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
Why is cancer on the rise?
- life expectancy increasing and cancer risk increases w/ age
What governs progression through the cell cycle?
- cyclin-dep kinases and their cyclins
What is the role of CDK-cyclin complexes?
- each activates multiple cellular components
- diff CDKs and cyclins can bind to one another to form diff complexes
What does the thickness of bands in the cell cycle show, and what does the thickest represent?
- how active CDK is
- thickest = peak activity
What is the cyclin + CDK involved in the G1/S checkpoint, and what is being checked for?
- CDK4/6 + cyclin D
- cell size and DNA integrity
What is the cyclin + CDK involved in the G2/M checkpoint, and what is being checked for?
- CDK1 + cyclin B
- completion of DNA rep and DNA damage
What is the cyclin + CDK involved in the M checkpoint, and what is being checked for?
- APC (anaphase promoting complex)
- formation of spindle fibres and attachment of spindle fibres to kinetochores (centromeres)
What experiment did Hartwell carry out to discover checkpoints?
- gen Ts mutant pop (as correctly guessed null alleles in cdc genes would be lethal
- used screens to identify 146 inter-dep functioning genes asso w/ control of cdc in yeast
- defined cdc mutants
- described each stage of cdc like clock that had to be completed before moving onto next step
How can you look at cell cycle in S. cerevisiae and S. pombe?
- visible so can tell which point its at
What would cdc mutants look like in yeast?
- growth in yeast arrests at end of cell cycle in starvation conditions (low nutrient media)
- mutant carries on through cdc until checkpoint defective in, then arrests
- cdc mutants all arrest at same point in cdc = synchronised (IF have media w/ all nutrients in it)
Is cell division synchronised in WT cells?
- no
How were Ts experiments carried out to isolate cdc mutants in yeast?
- DIAG*
- starve cells to synchronise all cells at 25° so all behave like WT
- add nutrient medium and shift to restrictive temp (37°)
- replica plating –> stamp 2x and grow in 2 conditions and Ts mutants present at 25° but not 37°
- cdc mutants arrest at specific point in cdc
- eg. cdc28 fail to bud and cdc8 as 2 joined cells w/ elongated nucleus
What did screens carried out in yeast identify about checkpoints?
- identified many cdc mutants
- select mutants arrested at diff stages in cdc
- group similar mutants and assign complementation groups
- now 22 cyclins and 5CDKs identified in yeast genome
How would you test whether new cdc phenotype caused by mutation in single gene?
- cross mutant to WT haploid –> get heterozygous diploid
- induce meiosis and look for 2:2 of mutant:WT phenotype in progeny
How does speed of cell division affect S. pombe cells?
- if cells divide too quickly, get lots of short cells = wee cells
- if cells divide too slowly, get really long cells = cdc cells
How was screening by cross-species complementation carried out for cdc28?
- took S. cerevisiae cdc28 Ts mutant and transformed w/ S. pombe cDNA lib (each w/ diff gene from S. pombe)
- occasionally get complementation and colony WT so can grow at restrictive temp
- seq insert to find gene
- translate into protein (virtually)
- compare to known protein databases
- identified as kinase –> then tested for in vitro kinase activity by getting it to phosphorylate things
- found cdc28 complemented by cdc2 in S.pombe
How was human Cdk1 discovered?
- same way as cdc2 in S. pombe (cross-species complementation)
How was a model for CDKs dev?
- cloning of cdc25 showed it was a phosphatase
- wee1 is a kinase and overexpression makes big cells
- cdc25 overexpression makes cell small
- wee1 phosphorylates cdc2 at tyrosine 15 (ATP binding site)
What is the model for CDKs?
DIAG
cdc25
- cdc2-P (inactive) cdc2 (active)
wee1
What has comparative genomics shown about CDKs in humans?
- functionally conserved
- many of these genes defective in cancers
How are classical genetic screens carried out in yeast?
- identify process of interest
- predict likely phenotype of mutant unable to carry out process
- devise method of identifying such mutants
- haploid life cycle facilitates recovery of recessive mutations
- use conditional mutants to identify genes in essential processes
- identify and test homologues in other species
Why were complementation screens used in cdc experiments?
- cloning gene wasn’t as easy at time
- so focussed on characterising mutants, seeing how inherited and whether single gene etc.
What is autophagy?
- self degradative process
- balances energy sources at critical times of dev and nutrient stress
- housekeeping role in removing misfolded/aggregated proteins and damaged organelles
What was the mutant phenotype of yeast unable to carry out autophagy, and how were these mutants identified?
- don’t make autophagomsomes in starving conditions
- autophagosomes not visible under microscope in WT
- when autophagy active but degradation process blocked, accum in vacuole and become visible (not in mutants)
- DIAG*
How did Oshumi carry out experiments into autophagy in yeast?
- KO gene for vacuolar degradation enzymes
- cultured mutants whilst starving cells to stimulate autophagy
- used chem mutagen to introd random mutations in many genes, then induced autophagy
- identified 15 APG genes
- characterised proteins encoded –> suggested mechanism of cascade of proteins and protein complexes, each regulating distinct stage of autophagosome initiation and formation
Why did the Nobel judges think Oshumi’s work on autophagy was of ‘outstanding signif’
- important in health and disease
- no autophagy results in amyloid aggregation in Alzheimer’s
- also linked to Parkinson’s, type II diabetes and genetic diseases
- important for eliminating invading intracellular bacteria and viruses post einfection
- contributes to embryo dev and cell differnentiation
- allowed dev of drugs that can target autophagy in various diseases
What are 2 examples of yeast being used as a model for human health and disease?
- yeast as platform for human genetic variants (using complementation)
- yeast as model for ageing in cells (using library of single non-essential gene deletions
What is CIN (chromosome instability) and what is it a sign of?
- hallmark of cancer
- change in chromosome structure or no. leading to chromosome gain/loss
What causes CIN?
- failure in mitotic chromosome transmission or defects in mitotic spindle checkpoint
What kind of offspring does CIN result in, and why?
- aneuploidy
- chromosomes split up unevenly, usually due to translocation
- normal cells have spindle cell checkpoint to abort if aneuploidy occurs, but CIN don’t
Why does CIN increase cancer risk?
- exacerbates tumorigenesis
- as accumulative large scale genome rearranges increases likelihood of disrupting expression or function of oncogene or tumour supressor gene
What are variants of uncertain significance (VUS’s) caused by?
- problem assoc w/ big seq experiments
- more tumours seq = more variants identified
- cancer cells will have many abnormal cDNAs (normal in expression level and abnormal in seq)
Why are VUS’s a problem?
- identifying their functional consequence has become rate limiting
- need to work out which, if any, variant is driving tumour progression and which are passenger mutations
How can CIN models translate to humans?
- human orthologs of genes likely to be important in tumour progression
What is an orthologue?
- genes inherited from common ancestor w/ same role in diff organisms
What is a paralogue?
- genes resulted from gene duplication events w/in a genome
How were human orthologs of yeast CIN genes found?
- performed large scale cross-species complementation screens
How was a one-to-one complementation screen carried out for CIN genes using yeast?
- when already know pair and asking if human gene can do job of yeast gene
- 322 essential yeast genes conferring CIN phenotypes paired w/ their human ortholgs
- diploid yeast heterozygous null mutants for CIN mutations transformed w/ plasmids containing functional human orthologs
- sporulation
- 2:2 ratio of individual spores expected (if no complementation)
- if complementation then all survive –> this means human gene CAN do job of yeast gene
How was a pool-to-pool screen carried out for CIN genes using yeast?
- took pools of heterozygous, null mutants for 622 essential yeast genes
- transformed w/ mixture of plasmids expressing 1010 human cDNAs
- looked for viable spores
- unbiased strategy, identified unknown orthologs and complementation between non-orthologous genes
What were the key findings from the 2 complementation screens for CIN genes?
- identified 65 human cDNAs that complement 58 yeast null mutants
- 20 of yeast-human pairs novel
- looked for patterns in data set and concluded “mostly encode cyto proteins w/ few physical interacting partners and unlikely to have regulatory roles
After the complementation screens, what was the next stage of the experiment?
- chose 3 yeast genes w/ human orthologs to investigate further
- tested tumour-specific missense mutations by making haploid strains containing null mutant yeast gene and missense mutant human gene
- tested for viability –> 4/35 lethal
- tested for alt growth rates –> 18 grew slowly, 1 fast and 12 unchanged
- tested for fitness (= resistance to DNA damaging agents) –> most showed alt sensitivity
Why is age a global problem?
- people living longer everywhere
- age is greatest risk factor of most major causes of death in developed nations
How can you tell a yeast is old?
- chronological life span (CLS) –> amount of time can survive in stat phase
- replicative life span (RLS) –> no. daughter cells prod by 1 mother cell
How can chronological life span be calc in yeast?
- viability calc by fraction of culture able to re-enter cell cycle after extended state of quiescence
- at various time points extract bit of cell culture and see how many can re-enter life cycle, and at some point can’t re-enter anymore
How can replicative life span be calc in yeast?
- replicative viability calc as mean no. daughters prod from mothers of particular strain before senescence
How do we know our models for telling if yeast are old are reliable?
- growing evidence that ageing at least partly determined by ancestral evolutionary origin
- suggests genetic basis of ageing
What did the authors of the study looking at cell ageing using yeast models predict?
- if yeast good models, then find some genes in screen already know to influence life span in other species and hopefully some new ones
How were yeast mutants w/ increased longevity isolated?
- used small needle attached to microscope to tease daughter cell away from mother after every cell division
- counted how many times mother cell divides
What big screen was carried out to look at mutants w/ increased longevity in yeast?
- screened single gene deletion strain library (containing null mutations in non-essential genes)
- measured replicative age of each null mutant
- 238/4698 (≈5%) showed longer lifespan than WT
Why did some genes identified in yeast screen seem to act to shorten cells lifespan?
- don’t want to pass on age assoc problems to offspring
What were the 5 categories of activity which identified genes fell into, which were known already and which were new discoveries?
- translation (cytosolic and mitochondrial) –> known to be important from worm ageing studies
- TCA cycle (almost universal metabolic pathway –> known from worm ageing studies
- proteasomal activity –> novel ageing pathway
- mannosylation –> novel ageing pathway
- SAGA complex –> SAGA type HAT complex that recruits basal transcrip machinery, not in worms, STAGA in humans
What is the role of LOS1 (loss of suppression 1) in yeast?
- does not function in any of pathways identified
- but deletion has biggest effect on increasing yeast lifespan
- Los1p encodes nuclear pore protein, involved in nuclear export of pre-tRNA and re-export of mature tRNAs after retrograde import from cyto
- deletion mutation extends replicative lifespan, as does exclusion of Los1p from nucleus in response to caloric restriction
What are the characteristics of los1 mutants in yeast?
- accum tRNA in nucleus, as Los1p not there to export them
- increases replicative lifespan
- DIAG*
What is the effect of MTR10 overexpression in yeast?
- increases lifespan
- due to increased nuclear tRNAs
- as transports tRNA in other direction across nuclear membrane
- DIAG*
Why does increased nuclear tRNA levels extend lifespan in yeast?
- dietary restriction (DR) extends lifespan in all organisms tested (inc yeast)
- growing los1 deletion strains in DR conditions (low glucose) did not further extend lifespan
- -> does life extension by DR function entirely via nuclear tRNA levels?
- -> OR is lifespan of los1 deletion strain the max lifespan of any yeast cell?
- authors believe Los1 is conversion point for multiple age signalling pathways inc DR master switch (TOR) and DNA damage coordinator Gcn4
- but no one really knows…
Can yeast survive w/o mito, and why is this significant?
- yes
- so experiments can be done which are not poss in other organisms
What is Caenorhabditis elegans?
- small transparent nematode round worm
Why is C. elegans a good model species?
- transparent so can observe development
- 1mm long and 900 cells
- grows quickly (egg-to-egg in 3.5 days) and lives for 3 weeks
- genome 1000mb, 5 autosome pairs and sex chromosome
- can reproduce sexually (1000 progeny) or by selfing (300-350)
- efficient transgenics
- genomic resources
How does wild C. elegans differ to lab-grown?
- in wild lives in soil and eats bacteria
- can be lab-grown on plates or in liquid culture (and fed E. coli)
What are the roles of cells in C. elegans?
- 1/3 nerve cells
- 1/3 gametes
- 1/3 something else
What is the life cycle of C. elegans, and how long do these stages take?
- DIAG*
- egg to larvae 1 (L1) takes 8 hours from laying of eggs to L1
- whole embryogenesis from sperm entry to hatching takes approx 14 hours
- dev into Dauer stage if under starvation conditions –> reach L4 stage after placing in food
What are the 2 sexes of worms and how do they differ?
Males:
- sex chromosomes XO
- rare in wild but can be bred in lab
- prod only sperm
- mate w/ hermaphrodite to reproduce
Hermaphrodites:
- sex chromosomes XX
- mostly self fertilise
- prod eggs and sperm
- prod 99% XX and 1% XO
What has C. elegans been a model for?
- euk dev
- post genomic seq
- apoptosis (in context of dev)
- cell signalling (in context of dev)
- ageing
- RNAi
- post translational gene reg discovery
How is a classical genetic screen carried out in C. elegans?
- take hermaphrodites and expose to mutagen (eg. EMS) –> this is P0 and all +/+
- allow F1 gen to self fertilise –> all m/+
- look for mutagens in F2 gen –> 25% +/+, 50% m/+ and 25% m/m
- collect and maintain mutants as indep lines from F3 onwards
What does the classic genetic screen carried out in C. elegans rely on?
- careful dev of phenotypic analysis and interpretation in relevant biological context
How can transgenic worms be created?
- DNA can be directly injected w/ selectable marker into gonad
- DNA can be linear or circular
- can KO gene or add transgene (or use RNAi)
- in worms selectable marker is phenotypic (not antibiotic)
- 1 common marker is dominant collagen mutant rol-6
Why does marker in transgenic worm need to be dominant?
- 1st gen always heterozygotes so needs to be dominant in order to identify mutants
How was RNAi characterised in worms?
- injecting worms w/ dsRNA complementary to exon seqs results in specific silencing of gene
- silencing spreads through organism
- silencing inherited by progeny (for few cell divisions while still in mother
- silencing does not occur in animals defective for RNAi
What is RNAi and how can it be carried out in worms?
- dsRNA specifically silences expression of homologous genes through degradation of their cognate mRNA
- in worms gene can be selectively disabled and phenotype determined by feeding WT animals dsRNA
What can RNAi be used for in worms, and what method is used?
- gene knockdowns
- for specific gene knockdowns more efficient to use injection method
How does feeding of dsRNA result in silencing of gene in RNAi in worms?
- for large scale screens, long dsRNAs fed to worms in E. coli
- once eaten meets dicer
- chops it up into 21nts w/ 2 base overhang and loads it onto risc (RNAi silencing complex)
- then each risc hold ss bit of RNA, goes off to find mRNA exactly complementary and binds
- dicer chops target RNA into lots of little bits
- resulting in silencing of that gene
What were these RNAi screens used to characterise?
- cell-to-cell signalling during dev
How was a large amount of dsRNA able to be fed to worms in E. coli?
- plasmid w/ cDNA encoded into
- T7 promoter at each end, 1 going CW and other CCW
- so if clone cDNA get sense and antisense strand expressed
Are RNAi screens for worms genome wide, why?
- yes
- can purchase RNAi feeding libraries
- 16, 757 clones
- covers 94% C elegans genes
Once selected mutants from RNAi screens, how do you identify the gene?
- seq insert from plasmid
- know flanking seq and T7 promoter, so can start from here and doesn’t matter that don’t know unknown seq
What are the advantages of RNAi over classical genetic screens?
- gene seq known immediately, compared to laborious cloning stage
- can introd dsRNA at diff dev stages, bypassing earlier reqs, compared to maternal effect genes w/ zygotic req being hard to identify
multiple genes w/ shared seq can be knocked down to recover redundancy, compared to mutations usually only affecting single genes
What are the advantages of a classical genetic screen over RNAi?
- gain of function alleles can be isolated to uncover regulatory mechanisms, tissue specific alleles can be recovered and insights into structure-function relationships can be obtained from point mutations, compared to RNAi-mediated knockdown resulting in decreased level of WT product
- every gene should be mutateable, but not every gene susceptible to RNAi, some tissues resistant and genes encoding proteins w/ long half lives hard to knockdown effectively
- mutant alleles heritable so can maintain, but RNAi knockdown not usually heritable, except when silencing construct expressed as transgene(s)
How are worms formed?
- through highly deterministic dev programme
Do worms have simple construction?
- yes
- adult hermaphrodite has 959 somatic cells
- male has over 1000
How is dev described using cell lineage charts?
- dev stages and times along y-axis
- vertical lines represent cell division
- horizontal line links 2 cells formed from cell division
What is the vulva and where is it?
- egg laying organ
- approx half way along adult worm
What occurs during round 1 of vulval dev?
- vulva formed in stages during larval dev
- coord by several rounds of cell-cell interactions
- 2 developmentally equivalent cells communicate to decide which one becomes gonadal anchor cell (and goes on to make vulva) and other becomes precursor to uterus
How do we know lin12 gene involved?
- lin12/lin12 mutants both become anchor cells (genes in italics)
- lin12(d) both become uterine precursors (dominant mutation)
- lin12 encodes peptide receptor
How is cell-to-cell communication involved in anchor cell determination?
- both cells secrete a little Delta signal protein (binds to notch receptor) –> encoded by lag-2
- both cells express a little of receptor protein –> LIN-12
- by chance, then +ve feedback loops, 1 cell becomes signal expressing cell, other becomes receptor expressing cell
- ensures only 1 cell becomes anchor cell and 1 uterine precursor cell
What happens during further rounds of vulval dev?
- several more rounds of cell-to-cell communication, signalling and programmed cell death events that control it
What would the phenotype be if worm couldn’t express LIN-12 receptor protein?
- neither cell becomes uterine precursor
- both become anchor cells (expressing the signal)
- no vulva forms and get egg-laying defective mutants
What happens with maternal effect lethal mutants in worms?
- earliest divisions of embryo directed by RNAs and proteins that are deposited by mother (hermaphrodite)
- so homozygous embryo that lacks gene for these components can still dev normally, due to contribution form heterozygous mother
- but F3 die as lack of maternal contribution from F2 parent
What can mel screens be used to find?
- genes involved in early dev
What are the phenotypes of lin-2 and mel mutants?
- lin-2/lin-2 and +/+ = ‘bag of worms’, lin-2 homozygote consumed by her progeny as cuticle filled w/ L1 larvae
- lin-2/lin-2 and mel/mel = dead mother filled w/ dead eggs
If mutagenised lin-2/lin-2 mutants in which gen would you see first mel/mel mutant?
- would expect 1:3 ratio in rare plates in F2 gen to contain F2 mothers w/ dead F3 eggs filling cuticle
What did mel screens identify?
- 2 genes essential for early dev
- skn-1 (skinhead-1) –> specifies blastphere fate
- par (embryonic partitioning abnormal) –> req for embryonic asymmetry
How were maternal effect screens carried out in worms?
- expose lin2 homozygous hermaphrodite worms to mutagen (eg. EMS)
- allow P0 to lay eggs and separate on diff plates
- allow F1 to self fertilise –> all will explode as bag of worms and some will carry mel mutation
- most F2 are bag of worms but look for occasional no-bag mel homozygous recessive mutants
- collect no-bag siblings to maintain mel mutation as heterozygote
- F2
- -> 25% +/+ ; lin-2/lin-2
- -> 50% mel/+ ; lin-2/lin-2 (bred to maintain line)
- -> 25% mel/mel ; lin-2/lin-2
- but can’t tell +/+ and mel/+ apart, so take few onto new plates and wait till next gen (maintain in isolation)
What is arabidopsis?
- small weed in brassica family
What is the structure of the genome of arabidopsis, and its genes?
- genome 135Mb
- 5 chromosomes (no sex chromosomes)
- encodes about 27,000 genes
- each gene approx 2kb w/ 4 introns
How does arabidopsis genome density compare to humans, and why is this important?
- similar gene no. to humans, but 25x smaller
- compact genome for a plant (not compared to E. coli etc.)
- made it convenient for gen tools in post genomic era
How and why is the wheat genome extreme in size?
- v extensively bred
- hexaploid, so has 3 genomes (A, B and D)
What is arabidopsis a model for?
- evo and adaption
- pop genetics
- dev of more complex plants, eg. maize, wheat, rice
- env interactions
- plant genomes
- gene reg
Why is arabidopsis a good model?
- short life cycle (6 weeks seed to seed)
- large no.s progeny (see rare events)
- can be grown in restrictive conditions
- can be efficiently transformed using Agrobacterium tumefaciens
- genome seq available (since 2000)
- large collection of mutant stocks, natural ecotypes and genomic resources
What is the life cycle of arabidopsis?
- germination
- seeding establishment
- vegetative growth
- flowering
- senescence
How does life cycle of arabidopsis vary in diff envs?
- if somewhere cold, eg. Sweden, stays in vegetative state for long time
- if hot then flowers grow, Cape Verde
How does the genetics of plant life cycles differ from other organisms?
- no germline
- both diploid and haploid cells undergo mitosis
How does haploid phase (gametophyte) vary between higher and ancient plants?
- in higher (inc arabidopsis), reduced to just few divisions in ovule or pollen
- in ancient (eg. ferns/mosses), bigger and sometimes dominant
How does arabidopsis reproduce?
- mostly self fertilises, but amenable to crossing
How can arabidopsis be crossed in the lab?
- male is flower chosen to be pollen donor –> wide open flower
- female is flower chosen to be ovule donor –> younger flower
- demasculate female by removing anthers and marked w/ thread to distinguish
- then cross polinate
- growth of silique
- drying down and maturation of seed
Why is it useful for geneticists that arabidopsis can be crossed?
- homozygous lines can be stably maintained
- crosses can be performed when req (eg. mapping genes, backcrossing to parental lines, looking at interaction between genes)
What are strains from diff place generally, and what are they called?
- homozygous
- ecotypes or accessions
What was the aim of the 1001 genome project in arabidopsis?
- discover detailed whole genome seq variation in at least 1001 arabidopsis strains
- naturally inbred lines used, that are products of natural selection under diverse ecological conditions
How are arabidopsis used in evolutionary diversification and adaption studies?
- can study and quantify natural variation
- 1001 genomes project
What does Agrobacterium tumefaciens cause in the wild, and how was this adapted to use in arabidopsis?
- tumours
- removed virulence genes and kept ones for injection and integration into host genome
How is T-DNA inserted into plant genome by bacteria?
- bacteria grown to certain OD, then resuspended in sucrose
- dip flowers into this mixture
- can wrap plants for 12 hours
- stand them up and allow to grow as normal
- collect seeds
- have selectable marker in petri dish –? herbicide or kanomycin
- only transformants survive
How can a T-DNA insertion pop be gen?
- each seedling will have indep T-DNA insertion in diff location, as insertions random
- so some will be in genes
- likely to abolish gene function in gene inserted into (makes null mutant)
- can insert tagged genes, eg. GFP
What plants is arabidopsis commonly used as a model for?
- crop plants
Why is arabidopsis useful for looking at dev of multicellular organisms?
- cells never migrate or move, unlike animal cells
What arabidopsis resources are available?
- mutant collections
- stock centres
- genome info
- epigenome info
- expression profiling
What is vernalisation?
- flowering after prolonged cold (winter)
How have arabidopsis been used as an example of environmentally-mediated epigenetic silencing, and how can this be quantified?
- use progressive silencing of expression of floral repressor gene, FLC
- FLC on in autumn and stops flowering
- if winter long enough then stays off and flowers
- if not long enough then expression quickly increases again and no flowering
- in Arctic circle need lot more winter to flower than, eg. Edinburgh, and if from Cape Verde don’t need any
- can measure how silenced gene is by how many days takes to flower
Why can’t plants measure how long winter has been by measuring day length?
- Sep and March day length same
- so couldn’t distinguish between autumn and spring
What are small interfering (si)RNAs, what is their role and where are they prod?
- ds RNAs
- 20-25nts long
- 2 unpaired bases at 3’ ends of each strand
- an immune response
- mediators of seq specificity in RNA silencing
- prod by DICER protein complex
How have arabidopsis been used as an example of RNA silencing?
- when siRNAs bound to argonaute protein (a nuclease) they can degrade targeted mRNA
What are the general steps for functional analysis by gene discovery?
- amass mutants affecting biological property of interest
- cross mutants to WT to see if descendants show ratios of WT to mutant that are characteristic of single gene inheritance
- deduce functions of gene
- deduce how gene interacts w/ other genes to prod progeny in question
Why can many arabidopsis mutants be grown in a small space?
- long stems so easy to collect seeds, even though densely pop
How are forward genetic screens carried out in arabidopsis?
- introd random mutations by treating seeds w/ chem mutagen (eg. EMS, X-rays) or using random T-DNA insertions –> to cause mutations in embryos
- grow up chimeric M1 pops –> only get mutants in embryo if in 2 cells in shoot apical meristem as they go on to form the embryo
- screen for mutant phenotypes in M2 pop
- expect mutant phenotype from recessive gene in 1 in 8 M2 individuals (7:1) –> 4 WT form healthy cell and 3/4 WT from mutant cell
Once have mutant, by what method do you identify gene responsible for phenotype?
- mapping
What is the principle of mapping?
- the closer a marker is to a mutation responsible for a phenotype (the gene), the more likely that you observe parental seq at that loci
- due to recombination in both parents
How are mutant recessive genes in arabidopsis mapped?
- need to use diff ecotype (WT from diff place than one mutagenised to find mutant)
- will have small seq diffs (often SNPs) throughout genome (these diff already characterised)
- can use SNPs to tell which ecotype each portion of genome came from
What are 2 common strains used in arabidopsis mapping?
- Columbia
- Landsberg erecta
In mapping of arabidopsis how do you know which parent each seq came from?
- diagnostic PCR test for each marker
- cleaved amplified polymorphic sequence (CAPS)
- pick parts of genome w/ diff restriction sites
- 1st amplify region containing SNP by PCR –> design primers across region containing SNP
- then digest using restriction enzyme
- eg. if EcoRI only cuts Col, then 2 fragments on gel is Col, 1 fragment is L. er and all 3 bands shows a heterozygote
How are dCAPs markers found in arabidopsis mapping?
- online programme
- enter WT seq, mutant seq and how many mismatches in primer
What assumptions are made in the strategy for mapping in arabidopsis?
- parent must be (near) homozygous
- Col and L. er parents have polymorphic markers
- g = mutant gene, recessive phenotype
- G = WT copy of g
How is a mutation identified through mapping in arabidopsis?
- DIAG*
- heterozygote F2 ignored as looks like WT
- other 3 exhibit mutant phenotype, so can pool them, then test for which parental marker present at each loci (A-D)
- area around causative gene will always be homozygous for mutant parent
After mapping has been carried out in arabidopsis, how is the gene found?
- when have candidate area of genome, can go online to check for ‘candidate gene’ in that area
- Sanger seq can be used to identify exact mutation
Why might mapping fail in arabidopsis?
- if mutation in area of genome w/ v low recombination rates (as recomb rate not equal along genome, there are hotspots)
- if more than 1 mutation (in multiple genes at multiple loci) causing phenotype
In arabidopsis mapping, how could you test whether mutation is in single gene (assuming phenotype caused by homozygous recessive allele)?
- backcross mutant to parental line
- F1 should be heterozygous
- look for segregation patterns in F2 following selfing
Why don’t you just seq whole arabidopsis genome instead of carrying out mapping?
- can, but…
- mutagen will cause 100s of addition SNPs throughout genome
- need many gens of backcrossing to ‘clear up’ background and find right SNP (each bx takes at least 6 weeks)
- better bioinformatics tools have made this a better option, but still not quick or easy
After mapping in arabidopsis, how can you prove ‘new’ mutant is causing phenotype of interest?
- order T-DNA insertion line(s) from stock centre
- see if homozygous T-DNA null mutants has same phenotype as your mutant
What is Bio-Analytical Resource (BAR) used for?
- can learn lots about when and where gene of interest expressed, w/o doing experiments
- eg. is gene stress responsive
Where did all the expression date for arabidopsis come from?
- donated by community –> still donate any extra unneeded date, as may still be useful to a diff experiment
- most data from microarrays
- commonly used Arabidopsis Affymetrix microarray chip measures 28,000 genes w/ up to 26 probes per gene
What is the role of microarrays?
- measures relative expression of many genes at once
