Yeast Genetics Flashcards
What makes Yeast an ideal model organism?
- Strong genetic and coding gene homology to humans and eukaryotes
- Useful for studying cell cycle
- Unicellular, few cell types, cheap, smaller genome and fast to grow
- Haploid and diploid lifecycle allows cross over, recombination and haploid experimentation
What are the core steps in forward genetics?
- Recessive or dominant
- How many mutations per cell for phenotype
- How many unique genes linked to phenotype
- Metabolic/genetic heirarchy
What are the core steps in reverse genetics?
- Fluoresce protein
- Knock out or down
- Overexpress
- Chip-seq
- Protein-protein interactions
- Crossing mutants
How do you study essential genes?
- Conditional mutants: temperature sensitive
What are the two species of yeast used for?
- S. cerevisiae (budding): tetrad analysis, forward genetics,
- S. pombe(fission): similar to higher eukaryotes, RNAi, centromeric structure, mitosis regulation
What are complementation tests?
- Test whether two mutants are in the same gene
- cross two recessive mutants
- If cross still KOs same gene in all offspring then they are same gene
What does a cdc2 complementation screen show?
- Recessive mutants show cdc phenotype, failure to divide leads to elongation
- Dominant mutants show wee phenotype, divide at smaller size
- When inactivated cannot enter mitosis but dominant mutant disrupts regulation instead
What are the stages of S. c meiosis called?
- Haploid spores: Ascus
- Haploid cells: Tetrads
What is a tetrad dissection?
- As each division creates a spore they are distinguishable form other divisions
- Seperated by micromanipulator, needle and microscope, and digestive enzyme
- Moved to defined place to investigate phenotype ratios
What ratios will arise from tetrad analysis of unlinked genes?
- 1:1 parental ditype and non-parental ditype
- Each combination equally likely in random combination
What ratios arise from tetrad analysis of linked genes?
- Linked means they are on same chromosome
- If very tightly linked only PDs will be present
- If recombination is allowed the ratio will be 1:1:4 and the distance is derived from the equation
How can double mutants be utilized?
- Tell you how and in which order mutants interact with eachother
- Generated via tetrad analysis, take the NPDs
- Two genes in same pathway double mutant phenotype similar to upstream gene
How are nutrient markers used?
- Some mutants prevent yeast from producing necessary compounds, auxotrophic
- Can screen via selection(survive) and counterselection(cannot grow) media
How are genetic nutrient markers to used to isolate diploid cells?
- Complementation using recessive auxotrophic mutations which together grow in SD
- Using dominant markers to select ones that can grow in two drugs
How do yeast vectors work?
- Yeast plasmids can propagate and have markers in both yeast and E. coli
How many yeast plasmids are lost per divide?
1-10%
How are ts yeast vectors made for cloning?
- Create library with thousands of plasmids with different fragments of genomes and markers
- Plate ts mutant with library
- At low temperature they should all grow
- At high temperature only ones with WT gene will grow
How does targeted deletion of yeast genes work?
- Only works with dominant genes as wt copy remains
- Yeast recombination machinery makes use of flanking homology
- Use tetrad analysis to test success 2:2 ratio
How do epitope markers work?
- Specific antibodies hard to make
- So attach epitope with known antibody to gene end to mark protein
- GFP is a common example
What is a regulateable promoter(for experiments)?
- Normal promoter replaced by regulateable which can be regulated by addition of transcription factor
What is a heat inducible degron?
- Tagged with degron which targets protein for degredation
- Only acts as degron at high temperature
What are suppressors?
- A further change in genome (plasmid, mutantion) that counteracts phenotype of mutant
What are intragenic suppressors?
- Mutations in same gene that counteract mutant phenotype
- Means that supressor and mutation are highly linked
What are some types of information maps that can been collected in yeast functional genomics?
- genome wide seq such as ChIP, RNA-seq, ATAC
- Deletion mutants for phenotypes and genetics interactions
- Epitope tagging for protein-protein interactions
- GFP for protein localization
What can deletion libraries be used for?
- Either individual analysis or competitive growth
What are the possible differences in phenotype between single and double mutants?
- Positive interactions: double mutant weaker
>Mutation to both regulator and transcription factor stops toxin production - Negative interactions: phenotype gets worse
>Redundancy: single mutant no effect due to redundancy
>Hypomorphic mutation essential pathway: Single mutant weak phenotype, double essential - Null: no difference
What is a synthetic gene array?
- Make double mutant between gene of interest and all other mutants in library
- Slow and labour intensive
What are HIP, HOP and HIPHOP?
- HIP: Assumes single copy of gene makes hypersensitive to drug
- HOP: Assumes genes that regulate target will be sensitive to drug
- HIPHOP: Looks at drugs and finds both direct and indirect targets of drugs
How can yeast be used to study human mutants?
- In cases lacking homology you can just insert human diseased gene in yeast to see what happens
What is chronological life span (CLS) concept in yeast?
- How long cell survives in non replicative environment
- Assessed by colony formation capacity over time
Why are daughter cells younger than mother cells?
- Ageing factors segregate to mother
- ERCs: extra chromosomal rRNA circles
- Defective mitochondria
- Aggregated proteins
- Enlarged highly acidic vacuoles
What are some drugs produced synthetically in yeast?
- Artemisinin antimalaria drug has a complex pathway that had to be reproduced
- Morphine
What are some examples of long ncRNAs in yeast?
- Stable Unannortated Transcripts (SUTs): structurally similar to mRNAs
- Cryptic Unstable Transcripts: (CUTs): expressed at low levels, overlap with coding sequences
