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MBB262 > Alvey (Model organisms) > Flashcards

Alvey (Model organisms) Flashcards

1
Q

Why is Saccharomyces cerevisiae a good model organism?

A
  • 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
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2
Q

What are the feature of the life cycle of yeast?

A
  • sexual and asexual phases
  • cell division by budding following mitosis
  • 2 mating types, determined by MATa and MATα
  • stable haploid and diploid phases
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3
Q

What happens when 2 haploid cells of opp mating types of yeast mate?

A
  • can undergo mitosis and remain diploid or meiosis and return to haploid
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4
Q

Why is it useful to have stable haploid and diploid phases in yeast for mutational studies, ie. in what circumstances could each be used?

A
  • 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
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5
Q

What was the aim of the Saccharomyces genome deletion project?

A
  • to systematically KO every ORF in genome
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6
Q

How was the Saccharomyces genome deletion project carried out?

A
  • 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
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7
Q

Why were approx 5% of genes in Saccharomyces genome deletion project not knocked out?

A
  • 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
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8
Q

What research is underway in beer labs?

A
  • 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
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9
Q

What areas of research has yeast made contributions to?

A
  • cell cycle
  • trafficking
  • recombination
  • gene interactions
  • mito genetics
  • genetics of mating types and switching
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10
Q

What are the aims of genetic screens?

A
  • identify process of interest
  • predict likely phenotype of mutant unable to carry out process
  • devise method of identifying mutants w/ that phenotype
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11
Q

How does the life cycle of yeast lend it to genetic experiments?

A
  • 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
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12
Q

Why is secretion important?

A
  • 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
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13
Q

How did Schekman use genetic screens to solve a physiological problem using yeast?

A

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
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14
Q

How are recessive lethal mutations investigated?

A
  • in diploids maintained in heterozygous state

- in haploids heat sensitive lethal alleles used

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15
Q

What are Ts alleles thought to be caused by?

A
  • mutations that make protein prone to misfolding into inactive form at restrictive temp
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16
Q

How can an assay for secretion and cell surface growth be carried out?

A
  • 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
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17
Q

How were the 1st sec mutants isolated in yeast?

A
  • 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
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18
Q

What did the second mutant screen involve, for isolating sec mutants in yeast?

A
  • 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
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19
Q

What were the diff complementation groups that sec mutants were organised into in yeast?

A
  • 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
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20
Q

How do complementation tests work?

A
  • 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
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21
Q

How would you know if a Ts mutant had mutation in acid phosphatase gene unrelated to secretion in yeast?

A
  • 2nd assay (eg. chromate uptake)
  • characterise it phenotypically
  • but unlikely as essential gene
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22
Q

Can you still do a complementation test if Ts mutation is dominant?

A
  • usually recessive lethal, so unlikely

- but could prove by going to F2 gen

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23
Q

What role did Hartwell, Hunt & Nurse play in their Nobel Prize for “discoveries of key regulators of cell cycle”?

A
  • 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)
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24
Q

Why is the cell cycle so important?

A
  • culminates in mitosis
  • fundamentally same in all euks
  • governed by genetically reg programme (presumably conserved throughout euks
  • disruption of this underlies malignancy and cancer
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25
Q

Why is cancer on the rise?

A
  • life expectancy increasing and cancer risk increases w/ age
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26
Q

What governs progression through the cell cycle?

A
  • cyclin-dep kinases and their cyclins
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27
Q

What is the role of CDK-cyclin complexes?

A
  • each activates multiple cellular components

- diff CDKs and cyclins can bind to one another to form diff complexes

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28
Q

What does the thickness of bands in the cell cycle show, and what does the thickest represent?

A
  • how active CDK is

- thickest = peak activity

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29
Q

What is the cyclin + CDK involved in the G1/S checkpoint, and what is being checked for?

A
  • CDK4/6 + cyclin D

- cell size and DNA integrity

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30
Q

What is the cyclin + CDK involved in the G2/M checkpoint, and what is being checked for?

A
  • CDK1 + cyclin B

- completion of DNA rep and DNA damage

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31
Q

What is the cyclin + CDK involved in the M checkpoint, and what is being checked for?

A
  • APC (anaphase promoting complex)

- formation of spindle fibres and attachment of spindle fibres to kinetochores (centromeres)

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32
Q

What experiment did Hartwell carry out to discover checkpoints?

A
  • 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
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33
Q

How can you look at cell cycle in S. cerevisiae and S. pombe?

A
  • visible so can tell which point its at
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34
Q

What would cdc mutants look like in yeast?

A
  • 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)
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35
Q

Is cell division synchronised in WT cells?

A
  • no
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36
Q

How were Ts experiments carried out to isolate cdc mutants in yeast?

A
  • 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
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37
Q

What did screens carried out in yeast identify about checkpoints?

A
  • 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
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38
Q

How would you test whether new cdc phenotype caused by mutation in single gene?

A
  • cross mutant to WT haploid –> get heterozygous diploid

- induce meiosis and look for 2:2 of mutant:WT phenotype in progeny

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39
Q

How does speed of cell division affect S. pombe cells?

A
  • if cells divide too quickly, get lots of short cells = wee cells
  • if cells divide too slowly, get really long cells = cdc cells
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40
Q

How was screening by cross-species complementation carried out for cdc28?

A
  • 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
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41
Q

How was human Cdk1 discovered?

A
  • same way as cdc2 in S. pombe (cross-species complementation)
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42
Q

How was a model for CDKs dev?

A
  • 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)
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43
Q

What is the model for CDKs?

A

DIAG
cdc25
- cdc2-P (inactive) cdc2 (active)
wee1

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44
Q

What has comparative genomics shown about CDKs in humans?

A
  • functionally conserved

- many of these genes defective in cancers

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45
Q

How are classical genetic screens carried out in yeast?

A
  • 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
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46
Q

Why were complementation screens used in cdc experiments?

A
  • cloning gene wasn’t as easy at time

- so focussed on characterising mutants, seeing how inherited and whether single gene etc.

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47
Q

What is autophagy?

A
  • self degradative process
  • balances energy sources at critical times of dev and nutrient stress
  • housekeeping role in removing misfolded/aggregated proteins and damaged organelles
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48
Q

What was the mutant phenotype of yeast unable to carry out autophagy, and how were these mutants identified?

A
  • 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*
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49
Q

How did Oshumi carry out experiments into autophagy in yeast?

A
  • 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
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50
Q

Why did the Nobel judges think Oshumi’s work on autophagy was of ‘outstanding signif’

A
  • 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
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51
Q

What are 2 examples of yeast being used as a model for human health and disease?

A
  • yeast as platform for human genetic variants (using complementation)
  • yeast as model for ageing in cells (using library of single non-essential gene deletions
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52
Q

What is CIN (chromosome instability) and what is it a sign of?

A
  • hallmark of cancer

- change in chromosome structure or no. leading to chromosome gain/loss

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53
Q

What causes CIN?

A
  • failure in mitotic chromosome transmission or defects in mitotic spindle checkpoint
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54
Q

What kind of offspring does CIN result in, and why?

A
  • aneuploidy
  • chromosomes split up unevenly, usually due to translocation
  • normal cells have spindle cell checkpoint to abort if aneuploidy occurs, but CIN don’t
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55
Q

Why does CIN increase cancer risk?

A
  • exacerbates tumorigenesis
  • as accumulative large scale genome rearranges increases likelihood of disrupting expression or function of oncogene or tumour supressor gene
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56
Q

What are variants of uncertain significance (VUS’s) caused by?

A
  • 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)
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57
Q

Why are VUS’s a problem?

A
  • 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
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58
Q

How can CIN models translate to humans?

A
  • human orthologs of genes likely to be important in tumour progression
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59
Q

What is an orthologue?

A
  • genes inherited from common ancestor w/ same role in diff organisms
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60
Q

What is a paralogue?

A
  • genes resulted from gene duplication events w/in a genome
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61
Q

How were human orthologs of yeast CIN genes found?

A
  • performed large scale cross-species complementation screens
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62
Q

How was a one-to-one complementation screen carried out for CIN genes using yeast?

A
  • 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
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63
Q

How was a pool-to-pool screen carried out for CIN genes using yeast?

A
  • 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
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64
Q

What were the key findings from the 2 complementation screens for CIN genes?

A
  • 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
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65
Q

After the complementation screens, what was the next stage of the experiment?

A
  • 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
66
Q

Why is age a global problem?

A
  • people living longer everywhere

- age is greatest risk factor of most major causes of death in developed nations

67
Q

How can you tell a yeast is old?

A
  • chronological life span (CLS) –> amount of time can survive in stat phase
  • replicative life span (RLS) –> no. daughter cells prod by 1 mother cell
68
Q

How can chronological life span be calc in yeast?

A
  • 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
69
Q

How can replicative life span be calc in yeast?

A
  • replicative viability calc as mean no. daughters prod from mothers of particular strain before senescence
70
Q

How do we know our models for telling if yeast are old are reliable?

A
  • growing evidence that ageing at least partly determined by ancestral evolutionary origin
  • suggests genetic basis of ageing
71
Q

What did the authors of the study looking at cell ageing using yeast models predict?

A
  • if yeast good models, then find some genes in screen already know to influence life span in other species and hopefully some new ones
72
Q

How were yeast mutants w/ increased longevity isolated?

A
  • used small needle attached to microscope to tease daughter cell away from mother after every cell division
  • counted how many times mother cell divides
73
Q

What big screen was carried out to look at mutants w/ increased longevity in yeast?

A
  • 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
74
Q

Why did some genes identified in yeast screen seem to act to shorten cells lifespan?

A
  • don’t want to pass on age assoc problems to offspring
75
Q

What were the 5 categories of activity which identified genes fell into, which were known already and which were new discoveries?

A
  • 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
76
Q

What is the role of LOS1 (loss of suppression 1) in yeast?

A
  • 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
77
Q

What are the characteristics of los1 mutants in yeast?

A
  • accum tRNA in nucleus, as Los1p not there to export them
  • increases replicative lifespan
  • DIAG*
78
Q

What is the effect of MTR10 overexpression in yeast?

A
  • increases lifespan
  • due to increased nuclear tRNAs
  • as transports tRNA in other direction across nuclear membrane
  • DIAG*
79
Q

Why does increased nuclear tRNA levels extend lifespan in yeast?

A
  • 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…
80
Q

Can yeast survive w/o mito, and why is this significant?

A
  • yes

- so experiments can be done which are not poss in other organisms

81
Q

What is Caenorhabditis elegans?

A
  • small transparent nematode round worm
82
Q

Why is C. elegans a good model species?

A
  • 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
83
Q

How does wild C. elegans differ to lab-grown?

A
  • in wild lives in soil and eats bacteria

- can be lab-grown on plates or in liquid culture (and fed E. coli)

84
Q

What are the roles of cells in C. elegans?

A
  • 1/3 nerve cells
  • 1/3 gametes
  • 1/3 something else
85
Q

What is the life cycle of C. elegans, and how long do these stages take?

A
  • 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
86
Q

What are the 2 sexes of worms and how do they differ?

A

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
87
Q

What has C. elegans been a model for?

A
  • euk dev
  • post genomic seq
  • apoptosis (in context of dev)
  • cell signalling (in context of dev)
  • ageing
  • RNAi
  • post translational gene reg discovery
88
Q

How is a classical genetic screen carried out in C. elegans?

A
  • 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
89
Q

What does the classic genetic screen carried out in C. elegans rely on?

A
  • careful dev of phenotypic analysis and interpretation in relevant biological context
90
Q

How can transgenic worms be created?

A
  • 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
91
Q

Why does marker in transgenic worm need to be dominant?

A
  • 1st gen always heterozygotes so needs to be dominant in order to identify mutants
92
Q

How was RNAi characterised in worms?

A
  • 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
93
Q

What is RNAi and how can it be carried out in worms?

A
  • 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
94
Q

What can RNAi be used for in worms, and what method is used?

A
  • gene knockdowns

- for specific gene knockdowns more efficient to use injection method

95
Q

How does feeding of dsRNA result in silencing of gene in RNAi in worms?

A
  • 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
96
Q

What were these RNAi screens used to characterise?

A
  • cell-to-cell signalling during dev
97
Q

How was a large amount of dsRNA able to be fed to worms in E. coli?

A
  • 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
98
Q

Are RNAi screens for worms genome wide, why?

A
  • yes
  • can purchase RNAi feeding libraries
  • 16, 757 clones
  • covers 94% C elegans genes
99
Q

Once selected mutants from RNAi screens, how do you identify the gene?

A
  • 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

100
Q

What are the advantages of RNAi over classical genetic screens?

A
  • 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
101
Q

What are the advantages of a classical genetic screen over RNAi?

A
  • 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)
102
Q

How are worms formed?

A
  • through highly deterministic dev programme
103
Q

Do worms have simple construction?

A
  • yes
  • adult hermaphrodite has 959 somatic cells
  • male has over 1000
104
Q

How is dev described using cell lineage charts?

A
  • dev stages and times along y-axis
  • vertical lines represent cell division
  • horizontal line links 2 cells formed from cell division
105
Q

What is the vulva and where is it?

A
  • egg laying organ

- approx half way along adult worm

106
Q

What occurs during round 1 of vulval dev?

A
  • 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
107
Q

How do we know lin12 gene involved?

A
  • lin12/lin12 mutants both become anchor cells (genes in italics)
  • lin12(d) both become uterine precursors (dominant mutation)
  • lin12 encodes peptide receptor
108
Q

How is cell-to-cell communication involved in anchor cell determination?

A
  • 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
109
Q

What happens during further rounds of vulval dev?

A
  • several more rounds of cell-to-cell communication, signalling and programmed cell death events that control it
110
Q

What would the phenotype be if worm couldn’t express LIN-12 receptor protein?

A
  • neither cell becomes uterine precursor
  • both become anchor cells (expressing the signal)
  • no vulva forms and get egg-laying defective mutants
111
Q

What happens with maternal effect lethal mutants in worms?

A
  • 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
112
Q

What can mel screens be used to find?

A
  • genes involved in early dev
113
Q

What are the phenotypes of lin-2 and mel mutants?

A
  • 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
114
Q

If mutagenised lin-2/lin-2 mutants in which gen would you see first mel/mel mutant?

A
  • would expect 1:3 ratio in rare plates in F2 gen to contain F2 mothers w/ dead F3 eggs filling cuticle
115
Q

What did mel screens identify?

A
  • 2 genes essential for early dev
  • skn-1 (skinhead-1) –> specifies blastphere fate
  • par (embryonic partitioning abnormal) –> req for embryonic asymmetry
116
Q

How were maternal effect screens carried out in worms?

A
  • 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)
117
Q

What is arabidopsis?

A
  • small weed in brassica family
118
Q

What is the structure of the genome of arabidopsis, and its genes?

A
  • genome 135Mb
  • 5 chromosomes (no sex chromosomes)
  • encodes about 27,000 genes
  • each gene approx 2kb w/ 4 introns
119
Q

How does arabidopsis genome density compare to humans, and why is this important?

A
  • 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
120
Q

How and why is the wheat genome extreme in size?

A
  • v extensively bred

- hexaploid, so has 3 genomes (A, B and D)

121
Q

What is arabidopsis a model for?

A
  • evo and adaption
  • pop genetics
  • dev of more complex plants, eg. maize, wheat, rice
  • env interactions
  • plant genomes
  • gene reg
122
Q

Why is arabidopsis a good model?

A
  • 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
123
Q

What is the life cycle of arabidopsis?

A
  • germination
  • seeding establishment
  • vegetative growth
  • flowering
  • senescence
124
Q

How does life cycle of arabidopsis vary in diff envs?

A
  • if somewhere cold, eg. Sweden, stays in vegetative state for long time
  • if hot then flowers grow, Cape Verde
125
Q

How does the genetics of plant life cycles differ from other organisms?

A
  • no germline

- both diploid and haploid cells undergo mitosis

126
Q

How does haploid phase (gametophyte) vary between higher and ancient plants?

A
  • in higher (inc arabidopsis), reduced to just few divisions in ovule or pollen
  • in ancient (eg. ferns/mosses), bigger and sometimes dominant
127
Q

How does arabidopsis reproduce?

A
  • mostly self fertilises, but amenable to crossing
128
Q

How can arabidopsis be crossed in the lab?

A
  • 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
129
Q

Why is it useful for geneticists that arabidopsis can be crossed?

A
  • homozygous lines can be stably maintained
  • crosses can be performed when req (eg. mapping genes, backcrossing to parental lines, looking at interaction between genes)
130
Q

What are strains from diff place generally, and what are they called?

A
  • homozygous

- ecotypes or accessions

131
Q

What was the aim of the 1001 genome project in arabidopsis?

A
  • 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
132
Q

How are arabidopsis used in evolutionary diversification and adaption studies?

A
  • can study and quantify natural variation

- 1001 genomes project

133
Q

What does Agrobacterium tumefaciens cause in the wild, and how was this adapted to use in arabidopsis?

A
  • tumours

- removed virulence genes and kept ones for injection and integration into host genome

134
Q

How is T-DNA inserted into plant genome by bacteria?

A
  • 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
135
Q

How can a T-DNA insertion pop be gen?

A
  • 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
136
Q

What plants is arabidopsis commonly used as a model for?

A
  • crop plants
137
Q

Why is arabidopsis useful for looking at dev of multicellular organisms?

A
  • cells never migrate or move, unlike animal cells
138
Q

What arabidopsis resources are available?

A
  • mutant collections
  • stock centres
  • genome info
  • epigenome info
  • expression profiling
139
Q

What is vernalisation?

A
  • flowering after prolonged cold (winter)
140
Q

How have arabidopsis been used as an example of environmentally-mediated epigenetic silencing, and how can this be quantified?

A
  • 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
141
Q

Why can’t plants measure how long winter has been by measuring day length?

A
  • Sep and March day length same

- so couldn’t distinguish between autumn and spring

142
Q

What are small interfering (si)RNAs, what is their role and where are they prod?

A
  • 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
143
Q

How have arabidopsis been used as an example of RNA silencing?

A
  • when siRNAs bound to argonaute protein (a nuclease) they can degrade targeted mRNA
144
Q

What are the general steps for functional analysis by gene discovery?

A
  • 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
145
Q

Why can many arabidopsis mutants be grown in a small space?

A
  • long stems so easy to collect seeds, even though densely pop
146
Q

How are forward genetic screens carried out in arabidopsis?

A
  • 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
147
Q

Once have mutant, by what method do you identify gene responsible for phenotype?

A
  • mapping
148
Q

What is the principle of mapping?

A
  • 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
149
Q

How are mutant recessive genes in arabidopsis mapped?

A
  • 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
150
Q

What are 2 common strains used in arabidopsis mapping?

A
  • Columbia

- Landsberg erecta

151
Q

In mapping of arabidopsis how do you know which parent each seq came from?

A
  • 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
152
Q

How are dCAPs markers found in arabidopsis mapping?

A
  • online programme

- enter WT seq, mutant seq and how many mismatches in primer

153
Q

What assumptions are made in the strategy for mapping in arabidopsis?

A
  • parent must be (near) homozygous
  • Col and L. er parents have polymorphic markers
  • g = mutant gene, recessive phenotype
  • G = WT copy of g
154
Q

How is a mutation identified through mapping in arabidopsis?

A
  • 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
155
Q

After mapping has been carried out in arabidopsis, how is the gene found?

A
  • 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
156
Q

Why might mapping fail in arabidopsis?

A
  • 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
157
Q

In arabidopsis mapping, how could you test whether mutation is in single gene (assuming phenotype caused by homozygous recessive allele)?

A
  • backcross mutant to parental line
  • F1 should be heterozygous
  • look for segregation patterns in F2 following selfing
158
Q

Why don’t you just seq whole arabidopsis genome instead of carrying out mapping?

A
  • 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
159
Q

After mapping in arabidopsis, how can you prove ‘new’ mutant is causing phenotype of interest?

A
  • order T-DNA insertion line(s) from stock centre

- see if homozygous T-DNA null mutants has same phenotype as your mutant

160
Q

What is Bio-Analytical Resource (BAR) used for?

A
  • can learn lots about when and where gene of interest expressed, w/o doing experiments
  • eg. is gene stress responsive
161
Q

Where did all the expression date for arabidopsis come from?

A
  • 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
162
Q

What is the role of microarrays?

A
  • measures relative expression of many genes at once

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