11. Yeast functional genomics Flashcards by Barbora Buc

What are the main yeast species used in genetic research?

Saccharomyces cerevisiae (budding)
Saccharomyces pombe (fission)

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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

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What are the main differences between S. cerevisiae and S. pombe?

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What are the uses of yeast in culture?

For bread, beer, wine + also important for model for basic biological sciences

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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

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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

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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

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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

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Explain temperature sensitive gene disruption

Temperature sensitive (ts)

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Explain temperature-inducible degron in gene disruption

Degron - unstable in certain conditions

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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

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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

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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

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How can the function of whole genome be investigated in a functional genomics approach?

Steps for whole genome functional analysis:

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Differentiate between forward and reverse genetics

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What are the advantages and disadvantages of using random mutagenesis in forward genetics in functional genomics screen?

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What are the key requirements for a systematic functional genetic screen?

accurate and complete mutant libraries
convenient and sensitive phenotypic assay

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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

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What proportion of yeast genome is essential genes?

20%

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How is phenotype of interest screen after mutation?

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What are the aprpoaches for yeats screening for a function?

reporter gene
fitness profiling
genetic interactions

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

What are the techniques used for RNA expression prifiling?

- microarrays - RNA-seq

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

What are the approaches for performing protein expression analysis?

- epitope-tagging - mass spectrometry

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

What are the methods used for studying protein interactions?

Protein interactions studied using: - yeast 2 hybrid assay (Y2H) - affinity purification and mass spectrometry (AP-MS)

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 do protein interaction tsudies help to identify in genomics?

Protein interactions allow to figure out protein functional networks more easily

What are the two complementary approaches to studying protein inetractions?

Y2H + AP-MS

What do protein inetraction studies help to identify in genomics?

Protein interactions allow to figure out protein functional networks more easily

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