gene with a mutated version Marker or mutation Gene X Gene X Plasmid Homologous Recombination Chromosome Gene X Gene X

4. Homologous Recombination Sister chromatids (dsDNA) Enzyme nicks Strand Exchange Strands Join A Enzyme nicks on axis A B Enzyme nicks on axis B

5. Homologous Recombination – Double Crossover • If a double crossover event occurs, only DNA between the • two recombination sites is exchanged: • We can introduce a plasmid into yeast and exchange can • occur:

6. Gene Targeting • Homologous recombination can be used to “tag” a gene with • a marker in order to detect its inheritance • For example  Let’s say we want to be able to detect the • presence of a specific allele of the gene YFG, which we will • call YFG* • If YFG* has no easily measurable phenotype associated with • it, we can tag it with a marker that we can detect • In our example, we will tag YFG* with a URA3 and transform • it into a yeast strain that can’t produce uracil • If we linearize a plasmid that has URA3 and YFG*, the end • sequences will recombine with their identical counterpoint on • the yeast chromosome

7. Gene Targeting * * * * * *

8. Gene Targeting – A Practical Example • So we’ve tagged YFG* with a URA3 gene and inserted it into • one chromosomal copy in a URA3- mutant • As an example, let’s say we suspect that YFG* causes • resistance to hygromycin and that yeast with YFG only is • susceptible to hygromycin YFG*/ YFG* YFG* From the gene targeting on the previous slide we already have a yeast of the genotype YFG* / YFG. YFG* / YFG YFG YFG / YFG When this yeast reproduces, three genotypes will result. w/o uracil w/ hygroymicin 100% of hygromycin resistant yeast have URA3. YFG* must be a hygromycin resistance gene! Now let’s plate these with hygromycin. YFG* yeast also have URA3. Let’s replica plate on media w/o uracil.

9. Creating Gene Knockouts by Transplacement • We can use HR to create gene • knockouts by replacing a wild type • copy of a gene with a gene that has • an insertion • Insertion is a selectable marker • gene so we can identify knockouts • Problem: selectable markers are • dominant, so how do we get stable • knockouts that won’t segregate

10. Tetrad Analysis • Yeast cells can be either haploid or diploid; when in the • diploid state they don’t mate • Yeast can be induced to produce haploid spores under low nutrient conditions • By microdissection, we can separate the four haploid spores • (called a tetrad) and culture each one separately • This allows us to isolate mutants that are hemizygous for a • given knockout or mutation • If the knockout is lethal, half of the spores will not survive to • form colonies

11. Tetrad Analysis Here we continue the example of the YFG gene with the URA3 insert. Our yeast was heterozygous, but if we isolate spores we can get a hemizygous mutant. If the YFG gene is essential, all yeast with URA3 will not survive.

12. Studying Higher Eukaryotic Genes in Yeast • Commonly conserved genes, like cell cycle genes, are so • similar between yeast and mammals, that they can be • switched • This allows us to study the function of mammalian genes • without having to use a mammalian system • Ex: If we wanted to study the DNA binding domain of a human • transcription factor, we could mutant it, tag it with a marker • and replace a similar yeast gene with it • A technique called plasmid shuffle can be used to replace • essential genes in yeast with their mammalian counterparts

13. Plasmid Shuffle We’ve created a yeast knockout of TPK1 gene. Because TPK1 is essential, yeast needs a plasmid with TPK1 to stay alive. TPK homolog Transform with mouse TPK gene in another plasmid. Let these yeast cells lose one plasmid by removing selection criteria. Plate on media to show that one kinase gene is active. Replica plate w/o leu2 to show that mouse TPK gene is in yeast.