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Lecture 9

Lecture 9. Assignment 3 Yeast complementation analysis Discussion of Article 6. The article for the final examination is posted. Remember I do not discuss this article with you. Final Examination Date and Time Wednesday Dec. 17, 2008 7:00 PM. Assignment 3.

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Lecture 9

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  1. Lecture 9 • Assignment 3 • Yeast complementation analysis • Discussion of Article 6

  2. The article for the final examination is posted. • Remember I do not discuss this article with you.

  3. Final Examination Date and Time Wednesday Dec. 17, 2008 7:00 PM

  4. Assignment 3 • Page 1 picture of your gel of total RNA with a figure legend. • Page 2 picture of your RT-PCR with a figure legend. • Page 3-4 see the assignment page for instructions.

  5. Complementation analysis • One of the pillars of genetic analysis. • Where have we talked about complementation before?

  6. Genetic screens • If I do a screen for proline auxotrophs and find 10 mutants, does this mean there are 10 genes required for proline biosynthesis? • If I do a screen for arginine auxotrophs and find 100 mutants, does this mean there 100 genes required for arginine biosynthesis?

  7. 4 Example of arginine biosynthesis. There are several enzymes involved in this process, and we want to generate mutants in all of them. So, we mutagenize yeast and look for auxotrophs of arginine. Generate several mutants and cross them all with each other

  8. 4 Can discover a series of complementation groups.    

  9. 4 Mutants 1 and 4 can complement each other. They are therefore in different genes. Mutants 1 and 2 do not complement each other. They are in the same gene and in the same complementation group.

  10. Mating and complementation analysis • Complement implies the number two. • To do complementation in yeast you need to mate two haploids to make a diploid. • Why do we need to do genetics in heterothallic yeast strains?

  11. Heterothallic life cycle • Stable haploid mating types a and alpha. • Haploids can mate but not sporulate. • Diploids can sporulate but not mate. • Sporulation is meiosis and the encapsulation of the 4 haploid products.

  12. Heterothallic Yeast Life Cycle

  13. A. Haploid yeast cells budding B. Haploid cells forming shmoos and zygotes C. Zygote budding off diploid D. Diploid budding E. Diploid forming asci (spore-containing sacs) & freed haploid spores

  14. Our screen • You have isolated haploid yeast that do not express the HO gene because of mutations in genes required for HO expression: the swi and she genes. • We want to do a complementation analysis. • What will we be measuring/assaying in a complementation analysis?

  15. What in HO biology makes it impossible to do regular complementation analysis? • HO is a haploid specific gene; it is not expressed in a/a diploids. • HO is expressed in mother cells and not daughter cells. • HO is expressed at G1 of the cell cycle.

  16. How were they able to do complementation analysis?

  17. Genotype of haploids • mat del::LEU2, leu2, trp1, ade2-1, his3-11, ura3::C2791::URA3, HO-ADE2, HO-CAN1. C2791=YIplac211+HO/GAL-lacZ plus a mutation in a gene required for HO regulation. • MATa, swi5::LEU2, leu2, trp1, ura3, his3, ade2-1, can1, pCEN TRP1.

  18. Mating type locus

  19. Mating type genetics • What happens when you delete MATa or MATa? • How do they mate as haploids, and what happens when they become dipoids. Can they mate; can they sporulate?

  20. Mating type locus

  21. Solution to the problem. • matdel mate as a cells because a-specific genes are expressed. Called a-like fakers. • When mated with a Mata strain, the diploid mates as an a cell and does not sporulate because there is no Mata1 protein in the cell to suppress expression of haploid specific genes. • Therefore, we can have expression of haploid specific genes in a diploid for complementation analysis

  22. Your complementation analysis 1 2 5 8 7 3 4 6 Parental strain 4570 swi5 tester strain Your mutants This week you will cross your mutants and the parental to the swi5 tester strain on a YEPD rich plate. Next week you will put the mated cells on diploid selection plates; score red/white a week later.

  23. How do we select diploids?

  24. Genotype of haploids • mat del::LEU2, leu2, trp1, his3-11, ura3::C2791::URA3, HO-ADE2, HO-CAN1. C2791=YIplac211+HO/GAL-lacZ plus a mutation in a gene required for HO regulation. • MATa, swi5::LEU2, leu2, trp1, ura3, his3, ade2-1, can1, pCEN TRP1.

  25. What do we need in the plates?

  26. Complementation analysis • What gene are we analyzing for complementation/ non complementation? • What do you expect to see for complementation/ non complementation?

  27. Genotype of haploids • mat del::LEU2, leu2, trp1, his3-11, ura3::C2791::URA3, HO-ADE2, HO-CAN1. C2791=YIplac211+HO/GAL-lacZ plus a mutation in a gene required for HO regulation. • MATa, swi5::LEU2, leu2, trp1, ura3, his3, ade2-1, can1, pCEN TRP1.

  28. What is happening in the Diploids. swi5::LEU2 SWI+/SHE+ HO-ADE2 WHITE SWI5+ swi/she ade2-1 swi5::LEU2 HO-ADE2 RED swi5 ade2-1

  29. So what does this tell you about where your mutant is?

  30. If you wanted to clone the gene identified by your mutant, what would you do?

  31. You have the following • mat del::LEU2, leu2, trp1, ade2-1, his3-11, ura3::C2791::URA3, HO-ADE2, HO-CAN1. C2791=YIplac211+HO/GAL-lacZ plus a mutation in a gene required for HO regulation. • A library of yeast wild-type DNA carried on a yeast replicating plasmid.

  32. Example of • Functional complementation • Reversing the positive negative screen

  33. Discussion of Article 6

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