11. Yeast functional genomics Flashcards
What are the main yeast species used in genetic research?
Saccharomyces cerevisiae (budding)
Saccharomyces pombe (fission)
What are the advantages of yeats as model organisms for genetic studies?
Adv:
- quick, easy, cheap to grow and maintain
- easy to genetically manipulate
- compact genome ~12MB
- haploid and diploid states
- unicellular eukaryote but essential cellular functions conserved in evolution
What are the main differences between S. cerevisiae and S. pombe?
What are the uses of yeast in culture?
For bread, beer, wine + also important for model for basic biological sciences
What are the approches for studying yeast gene functions?
Reverse geentics - mutate specific gene -> discover function - approaches:
- gene deletion
- gene disruption
- over-expression
- protein-tagging
By using genetic yeast transformation (plasmid / linear DNA) + high freq HR
Explain how gene deletion might be used to study gene function
Design a linear insert with homologous regions for HR - replace the target gene -> observe phenotype
Explain how protein-tagging might be used to study gene function
Tag attaches to an engineered linear DNA insert - HR to fuse gene of interest with - gene mRNA translation with the tag - visualisation of tagged protein expression
?? how is this functional analysis
Explain how gene disruption might be used to study gene function
If gene essential - KO non-effective to study function - won’t survive -> conditional alleles with the gene:
- genome-wide mutagenesis
- gene-specific random mutagenesis
-> screen for transformed colonies
Explain temperature sensitive gene disruption
Temperature sensitive (ts)
Explain temperature-inducible degron in gene disruption
Degron - unstable in certain conditions
Explain auxin-inducible degron in gene disruption
Auxin induces degradation of target proteins via ubiquitination if auxin-inducible tag (AID) added to target protein of target gene
Explain how gene over-expressoin might be used to study gene function
Gene function might not be evident from deletion/mutagenesis - to uncover use over-expression - add more copies of the gene transcripts by strong promoter / high gene copy plasmid -> overexpression can help study genetic interactors - ex: if over-expression compensates for mutation
Explain what is a high cipy supressor screen
High copy supressor screen - high gene copy plasmid inserted with engineered selectable marker - colonies plated - look for retained mutants which can grow at restricted conditions - high-copy gene might be compensating
How can the function of whole genome be investigated in a functional genomics approach?
Steps for whole genome functional analysis:
Differentiate between forward and reverse genetics
What are the advantages and disadvantages of using random mutagenesis in forward genetics in functional genomics screen?
What are the key requirements for a systematic functional genetic screen?
- accurate and complete mutant libraries
- convenient and sensitive phenotypic assay
How are deletion libraries constructed?
HR used to insert an engineered insert instead of a gene in yeast genome -> gene deletion - Bar codes on both ends for identification
What proportion of yeast genome is essential genes?
20%
How is phenotype of interest screen after mutation?
Explain how a reporter gene might be used to study gene function?
Crossing deletion library yeast with reported inserted yeast - HR -> product fused - ex: ade6+: if gene in heterochromatin - red colony / if white colony - gene required for heterochromatin
Explain how fitness profiling screening might be used to study gene function?
Different gene deletion strains with Bar codes up + down mixed - grown in chosen conditions - more fit grow faster - purify genomic DNA - PCR - tag quantities analsyed => screen for fitness of specific mutation
Explain how genetic interactions might be used to study gene function?
Genetic interactions between genes influence thei functionality - dependent on each other - ex: if one mutated other could take over - but if both mutated - functional disruption => pairing different mutations helps to figure out genetic interactions
Negative genetic interaction - phenotype is affected
What is fitness profiling good at identifying>
Gene srelated to survival - ex DNA damage response - can use UV as growth condition of choice - see which mutant colonies survive worst - mutations affect DNA damage response
Explain what are synthetic genetic arrays
Synthetic genetic arrays (SGA) - different mutants crossed - array of double mutants - colony size compared -> determined which affect growth
Explain quantitative SGA
Considers the degree of genetic inetraction of double mutants - negative / neutral / positive compared to expected effect
What genetic interaction helps to investigate?
Genetically interacting genes are oftne functionally similar - functional insights + identification of new genes
Explain microarray method in RNA profiling
ORFs amplified by PCR -> spotted on glass slide in single gene spots - RNA hybridised -> into cDNA by reverese transcriptase + dye (usually diff dyes for diff origin samples - ex: mutated / WT) - scanning to see dye mixture colour => determien relative RNA quantities
Explain RNA-seq method in RNA profiling
Extract RNA -> convert to cDNA in reverse transcription -> amplification and adaptor ligation -> create sequencing library -> sequencing -> genome assembly from reference genome => expression profile + expression levels
Analyse microarray result
What are the advantages of RNA-seq?
How can transcirptional analysis be mapped?
Explain how gene co-expression analysis is performed
What is ribosome profiling used for?
Gene transcription level NOT same as its translation into protein -> ribosome profiling helps to assess translation efficiency - identifies the # fo ribosomes on a transcript -> indicates rate of translation
Explain the methodology of ribosome profiling
Does translation efficiency equal transcription efficiency?
No - translation lower efficiency - less translated than transcribed
Explain epitope-tagging as a protein expression analysis approach
Protein tagged with visual tag - expression can be localised in the cell
Explain mass spectrometry as a protein expression analysis approach
Extract cell’s protein mixture - digest into peptides -> liquid chromatography to separate by size - no tag needed + no change in protein nature due to tagging
Explain Y2H method
Yeast two-hybrid (Y2H) - allows to test an interaction between 2 target proteins:
Gal4 natural yeast TF used - 2 domains: DBD + AD - when come together gene expressed - 2 target proteins fused with DBD / AD:
1) if the target proteins inetract - reproter gene expressed because DBD and AD end up on the gene
2) if teh target proteins don’t inetract - don’t bind - DBD and AD don’t come to the gene - no reporter gene expression
Explain AP-MS method
Affinity purification - mass spectrometry (AP-MS) allows to study protein inetraction in cells:
- epitope tagged with bait protein - binds / doesn’t bind other proteins - the AB pooled down - target protein + other which interact pooled together - mass spectrometry allows to identify the proteins
What are the two complementary approaches to studying protein inetractions?
Y2H + AP-MS
Which yeast species is better for studying epigentics and why?
S. pombe - has more conserved epigenetic mechanisms comapred to humans
What experiment helps identify genes important for heterochromatin in S. pombe?
What directs chromaitn assembly in S. pombe?
RNAi
Lecture summary
