Assignment 3

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Lecture 9

• Assignment 3
• Yeast complementation analysis
• Discussion of Article 6

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The article for the final examination is posted.

• Remember I do not discuss this article with you.

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Final Examination Date and Time Wednesday Dec.
17, 2008 700 PM
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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.

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Complementation analysis

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

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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?

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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
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Can discover a series of complementation groups.
                            
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                         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.
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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?

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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.

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Heterothallic Yeast Life Cycle
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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
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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?

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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.

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How were they able to do complementation analysis?
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Genotype of haploids

• mat delLEU2, leu2, trp1, ade2-1, his3-11,
  ura3C2791URA3, HO-ADE2, HO-CAN1.
  C2791YIplac211HO/GAL-lacZ plus a mutation in a
  gene required for HO regulation.
• MATa, swi5LEU2, leu2, trp1, ura3, his3, ade2-1,
  can1, pCEN TRP1.

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Mating type locus
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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?

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Mating type locus
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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

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Your complementation analysis
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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.
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How do we select diploids?
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Genotype of haploids

• mat delLEU2, leu2, trp1, his3-11,
  ura3C2791URA3, HO-ADE2, HO-CAN1.
  C2791YIplac211HO/GAL-lacZ plus a mutation in a
  gene required for HO regulation.
• MATa, swi5LEU2, leu2, trp1, ura3, his3, ade2-1,
  can1, pCEN TRP1.

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What do we need in the plates?
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Complementation analysis

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

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Genotype of haploids

• mat delLEU2, leu2, trp1, his3-11,
  ura3C2791URA3, HO-ADE2, HO-CAN1.
  C2791YIplac211HO/GAL-lacZ plus a mutation in a
  gene required for HO regulation.
• MATa, swi5LEU2, leu2, trp1, ura3, his3, ade2-1,
  can1, pCEN TRP1.

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What is happening in the Diploids.
swi5LEU2
SWI/SHE
HO-ADE2
WHITE
swi/she
SWI5
ade2-1
swi5LEU2
HO-ADE2
RED
swi5
ade2-1
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So what does this tell you about where your
mutant is?
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If you wanted to clone the gene identified by
your mutant, what would you do?
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You have the following

• mat delLEU2, leu2, trp1, ade2-1, his3-11,
  ura3C2791URA3, HO-ADE2, HO-CAN1.
  C2791YIplac211HO/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.

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Example of

• Functional complementation
• Reversing the positive negative screen

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Discussion of Article 6