Download 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
• 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.
Complementation analysis
• One of the pillars of genetic analysis.
• Where have we talked about
complementation before?
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?
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
4
Can discover a series of complementation groups.
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.
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?
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.
Heterothallic Yeast Life Cycle
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
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?
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.
How were they able to do complementation
analysis?
Genotype of haploids
• mat del::LEU2, leu2, trp1, ade2-1, his311, ura3::C2791::URA3, HO-ADE2, HOCAN1. C2791=YIplac211+HO/GALlacZ plus a mutation in a gene required
for HO regulation.
• MATa, swi5::LEU2, leu2, trp1, ura3,
his3, ade2-1, can1, pCEN TRP1.
Mating type locus
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?
Mating type locus
Solution to the problem.
• matdel mate as a cells because aspecific genes are expressed. Called alike 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
Your complementation analysis
1
2
5
4
6
3
8
7
Your mutants
This week you
will cross your
mutants and
the parental to
the swi5 tester strain on a
YEPD rich plate.
Parental strain
4570
swi5 tester
strain
Next week you will put the
mated cells on diploid selection
plates; score red/white a week later.
How do we select diploids?
Genotype of haploids
• mat del::LEU2, leu2, trp1, his3-11,
ura3::C2791::URA3, HO-ADE2, HOCAN1. C2791=YIplac211+HO/GALlacZ plus a mutation in a gene required
for HO regulation.
• MATa, swi5::LEU2, leu2, trp1, ura3,
his3, ade2-1, can1, pCEN TRP1.
What do we need in the
plates?
Complementation analysis
• What gene are we analyzing for
complementation/ non
complementation?
• What do you expect to see for
complementation/ non
complementation?
Genotype of haploids
• mat del::LEU2, leu2, trp1, his3-11,
ura3::C2791::URA3, HO-ADE2, HOCAN1. C2791=YIplac211+HO/GALlacZ plus a mutation in a gene required
for HO regulation.
• MATa, swi5::LEU2, leu2, trp1, ura3,
his3, ade2-1, can1, pCEN TRP1.
What is happening in the Diploids.
swi5::LEU2
SWI+/SHE+
HO-ADE2
WHITE
SWI5+
swi5::LEU2
swi/she
ade2-1
HO-ADE2
RED
swi5
ade2-1
So what does this tell you
about where your mutant is?
If you wanted to clone the
gene identified by your
mutant, what would you do?
You have the following
• mat del::LEU2, leu2, trp1, ade2-1, his311, ura3::C2791::URA3, HO-ADE2, HOCAN1. C2791=YIplac211+HO/GALlacZ plus a mutation in a gene required
for HO regulation.
• A library of yeast wild-type DNA carried
on a yeast replicating plasmid.
Example of
• Functional complementation
• Reversing the positive negative screen
Discussion of Article 6