Yeast as a Model Organism

Question 1

Forward genetics, S cerevisiae as a model and its life cycle

Answer 1

NB: Forward genetics= phenotype-> mutant-> gene (vs reverse)
S. cerevisiae model: unicellular euk, 16 chr, 6k genes. Mother cell-> bud-> daughter w/ genome copy.
Typical euk w/ all main cellular processes present, over ½ genes have human homologues (1281 involved in disease), many human genes can replace yeast counterparts. Unicellular, 3-4 cell types, compact genome (simple). Easy, cheap and quick to grow (divide <2hrs), versatile to media and temp, haploid or diploid, good for genetic screens, making double mutants, high transformation efficiency by homologous recombination. Certain env conditions, yeast diploid-> 4 meiotic products-> tetrad of spores in ascus.
Life cycle: mating types determined by MAT locus, a+ alpha conjugate-> diploid-> meiosis-> haploid spores-> germinate, grow on as haploids. Diploid more stable, so usually found as this.

Question 2

Efficient homologous recombination in S cerevisiae

Answer 2

Aside: Efficient Homologous recombination due to “mating type switching” in wt (in lab engineered so not)- mother switches types on budding so can mate directly w/ daughter, create diploids immediately. Mother exp “HO” endonuclease, cuts Mat locus, other Mat loci used to repair+ in this process switch types. Transforming w/ circular DNA causes recombination efficiency decrease (call can’t recognise break)

Question 3

S pombe as a model (+ lifecycle)

Answer 3

S pombe model: evolutionary distance to cerevisiae same as to human. Less common, shares experimental advantages w/ cerevisiae, resembles higher euk more in presence of RNAi pathway, centromere structure, regulation of entry into mitosis. Complementary, non-redundant model to cerevisiae. Cylindrical cells elongate to maximal size (~14 microns) before forming septum-> symmetric division. Exiting mitosis, can conjugate-> diploid-> meiosis and spore formation like cerevisiae.

Question 4

Naming conventions for genes and proteins

Answer 4

Naming conventions: Common gene names= three capitals+ number, sometimes related to function. Recessive mutants same as wt but lowercase w/ dash before #+ in italics. Proteins same as gene w/ lowercase p after. Common names often bad identifiers (different genes w/ same name, 2+ names for 1 gene, unstudied genes w/out common name). Systematic names essential for high throughput studies (e.g., YAL040C- yeast chromosome A (1st), left of centromere, position 40, Crick). Similar names of genes in different species doesn’t always ID homologues.

Question 5

Genetic screens

Answer 5

Genetic screens: start w/ phenotype likely related to process of interest, treat cells w/ mutagen, plate+ form colonies, examine phenotypes. Performed in haploids, so recessive mutations seen. If interested in essential gene, special strategy needed- isolate conditional mutations (e.g. ts mutations active @25, inactive @37). Done by plating @ permissive temp, replica plating @ non-permissive temp, examining Ts phenotypes. Characterisation may include: gene dominance, # of genes ID’d by mutations, if phenotypes cause by 1/ more mutations+ if mutations interact (double mutant analysis).

Question 6

Nurse et al’s pombe cdc screens

Answer 6

Nurse and s. pombe cdc screens: how isolation of cell division cycle mutants- used haploids to allow recessive mutation detection. Expect: genes for cycle ctrl essential, so ts mutants isolated; genes for cycle progression/ regulation prevent division but not affect growth (predicted phenotype elongated). Screen ID’d many cdc mutants showing cell elongation, no septa, defective in different cell cycle processes. Wee mutants divide @ smaller size, ID genes for mitosis entry timing.

Question 7

Scheckman’s cerevisiae sec screens

Answer 7

Schekman cerevisiae sec screens: secretory pathway. Yeast cells grow by increasing cell surface; require lipid, protein, carb synth+ vesicle delivery. Isolate sec mutants (haploid). Expect: genes for normal secretion essential (so use ts), mutations blocking secretion impede growth, cause accumulation of secreted proteins- acid phosphatase usually secreted, easily detected- used as marker. Sec mutants defective in several secretory steps- accumulation of vesicles, Golgi, ER.

Question 8

Testing for mutation dominance

Answer 8

Testing for mutation dominance (cerevisiae crosses)- wt and mutant m1 haploids crossed; heterozygous diploid phenotype shows if m1 dominant. Recessive mutations more common, usually LoF- many mutations tolerated if degenerate, or in insignificant part of protein. Dominant mutations rare, can arise bc: gene product not regulated, one copy of gene not sufficient for wt function (haploinsufficiency- useful in chemical genomics, e.g YFG1/yfg1 diploid), gene products form dimer (wt/mutant dimer not functional).

Question 9

Complementation tests

Answer 9

Complementation tests: isolate set of mutants in screen, find out how many genes defined by mutants by complementation. Cross m1 to m2- if mutations in different genes, wt phenotype (provided recessive mutations). To assign mutants to genes, do pair-wise analyses+ define complementation groups. Dominant mutations likely in cis elements, rarely in exons, characterised w/ different strategy.

Question 10

cdc2 mutant behaviours

Answer 10

Cdc2 mutants have different behaviours: recessive mutations-> elongated inviable cells (cdc phenotype), dominant mutations-> wee phenotype. Conclude: cdc2= major regulation of mitotic entry, if inactive, cell can’t enter mitosis, continue growing (elongated). Dominant mutations disrupt regulation-> premature mitosis.
NB: in meiosis, homologous chr segregated in 1st division, sister chromatids in 2nd.

Question 11

Tetrad analysis workflow and outcomes for unlinked genes

Answer 11

Dissection of ascus w/ enzyme to digest ascus wall+ micromanipulator (microscope+ fine needle). Spores picked w/ needle+ moved. Plate-> incubator-> colony from each spore. Phenotype investigated. Generate genetic maps, characterise mutations+ linkage, make double mutants.
2 unlinked genes a+b: assume no recombination between gene+ centromere. Cross haploid strains ab+AB-> diploid heterozygote. Segregation in meiosis I ab/AB-> parental ditype (PD) or aB/Ab-> non-parental ditype (NPD), then meiosis II. Segregation+ recombination in meiosis 2random, expect 1:1 PD:NPD

Question 12

Tetrad analysis for linked genes and calculating genetic distance; making double mutants and Scheckman sec mutants

Answer 12

2 linked genes a+b: unlikely to be separated by recombination. Cross ab+AB-> segregation in meiosis 1 only 1 way. Mostly PD (closer to 100% for closer linkage). If linked genes a bit further from each other, recombination possible in meiosis II. No crossover-> PD, single crossover-> tetratype (TT), 4-strand double crossover-> NPD. Genetic distance in centimorgans= 100(0.5TT+3NPD)/(TT+NPD+PD), valid up to ~35- after, probability of multiple crossovers increases. On average, 3kb=1cM- varies, frequency of recombination not constant across genome.
Making double mutants: show gene interactions, order of action in pathways. Cross 2 relevant mutants, select NPDs (double mutants), Compare to wt. Schekman sec mutants had 3 classes: I accumulate vesicles, II enlarged Golgi, III expanded ER. If 2 genes in same pathway, double mutant phenotype similar to inactivation of upstream gene. Cross results: I+ II= II phenotype. II+ III= III phenotype. II+ III= III phenotype. Therefore order of action III>II>I. Used to assign function to sec genes.

Question 13

Genetic markers and marker selection

Answer 13

Genetic markers: mutations used to follow plasmid/gene/chromosome, e.g., auxotrophic mutations. Yeast grown on defined/minimal medium where all component known, e.g., SD medium has Carbon, nitrogen, mineral and vitamin source. Wt cells prototrophic, i.e., synth most required compounds (except some vitamins). Auxotrophic mutants can’t do this. E.g., TRP2 mutants (recessive) can’t make Tryptophan, can’t grow on SD unless supplied w/ tryptophan.
Marker selection: some can be both selected (helpful for diploid selection)/counter-selected. Ura3 can’t make uracil, while wt URA3 sensitive to 5-FOA (Ura3p converts 5-FOA-> 5-fluoruacil (toxic). URA3 can’t grow on SD+uracil+5-FOA, ura3 can.

Question 14

Selecting yeast diploids, transformation and transformant selection

Answer 14

Selecting diploids: a and alpha mating not always successful. Can select diploids w/ auxotrophic mutation (e.g., ura3/LEU2 x URA3/leu2, diploid grows w/out uracil or leucine) or dominant marker (KanR x HygR diploids can grow on both hygromycin and kanamycin.
Transformation: directed modification of genome, most cells not transformed. Plasmid vectors most common. In yeast, shuttle plasmids (can be propagated and selected in both yeast+ E coli, purified, later transformed into yeast). Contain origin of replication, marker for bacteria+ marker for yeast (e.g., AmpR+URA3). Plasmid loss rate 1-10%, cells kept under selection to retain it. Loss can be useful.
Transformant selection: transform plasmid w/wt gene-> strain w/ recessive auxotrophic gene+ plate on SD. Or, transform wt strain w/ plasmid w/drug-resistance, plate on SD w/ drug.

Question 15

Cloning by complementation

Answer 15

Cloning by complementation: clone seqs ID’d by classical genetics. Make a library (collection of plasmids w/ genome fragments and selectable marker); transform into ts mutant, plate under permissive conditions+ select for plasmid; replica plate in restrictive conditions- cells w/ wt copy will grow; rescuing plasmid isolated, seq. e.g., Nurse&co isolated cdc2 gene. Similarly ID potential human homologue: human gene library-> human clones complements cdc2 mutation-> seq showed that human and S pombe cDNAs encoded related proteins (1st evidence of cell cycle ctrl machinery conservation yeast-> human)

Question 16

Targeted gene deletion

Answer 16

Targeted gene deletion: performed in diploids, need efficient homologous recombination machinery. Deletion construct: selectable marker (dom or rec) surrounded by regions homologous to those flanking target gene, usually made in vitro (PCR)+ transformed into yeast. 30-40bp homology sufficient for accurate+ efficient targeting. Flanking seq.s added to primers, PCR w/ plasmid containing marker as template. Get diploid w/ 1 wt+ 1 mutated copy, tetrad analysis to study phenotype (marker segregates 2:2). Essential genes ID’d by lack of spores w/ resistance gene (2-spore ascus), can’t be studied :(

Question 17

Epitope and fluorescent tagging

Answer 17

Epitope/ fluorescent tagging: to raise antibody, express+ purify protein, inject into host- costly, time-consuming, uses animals, success not guaranteed. Instead, add small seq (epitope/ fluorescent protein) to end of protein recognised by commercial antibody, introduced by homologous recombination, tagging construct made by PCR. GFP can be followed in vivo-> dynamic studies.

Question 18

Methods to study essential genes (3)

Answer 18

Studying essential genes: develop conditional mutants, targeted manner.
* Ts alleles- fast action but hard to construct. Plasmid shuffling= replacing wt w/ mutated form. Make random mutant alleles yfg1 w/ error-prone PCR, clone into plasmid w/ marker (say TRP1); make plasmid w/ wt YFG1 w/ other marker (URA3); transform URA3 plasmid into trp1/trp1 ura3/ura3 diploid heterpo for marked deletion of yfg1; sporulate, select haploids w/ deletion and YFG1 plasmid (kept alive by plasmid and wt gene); transform w/ TRP1 plasmid w/ yfg1 mutant library; grow on uracil+ 5-FOA @ permissive temp, select those that lost URA3 plasmid- will have deletion of chromosomal yfg1+ plasmid mutant yfg1- can now test for ts phenotype.
* Regulatable promoters: endogenous promoter replaced w/ regulatable one, e.g., TET switched off by doxycycline, by homologous recombination. Slow, as pre-existing mRNA+ protein needs degrading before see phenotype.
* Heat-inducible degrons: protein tagged w/ degron seqe (targts for degradation/ UQ pathway) by homologous recombination, inactive @ low temp-> temp-specific exp of tagged protein. Inactivation usually fast.

Question 19

Suppressor analysis:

Answer 19

Suppressor= change in genome (secondary mutation or plasmid) that cancels mutation-> revert to wt phenotype. ID interacting gene products, reveal common pathways. E.g.:
Interaction suppression: e.g., mechanism: original mutation disrupts interaction w/ protein A. Mutation in protein A gene allows interaction. Usually gene and allele-specific. Mutagenize ts mutant, plate at permissive temp; replica plate at restrictive temp, select survivors (i.e., suppressors), clone suppressor. Cloning can be tricky/ time-consuming as can’t complement to wt library, but often suppressors that show independent phenotypes (e.g., cold sensitivity) are selected, allowing cloning by complementation of 2nd phenotype. Alternatively, seq whole mutant genome, compare to parent strain- valid for all mutants, but usually strains have >1 mutation, need to ID right one (often requiring classical genetics).
Intragenic supressors: possible that new mutation in original gene causes suppression. Can check by back-crossing isolated suppressor to wt- if intragenic, all progeny wt; if mutation on 2nd gene, mutations won’t always co-segregate, some progeny show mutant phenotype.

Question 20

Functional genomics overview in yeast

Answer 20

Cerevisiae: protein genes+ classical noncoding RNAs; compact, few introns, small intergenic regions.
Pombe: ~5000 protein-coding genes, more introns than cerevisiae (43% coding genes), but also compact.
Genome seqs allowed “omic” approaches- proteomics/mass spec, DNA microarrays/ genomics, systematic genetic analyses.
Functional genomics aims to build a comprehensive map of the cell. Many maps already drawn in yeast through systematic collections of deletion mutants (phenotypes, genetic interactions), epitope and GFP tagged proteins (interactions, localisation, protein level determination), genome-wide exp analysis.

Question 21

Yeast deletion project and systematic analyses using deletion collections

Answer 21

Yeast deletion project: collection of strains carrying deletions in every gene, made by homologous recombination, deletions marked by dominant drug resistance gene. 16 labs (1 per chr), 3 collections: haploid (~5000 genes), homo diploid (~5000), hetero (~6000, including essential genes). Molecular barcodes ID each strain- each construct has 2 unique seqs (UP/DOWN), barcodes surrounded by seqs common to all strains, common seqs allow PCR amp/barcode, ID’d by seq/ using DNA microarrays- make parallel studies possible.
Systematic analyses using deletion collections- truly systematic (over forward genetics, all genes queried), complete null mutations (complete LoF by ORF deletion), but only non-essential genes. First set of deletions in diploids-> tetrad analysis- only 19%/genes essential under lab conditions (rich glucose medium), likely due to redundancy (minor), many genes required only in specific conditions/ non-essential processes, many cause subtle phenotypes. Collection used to see responses to stress, different media, drugs, cell shape/size, mating, sporulation, membrane trafficking, etc. Original papers cited >2500 times.

Question 22

Using deletion collections: individual strains and competitive growth in batch

Answer 22

Using deletion collections: individually or by batch analysis+ competitive growth.
* Individual strains: plated under condition of interest. Often automated. Phosphate depletion causes exp phosphatases (Pho1- increased activity-> dark red)- ID by colorimetric assay. Screen found known (PHO1) and new (PHO7- TF) genes in response- 13 plates enough to cover genome.
* Competitive growth in batch- uses barcodes. All deletion strains pooled in equal amounts, grown under competitive conditions, time course performed, DNA extracted @ different times, PCR and quantify barcodes, on microarray/ Bar-seq, relative amount reflects abundance+ fitness of strain. Allows phenotyping 1000s strains in 1 experiment.

Question 23

Interaction networks, genetic interaction and its causes

Answer 23

Interaction networks: Mendelian monogenetic mutations-> minority of human disease. Most conditions= complex inheritance. Studying mutation interactions difficult in complex genome. Yeast= simpler model. Genetic interaction= unexpected phenotype resulting from 2 mutations combining, e.g., no phenotype individually but synthetic sickness/lethality combined. Quantified by difference between expected and observed double mutant phenotype, measured in fitness (equivalent to growth rate) where 1=wt growth and 0=none. +ve interactions- double mutant phenotype weaker than expected, -ve= phenotype aggravated.
Causes:
* Redundancy- 2 genes, 1 function. Only if both mutated see phenotype
* Hypomorphic (weak) mutations in essential pathways- mutations each partially inactivate pathway, fully inactivate it together.
* Suppression: -ve regulation of signalling molecule inactivated by mutation, target deregulated-> lethality. Simultaneous mutation in regulator+ signalling molecule reverts this.

Question 24

Synthetic gene arrays- overview, procedure and analysis

Answer 24

Synthetic gene array (SGA)- systematic mapping of given gene’s interactions. Double mutant made w/ gene of interest x library. Standard methods (cross+ NPD selection) inadequate for high throughput studies, too slow, laborious. Can optimise, automate. Setup: Need a s query strain w/ deletion marked by antibiotic resistance x alpha w/ deletion marked by 2nd resistance. Obtain haploid double mutant pop of a type, eliminate diploids, haploid w/ 0/1 mutations, alphas. Target collection deletions tagged w/ different dominant markers, all his3 mutants. A train can HIS3 under MFA1 promoter ctrl, active in a but not alphas or diploids.
Procedure: haploids crossed, diploids selected on both drugs; sporulate; plate on SD without histidine; select double mutants by replica plating on both drugs. Easily automated.
Analysis: interactions visualised as computationally assembled network, genes w/ similar interaction profiles together- genes in same/related pathways cluster, function can be predicted from position.

Question 25

Chemical genomics, HIP and HOP and drug e.g.s (2)

Answer 25

Aims to ID functional relationships of specific genes+ chemical compounds. Many potential drugs/ targets poorly characterised bc: target unknown, a=unclear if additional targets exist, no drugs directed against known target. Deletion collections can be used to ID targets, action mechs of drugs. * HIP (Haploinsufficiency profiling)- use hetero collection, assume diploid has only 1 copy of gene encoding drug target->will be hypersensitive to drug (no compensation from other copy). Screen in presence of relevant drug, barcodes= resistant strains. Usually most sensitive mutant-> gene encoding direct drug target. Complements+ always done w/HOP: * HOP (homozygous profiling)-more so for finding essential gene targets- homo library, assume genes w/ functions close to target sensitive to drug. Screen w/ drug, barcode to ID resistant strains. * E.g., tunicamycin- inhibits protein glycosylation by targeting ALG7p-> unglycosylated proteins not properly folded, accumulate in ER-> stress response (UPR) mediated by protein kinase IRE1p+ TF HAC1p. HIP ID’d direct target ALG7, HOP ID’d genes that buffer drug effect (IRE1, HAC7) * Molsidomine- treats angina, metabolised-> NO-> vasodilatory. HIP ID’d ERG7 as most affected mutant- encodes lanosterol synthase (cholesterol biosynthesis). Molsidomine inhibits lanosterol synthase in vitro- hence HIPHOP IDs new targets/pathways affected by drugs. * Pros and cons: good for new drug target discovery, ID direct targets+ related activities, no a priori knowledge needed but target must be conserved in yeast for study to be useful.

Question 26

Yeast and human disease: Mitochondrial disease, humanised yeast, Huntington's and Alzheimers

Answer 26

Model for mitochondrial disease: can grow w/ or w/out mtDNA, but still need mt- not needed where they can ferment (glucose) but needed in media where need to respire (glycerol). Allows isolation+ study of mt LoF mutants. Mt encoded genes highly conserved yeast to human. E.g., Neuropathy Ataxia and Retinitis Pigmentosa (NARP)- maternally transmitted disease, nervous system, due to mutation in mt gene MT-ATP6. Yeast strains w/ mutations in ATP6 gene-> poor growth in glycerol, low ATP synth. Model used to ID candidate drugs to alleviate NARP (suppressors of ATP6 mutations), e.g., chlorhexidine (CH)- allele-specific suppression. Humanised Yeast: some conditions w/ no yeast gene homologue. Model by exp relevant disease gene in yeast. If yeast phenotype related to original condition obtained, other approaches applied. E.g.: * Huntington’s: dominant autosomal. Triplet expansion (CAG) in exon 1/ huntingtin (HTT) gene-> poly(Q) stretch- mutated protein toxic, aggregates. Expanded protein Htt103Q exp in yeast under regulatable promoter- exp toxic, slow growth. Exp in every mutant of haploid deletion collection-> mutants w/ improved growth, incl BNA4 gene deletion (encodes KTO, part of tryptophan degradation pathway, suppression of BNA4 pathway-> therapeutic target. KTO inhibitors relieve symptoms in mouse model. * Alzheimer’s: amyloid beta from cleavage of transmembrane precursor (APP), released into secretory pathway-> insoluble plaques. A beta1-42 fused to signal peptide to direct to secretory pathway in yeast, under regulatable promoter. Exp-> toxicity. Assay small molecules (140K)- clioquinol family (CQ) isolated- known drug candidate from mouse studies, indices Abeta degradation. Other candidates also found, not yet characterised.

Question 27

Yeast replicative ageing

Answer 27

Any age-specific decline in variable associated with fitness, esp mortality, reproduction, physiological performance. Yeast exhibits replicative ageing: newborn cell isolated, divides repeatedly, progeny removed, after 20-25 cycles, stops dividing+ dies (can’t be applied to large # cells, laborious). Budding leaves chitin “bud/birth scars” which can be visualised w/ some compounds. Proposed model for stem cell aging. # of times cell divides= replicative lifespan (RLS). Modern alternative to measure RLS: trap mother in microfluidics device (capture mother, fresh medium flows, wash daughters away, image @ regularly- automated, analyse 100s of cells) RLS modulated genetically by: * Sirtuins: flies, worms, mammals. Deacetylase activity. In yeast, SIR2 regulates RLS dose-dependently- overexp extends RLS + vice versa. * TOR (target of rapamycin): ser/thr kinase. Inactivation extends RLS. (SCH9= TOR downstream effector)

Question 28

Yeast Chronological ageing, ageing factors and ERCs

Answer 28

Chronological lifespan (CLS): cells kept under non-dividing conditions, viability measured regularly by assessing colony formation. Proposed model for non-dividing cells. Modulated genetically by TOR, but not SIR. Wt cells treated w/ rapamycin have extended CLS. Ageing pathways conserved: TOR inhibition and SIRT6 overexpression extends mouse lifespan. Calorie restriction in mice extends CLS, mimicked in yeast by low glucose conc- RLS extended Ageing factors: Daughter cells have full RLS (though rather old mothers have “older” daughters- some ageing factors slip through). Mothers accumulate aging factors that would damage daughter. Ageing factors: accumulate w/ age, segregate to mother, reducing it increases lifespan+ vice versa. E.g., ERCs (extrachromosomal rDNA circles), defective mt, aggregated proteins, enlarged highly acidic vacuoles. ERCs: circular DNA made by recombination between rDNA repeats (accumulate in SIR2 mutants!). ade2 pink mutants (accumulate intermediate of adenine biosynth pathway) transformed w/ ADE2 plasmid are white; if plasmid w/ ADE2 marker used, plasmid loss followed by colour. ERC labelled w/ ADE2 transformed into ade2. Single daughter isolated, divides. Mothers keep the ERC, except when quite old. Screen for potential aging factors: any molecule partitioned to mother= candidate. Screen library of GFP-tagged strains. 74 mother-enriched; deletion of 1/3 of them increased lifespan-> aging factors. Some proteins daughter-enriched- protective factors? Not yet analysed.

Question 29

Synthetic bio and production of artemisin

Answer 29

Design and construction of novel artificial bio pathways, organisms, devices, or redesign of existing natural bio systems. Yeast engineering: transplant and integrate pathways w. endogenous metabolism to increase yields. Production of artemisinin: anti-malarial from sweet wormwood. Long generation time-> price and supply unstable. Biosynth: acetyl co-A-> 5 enzymatic steps. Pathway transferred to cerevisiae-> produce artemisinic acid. Chemically convert to artemisinin- semisynthetic (precursor by biosynth isolated+ modified). Pathway transplant: overexp 9 cerevisiae genes (endogenous for artemisinic acid synth), deletion of 1, exp of 6 wormwood genes to optimise yield, reduce toxicity to yeast of by-products. Production in plants-14-18 months, yeast in weeks.

Question 30

Morphine production

Answer 30

Morphine: glucose+15-18 enzymatic steps (too complex for chemical synth). Poppy genome seq not available then, some endogenous enzymes in pathway still unidentified. Implement L-tyrosine to intermediate of S-reticuline pathway in yeast. R-reticuline to morphine in separate strain. ID enzyme for reticuline isomerisation in poppy-> whole synth from glucose in yeast. Exp 21 heterologous enzymes from planta, mammals, bacteria, yeast. Overexp 2 native enzymes, delete 1 native enzyme. Current yield <1ug/litre. Poppy-> morphine+ codeine farmed under licence- tech developments-> faster, more reliable production, higher product quality (purer)-> allow development of improved opiates (ethical/social/legal issues attached)

Question 31

Functional conservation yeast to humans, genome-wide studies and gene ontology

Answer 31

Functional conservation yeast to human-Similar results to Nurse cdc2 complementation obtained, then systematic study for S cerevisiae essential gene human homologues (functionally replacable)-> 43% essential genes could be replaced. Correlation between seq similarity and interchangeability weak. Best predictor= if protein in a complex interchangeable, whole complex usually is- suggests complementation of certain genes may require whole complex humanisation. Genome-wide studies-Computer-based automated approaches. Ideally, use all info possible on each gene for analysis. “Free text” info from scientific reports rewritten to controlled vocabularies of pre-defined terms for computer accessibility. E.g., to investigate if group of genes have common properties (e.g., pathway involvement, drug sensitivity, etc) Gene ontology (GO) most used- terms in 3 categories: cellular component, molecular function, biological process. Each term has unique ID. Info collected by curators-> databases like Pombase+ SGD (for cerevisiae)

Question 32

Non-coding genome: conventional, small and non-canonical RNAs (and their degradation pathways)

Answer 32

Non-coding genome Budding+ fission yeast have non-coding (nc)RNAs. RNA degradation by ribonucleases- Exo 5’-3’ (XRN1p in cytoplasm) and 3’-5’ (large multicopy complex= exosome). Classified non-coding when lacks ORF>100nt. Conventional- highly conserved, easy to ID. F(x) in translation and RNA processing, produced by different Pols, some present in multiple copies in yeast genome (140 rRNAs, 275 tRNAs)- needed in large amounts small (only pombe- contains conserved RNAi pathway including Dicer+ Ago (absent in cerevisiae, but present in related budding yeasts suggesting loss in cerevisiae), regulating formation of heterochromatin) non-canonical (aka long)- poorly conserved, can’t be predicted by seq only. ID experimentally w/ microarray/ high throughput seq. RNA Pol II. >200nt. Classified by pathway for degradation: * Stable unannotated transcripts (SUTs) detected in wt cells, like mRNA but don’t encode proteins, exp can be condition dependent. * Cryptic unstable transcripts (CUTs) exp at low levels, often overlap coding seqs, only detected when stabilised in exosome mutants * XRN1p-deoendent Unstable transcripts (XUTs)- low level exp, only detectable in xrn1 mutants. Functions of long ncRNAs mostly unaddressed, unclear if have function. Some ncRNAs may represent “pervasive” transcription (noise), w/ no function+ product down-regulation by rapid degradation. Some have well-defined roles.

Question 33

ncRNA and meiosis regulation: 2 examples

Answer 33

ncRNA meiosis regulation e.g. 1: IME1p TF= key factor in meiosis entry. Exp repressed indirectly by RME1p TF. Rme1 induces SUT IRT1 transcription, which induces formation of repressive chromatin, blocking positive activator binding (mediated by HDAC+ methylase recruitment) Haploid a cells produce a1 TF, alphas exp alpha2- diploids exp both, both inactive as monomers, form active heterodimer. A1/alpha2 represses RME1, IME1 exp (also requires +ve env signals), meiosis entered. ncRNA meiosis regulation e.g. 2: IME4p= 2nd activator of meiosis entry. Exp regulated by cis-acting ncRNA IME4-Antisense or RME2, transcribed from antisense IME4 in a or alpha haploids. In diploid, a1/alpha2 repressor complex binds RME2 promoter downstream of IME4 ORF, inhibits RME2 transcription.