1. Yeast as a Model System MBIOS 520/420 September 29, 2005

2. Advantages of Yeast as a Model Geneticists are always interested in humans, but it is impractical (and unethical) to do human studies Even mouse models & cell cultures can only take us so far: YeastMammalian Cell Culture 2 hour generation time Can be maintained as either haploid or diploid Small genome 24 hour generation time (after 4 week culturing) Always diploid Very large genomes Despite the differences, yeast cells are organized in a very similar manner to higher eukaryotes (organelles, chromosomes & proteins are similar)

3. The Yeast Genome THE MATH YeastHuman 14,000,000 bp 933 clones (1X λ library) 5600 clones (6X λ library) 70 clones (1X BAC library) 933 clones (6X BAC library) 3,500,000,000 bp 233,333 clones (1X λ library) 1,400,000 clones (6X λ library) 17,500 clones (1X BAC library) 105,000 clones (6X BAC library)

4. Identification of Yeast Biosynthetic Genes Cloning of biosynthetic genes aided both the study of cellular metabolism, and provided phenotypic genetic markers Biosynthetic genes = those involved in production of cellular metabolites like amino acids Because these are basic needs of all cells, the yeast genes are similar enough to the E. coli biosynthesis genes Researchers introduce random pieces of genomic DNA into E. coli mutants, until function is restored This is termed genetic complementation

5. Genetic Complementation – Cloning the LEU2 Gene Isolate yeast genomic DNA. Cut up the genome and ligate strands into a plasmid. Transform large numbers of E. coli with the entire library. Grow the cells on media lacking leucine. Only colonies with functional leucine-producing yeast genes will grow.

6. Identification of Yeast Biosynthetic Genes Once identified, a biosynthetic gene like LEU2 can be used to complement yeast cells that are leu - as well as E. coli A plasmid carrying LEU2 could then be used with leu - strains in the same way that ampicillin resistance is used as a selectable marker for E. coli But how do we identify biosynthetic mutants in the first place? Can we use selective media? You can’t isolate a colony that doesn’t grow. The answer  replica plating

7. Replica Plating Plate colonies on normal media with leucine. Place a piece of velvet (or silk) over the plate & the colonies will transfer. Press this “carbon copy” onto a new plate w/o leucine. See which colony doesn’t grow & pick it off the old plate. With Leuw/o Leu Leu - mutant replica

8. Yeast Vectors Three major types (each useful for a different application) Simplest LEU2 or similar marker Has bacterial ori & amp r (derived from pUC vector) Cannot replicate in yeast Limited to single copy (sometimes useful) Gene must integrate into yeast genome to be stably expressed Integrating Plasmids

9. Yeast Vectors Shuttle vectors (replicate in yeast & E. coli, has amp r & LEU2) One of two yeast ori sequences can be used 2μ = yeast plasmid ARS = autonomously replicating sequence (yeast chromosomal DNA) (less stable) CEN = centromere DNA, makes the yeast cell treat the plasmid like a chromosome at mitosis (1 copy only) Replicating Plasmids

10. Yeast Vectors Linear vector Has yeast telomere sequences at each end Replicates and is inherited like a chromosome Capable of large inserts (300 kb) Similar to BAC (just a very large plasmid) Yeast Artificial Chromosomes

11. Cloning Genes by Genetic Complementation Useful for identifying conditional lethal mutations Biosynthesis or temperature sensitive mutations for ex. After isolating mutants, transform them with the entire genomic library. Plate them w/o leucine to detect transformants. Replica plate them and grow under conditions to ID mutant (ex: higher temperature). Isolate & sequence the colonies that survive.

12. Sub-Cloning When we isolate a BAC or plasmid with a gene of interest, we must do sequencing to find the gene within the vector Modern sequencing only allows us to sequence 0.5 kb at a time (sequencing is like PCR, proceeds from a known primer) We need to sub-clone large inserts like BAC/YAC (~200 kb) or a lambda insert (~15 kb) by transferring them to plasmids The only other way would be to primer walk for our sequencing (very time consuming) We can either sequence all these, or do another genetic complementation on the sub-cloned sequences

13. Sub-Cloning To Find A Gene We’ve found a YAC with Gene X from our genetic complementation screen. Cut the YAC insert with restriction enyzmes. Put these smaller fragments into plasmids (< 4 kb) We would want to cut a 200 kb YAC fragment into 50 pieces to sequence get 4 kb inserts. Choose an appropriate enzyme. Introduce back into yeast & repeat complementation test. Sequence the survivor. Potential Problem? Restriction Digest Ligate into plasmids Transform Grow on leu-, high temp Sequence this one! Gene X

14. Yeast-Two-Hybrid Assay - Basis Used to detect genes that encode proteins that interact with each other (one is “bait” the other is “target”) Ex: Let’s say we’ve cloned Gene X for the first time. We know it binds to DNA, and we suspect it binds to another protein, but which one? We are using a transcription factor as an example, but this assay will work with any proteins that bind to each other The basis of this technique is the two domains of transcription factors  the DNA binding domain and the activation domain In domain swapping experiments, we saw that we could fuse the DNA binding domain of one TF with the activation domain of another

15. Yeast-Two Hybrid Assay - Background GAL4 promoter is fused with lacZ reporter gene. The binding domain (BD) & activation domain (AD) act together to start transcription. We can place this in a vector and create blue colonies when X-Gal is present. GAL4 DNA Binding Domain GAL4 Activation Domain RNA Polymerase LacZ GenePromoter MCS of plasmid vector GAL4 BDGAL4 AD

16. Yeast-Two Hybrid Assay - Procedure When we transform yeast cells with this, RNA polymerase is not activated. Protein X can’t bind to RNA polymerase. Transcription not activated. GAL4 DNA Binding Domain Protein X LacZ GenePromoter GAL4 BDGene X First we replace the GAL4 AD with Gene X. Colonies are white.

17. Yeast-Two Hybrid Assay - Procedure Next we fuse Gene Y to GAL4 AD. Then we transform yeast cells with two vectors: 1)Carrying GAL4BD-GeneX fusion 2)Carrying GeneY-GAL4AD fusion Let’s say Gene Y binds to Gene X. Protein X & Y bind to each other, bringing GAL4 AD close to RNA Polymerase. PromoterGAL4 BDGene X Vector 1 PromoterGene YGAL4 AD Vector 2 GAL4 BD Protein X LacZ GenePromoter Protein Y GAL4 AD Transcription occurs. Colonies turn blue.

18. Yeast-Two Hybrid Assay Summary Gene X and Gene Y bind to each other. Fuse Gene X to GAL4 BD & Gene Y to GAL4 AD. Transform yeast with two vectors, each carrying one of these fusion proteins. The GAL4 BD binds to DNA, along with the fused Protein X. Since Protein Y binds well to Protein X, it will bind near the promoter as well. Since GAL4 AD is fused with protein Y, it is brought close enough to the promoter to activate RNA polymerase & transcribe lacZ. This proves that Proteins X & Y bind together.

19. Yeast-Two Hybrid Assay Yeast-two hybrid is effective at demonstrating that two proteins interact, but what if we have no idea what protein Gene X binds to? How do we find that gene? STEP1. Make a cDNA library of your organism. STEP2. Fuse every cDNA in your library with the GAL4 AD. Isolate mRNA Reverse transcribe Put in vectors (thousands of cDNAs) NOTE: All vectors should have LEU2 selectable marker.

20. Yeast-Two Hybrid Assay STEP3. Transform yeast cells with the GAL4BD- GeneX fusion gene. NOTE: This vector should have a selectable marker different from LEU2! w/o tryptophan Transform TRP1 STEP4. Transform yeast cells again, this time with the GAL4AD-cDNA fusion genes. STEP5. One of the cDNAs fused with GAL4 AD will bind with Gene X. This one will turn activate transcription & create a blue colony. w/o trp or leu Isolate this colony. Sequence cDNA. (thousands of cDNA-GAL4 combinations) Transform LEU2