Yeast as Model Organism Flashcards
Why does having a diploid and haploid stage make yeast a good model organism?
Haploid - can identify a mutant phenotype immediately
Diploid - can complement with WT and maintain lines of lethal mutants as heterozygotes
When will a yeast cell undergo meiosis?
When starved
In nutrient rich conditions, is a yeast cell diploid or haploid?
Diploid
What is contained in the PCR product which is transformed into yeast cells in the Yeast Knockout Collection?
- 45bp complementary sequence to yeast genome
- Unique tags (UPTG/DNTG)
- Kanomycin resistance cassette
How was the yeast knockout collection created?
Transform PCR product into yeast cells using heat shock. Recombine with yeast genome via homologous recombination with host machinery
How can you select for a successfully transformed knockout cell?
Kanomycin resistance acts as selectable marker. Without transformation, cells will not be viable on kanamycin containing plate
What are the two mating types of s.cerevisae?
MATa and MATalpha
What is an essential factor to think about when designing a classical screen for yeast? (how will you know you can read the results)
You must imagine what phenotype the hypothetical null mutant of your gene of choice would look like. Is it assayable?
What is the function of sulfate permease in yeast cells?
Transported in vesicles to surface of cell. Allow uptake of chromate which is lethal to the cell.
What are two assayable compounds which should be secreted from yeast in wild type conditions?
invertase and acid phosphatase
What type of conditional mutant is used in the classic SEC mutant screen?
Temperature sensitive mutants
Why were conditional mutants used for SEC screens in yeast
Because it was predicted that a SEC mutant would be lethal.
What is thought to be the cause of heat sensitive temperature sensitive mutant phenotypes?
It is thought that at a restrictive temperature, the proteins expressed from TS alleles misfold and become inactive
What was the second technique used to screen for SEC mutants after it was confirmed that this genetic process existed?
A density gradient was used to enrich for sec mutant cells.
Mutant cells will have a much denser phenotype (full of small vesicles)
Why can yeast be used as model organisms for secretion?
Membrane trafficking is a structurally and functionally conserved process across all eukaryotes
