Alvey (Model organisms) Flashcards
Why is Saccharomyces cerevisiae a good model organism?
- can exist stably as haploids or diploids
- single celled euks
- form compact colonies on plates
- grown in large quantities in liquid emdium
- rapid life cycle (90 mins)
- small compact genome (12Mb)
- efficient homologous recombination
What are the feature of the life cycle of yeast?
- sexual and asexual phases
- cell division by budding following mitosis
- 2 mating types, determined by MATa and MATα
- stable haploid and diploid phases
What happens when 2 haploid cells of opp mating types of yeast mate?
- can undergo mitosis and remain diploid or meiosis and return to haploid
Why is it useful to have stable haploid and diploid phases in yeast for mutational studies, ie. in what circumstances could each be used?
- haploid when performing mutant screen, so can see phenotype if get new mutation
- diploid if want to organise mutants into complementation groups, as if 2 haploids w/ mutation in same gene will result in WT
- diploid if wanted to propagate strain w/ lethal mutation, as can maintain as heterozygote
What was the aim of the Saccharomyces genome deletion project?
- to systematically KO every ORF in genome
How was the Saccharomyces genome deletion project carried out?
- PCR based deletion strategy
- primers designed to add 45bp complementary seqs and unique ‘bar code’ (TAGs) to kanamycin resistance cassette
- gen 4 diff mutant collections
- -> haploid, mating type a
- -> haploid, mating type α
- -> diploid, homozygous for KO alleles in non-essential genes
- -> diploid, heterozygous for KO alleles in essential and non-essential genes
- did transformation in Matα diploid, as can derive other types from this
Why were approx 5% of genes in Saccharomyces genome deletion project not knocked out?
- relies on ATG and TAA being unique, so didn’t try and do it where genes duplicated, as didn’t want more kan cassettes being inserted
What research is underway in beer labs?
- some flavour is from yeast so using breeding to adjust aroma levels
- epigenetics to decrease sluggish growth following transfer to new food source (glucose in starter culture –> maltose in fermenter)
- make new hybrid strains that can ferment at lower temps (larger yeasts), but have more interesting flavours
What areas of research has yeast made contributions to?
- cell cycle
- trafficking
- recombination
- gene interactions
- mito genetics
- genetics of mating types and switching
What are the aims of genetic screens?
- identify process of interest
- predict likely phenotype of mutant unable to carry out process
- devise method of identifying mutants w/ that phenotype
How does the life cycle of yeast lend it to genetic experiments?
- recovery of recessive mutations poss
- even lethal mutations can be maintained if WT copy also present
- in pCR deletion collection, deletion construct introd into diploid strains, in case gene essential
Why is secretion important?
- essential for growth –> delivery to cell surface, as need more growth at periphery to expand
- essential for secretion of substances/proteins/enzymes outside of cell
- essential for message delivery at nerve cells
- essential for delivery of membrane proteins to cell membrane
- structurally and functionally conserved across euks
How did Schekman use genetic screens to solve a physiological problem using yeast?
1st dev assay for secretion:
- picked 2 enzymes secreted by healthy yeast (acid phosphatase and invertase) and assayed outside cell to see if secreted
- add chromate (needs membrane bound sulphate permease to get into cell), lethal if uptaken, so only mutants survive
Obtained Ts mutant collection to screen for sec mutants:
- used Ts as easier to do experiments w/ than diploids
- correctly predicted that mutations in secretion would be lethal
How are recessive lethal mutations investigated?
- in diploids maintained in heterozygous state
- in haploids heat sensitive lethal alleles used
What are Ts alleles thought to be caused by?
- mutations that make protein prone to misfolding into inactive form at restrictive temp
How can an assay for secretion and cell surface growth be carried out?
- grow mutant in permissive temp
- shift to non-permissive temp
- assay growth medium for enzyme (acid phosphatase) activity at various time points
- select mutants that fail to secrete acid phosphatase at non-permissive temps
How were the 1st sec mutants isolated in yeast?
- grow yeast and assay media for acid phosphatase activity
- sec1 and sec2 cells stop secreting acid phosphatase when shifted to 37°
- sec1 and sec2 more dense than WT cells when grown at restrictive temps, as vesicles blocked at delivery to pm, so cells cannot expand as they grow
What did the second mutant screen involve, for isolating sec mutants in yeast?
- used density-grad separation to enrigh for sec mutants from pop of Ts mutants
- shift cells to restrictive temp
- allow to grow
- separate on density grad and collect dense ones
- screen dense ones for acid phosphatase secretion
- this way can screen much bigger pop
- identified further 23 sec mutants
- used complementation and phenotypic analysis to organise mutant collection into groups
What were the diff complementation groups that sec mutants were organised into in yeast?
- group 1: 10 genes that accum secretory vesicles when mutated
- group 2: 9 genes that accum ER-like structures when mutated
- group 3: 2 genes that form strange golgi-like structures when mutated
How do complementation tests work?
- inter-crossing 2 independant indivs homozygous for diff recessive mutations
- check whether F1 indivs have WT or mutant phenotype
- if F1 not WT then 2 mutations must be recessive alleles of same gene
- if WT, 2 mutants said to have complemented, and mutations must be in diff genes
How would you know if a Ts mutant had mutation in acid phosphatase gene unrelated to secretion in yeast?
- 2nd assay (eg. chromate uptake)
- characterise it phenotypically
- but unlikely as essential gene
Can you still do a complementation test if Ts mutation is dominant?
- usually recessive lethal, so unlikely
- but could prove by going to F2 gen
What role did Hartwell, Hunt & Nurse play in their Nobel Prize for “discoveries of key regulators of cell cycle”?
- Hartwell = discovered cdc28 and coined concept of checkpoints
- Hunt = discovered cyclins (A+B) but didn’t work in yeast
- Nurse = discovered cdc2 in S. pombe and CDK1/2 in humans, which are cdc28 homologues and characterised them as cyclin-dep kinases (did screen by complementation)
Why is the cell cycle so important?
- culminates in mitosis
- fundamentally same in all euks
- governed by genetically reg programme (presumably conserved throughout euks
- disruption of this underlies malignancy and cancer