C. elegans and Arabidopsis

Question 1

give some basic details on C. elegans

Answer 1

small nematode - round worm,1 mm long, transparent, contains 900 cells

In the wild it lives in soil and eats bacteria. Can be lab-grown on plates or in liquid culture (and fed E. coli)
Grows quickly (egg-to-egg is 3.5 days, two generations in a week).

Lives for 3 weeks

Can reproduce sexually (1000 progeny, a hermaphrodite mates with a male) or by selfing (300-350, done by just a hermaphrodite)

Genome is 100 Mb, five autosome pairs and sex chromosomes

Efficient transgenics
Loads of info/genomic resources available

Question 2

C. elegans - hermaphrodites vs males?

Answer 2

hermaphrodites XX, males XO

herms produce eggs and sperm, males are rare in the wild but can be bred in the lab

herms mostly self-fertilise, males can only produce sperm so can’t

herms progeny - 99% XX, and the 1% XO are produced by non-disjunction
males mate with hermaphrodite to produce 50/50 (FYI O is literally zero, like they just have X)

Question 3

explain the life cycle of C. elegans

Answer 3

Adult hermaphrodite (has eggs) → sperm entry forms embryo and embryogenesis begins → lays an egg, embryo development continues for 8hrs (from sperm entry whole thing is 14 hrs), then hatches → now an L1 larva

DONT STRESS OVER THIS
route 1 - Grows for 12 hrs to become an L2 larva → 7 hrs later it’s an L3 → 8 hrs after that its an L4 larva → 10 hrs later you’ve got an adult

route 2 - If conditions are not favourable (starvation), the L1 larva grows for 12 hrs to become an L2 Dauer larva → after 13 hrs it becomes the Dauer larva, a resistant form of the worm that can survive for 4 months, hopefully conditions improve and it can re-enter the cycle by becoming an L4 larva

Question 4

what are worms a model for?

Answer 4

Eukaryotic development (has more than one cell type unlike yeast)
Post-genomic sequencing
Apoptosis (first discovered in worms)
Cell signalling
Aging
RNA interference (RNAi) a method of silencing genes initially identified here

Question 5

key important fact form John E. Sulston?

Answer 5

C. elegans has an invariable cell lineage - each division occurring in development happens every time the same way

Question 6

explain the steps in a classical genetic screen

Answer 6

Get a wildtype (+/+) hermaphrodite who will be the parent (P0)

Treat it with a mutagen that will cause mutations in all cells at random places in the genome - we only really care about ones in the eggs and/or sperm

F1 generation will be heterozygous for mutations in various genes, hopefully one we are interested in. we are unlikely to see a phenotype because most mutations are recessive

Self the F1 individual to get the F2. 25% (+/+), 50% (+/-), 25% (-/-) so this 25% will show the phenotype of the mutation - usually they appear curled, which is very easy to spot

Transfer these mutant phenotype worms to a separate plate. All the individuals form this hermaphrodite should show the mutant phenotype (F3)

Question 7

this classical screen is modified in what case? how is it modified?

Answer 7

when trying to identify maternal effect genes (lethal when mutated)

(The mother/hermaphrodite, pumps a load of gene-products and proteins and mRNA into the egg, via nurse cells surrounding the egg. Its this stuff that causes the divisions that occur
This is the maternal effect - the zygote’s phenotype is determined entirely by the mother’s genotype and her maternal effect genes
If mutated, the result is lethal)

In the screen, a mutant - lin2 - which has a vulvaless phenotype, is used. These hermaphrodites produce eggs and sperm, but cannot lay eggs

All offspring are trapped inside the hermaphrodite’s cuticle
So offspring can be matched with their maternal parent, allowing you to identify maternal effect lethal genes (i.e. if all the eggs die)

Its F3 - you are looking for death of F3 individuals. F2 may have some homozygous embryos lacking a certain gene, but because it’s the mother’s genotype that determine whether the embryos will die, it’s the offspring of the homozygous F2 embryos that will die (if the gene you mutated is a maternal effect gene involved in very early development)

Question 8

explain what the modified, maternal effect genetic screen looks like

Answer 8

So you are looking to see if you can identify a mel gene (maternal effect lethal gene)

P0 - Apply a mutagen to a WT (+/+) (hoping to hit a mel gene), and homozygous for lin2 (so all offspring in all generations won’t be able to lay eggs)

The common route - Your F1 individual is still WT for it’s mel genes, and ofc homo for lin2.

Self fertilise…
F2 will be a ‘bag of worms’ because of the lin2, but all-functional mel genes so the larvae still develop

The rare route. Your F1 individual is heterozygous for a mel gene (meaning the mutagen you applied caused a mutation in a mel gene) and ofc homo for lin2

F2 (cross two F1s?) will then have three possible outcomes -

Same as common route, a bag of worms, WT for mel genes (ofc they’ll all be homozygous for lin2 so whatever happens something’s gonna be trapped in them) F3 alive

Bag of worms, just heterozygous for the mel gene mutation. F3 alive

***bag of eggs - homozygous for the mel genes so it’s eggs get trapped and die without developing into larvae (what you wanted to see, but annoying because you cannot carry on this line). F3 dead (mothers genotype F2 was mutant)

you collect the siblings of the ‘bag of eggs’ (F2) so you can maintain the heterozygous mel mutant

Question 9

give two genes that were identified by the MEL screen (C. elegans)

Answer 9

Par genes - produces asymmetry by partitioning an embryo

Skn-1 gene, specifies blastomere fate

Question 10

what’s the easiest way to make transgenic worms, and what kind of marker is used?

Answer 10

directly inject DNA into the gonads
Can be linear or circular
You can knockout a gene, or add a transgene using homologous recombination as in mice and yeast

the marker - in worms, the selective markers are obviously not antibiotics, they have to be phenotypic
Most common is a dominant collagen mutant, rol-6, giving a phenotype of a rolled up worm (like a ‘c’ when the WT looks more like an ‘~’).
The marker needs to be dominant - homologous recombination is only going to target probably one copy of the gene. So it’s going to be hemizygous for your
Transgene - in order to see a hemizygous gene it needs to be dominant

Question 11

RNAi in worms - what is it used for and what happens when you inject dsRNA (complementary to an exon sequence) into worms?

Answer 11

RNAi is an endogenous cellular process by which messenger RNAs are targeted for degradation by double-stranded (ds) RNA of identical sequence, leading to gene silencing

Inject worms with dsRNA complementary to an exon sequence, resulting in specific silencing of that gene/any gene with that exon sequence
The silencing spreads through the organism, and is inherited into the progeny while the eggs are still in the mother

Question 12

RNAi is genetically controlled - what experimental evidence suggested this?

Answer 12

they made transgenic worms with GFP, then silenced it with the dsRNA
This didn’t work in animals defective for RNAi, showing the silencing itself is genetically controlled

Question 13

how is RNAi used in a screen?

Answer 13

Put the RNA sequence in a plasmid between two phage promoters (T7), causing transcription in either direction, so that you get dsRNA when it is replicated

Bacteria with this plasmid is fed to the worm

In the cell - double stranded RNA is cleaved into 21-25 nt fragments by nucleases of the Dicer family. These RNA fragments, short interfering (si)RNAs, are separated into single stranded molecules and one of the two strands then becomes bound to an Argonaute nuclease. This RNA then forms base pairs with longer endogenous mRNAs, allowing the argonaute nuclease to slice the mRNA
This silences the gene at the level of translation

Used to - make knock-down mutants in worms

Question 14

RNAi in C. elegans - when to use the injection method (easy answer) vs the bacteria method (longer answer)?

Answer 14

For specific gene-knockdowns it is more efficient to use the injection method

For large-scale screens, - bacteria
Long dsRNAs are fed to the worms in E. coli

Get a C.elegans RNAi library - a laid of different E.coli expressing different sequences of dsRNA - feed to worms - screen for phenotypes of interest

To identify the gene - you just sequence your gene directly from the E.coli plasmid, between the two T7 promoters
***These RNAi screens were used to characterise cell-to-cell signalling during development

Question 15

How else might you screen for maternal effect lethal mutation?

Answer 15

Using conditional mutants so you can see the phenotype in the restrictive conditions and maintain the line in the permissive conditions

Question 16

Why are worms a brilliant model for development geneticists?

Answer 16

Genome sequenced, good genetic tools, transparent cuticle, invariant lineage/deterministic cell fate, simple development, only 959 cells, can be used for studying things that require a multicellular organism

great personality

Question 17

compare RNAi screens with a classical genetic screen

Answer 17

RNAi knockdown reduces level of WT product. a classical screen can uncover tissue-specific alleles, and provide insight into structure-function relationships of point mutations, and gain-of-function mutations can be isolated

RNAi - the gene sequence is known immediately, but for classical the cloning stage is laborious

classical - every gene is mutable. RNAi - not every gene is susceptible, e.g. proteins with long half life are hard to knockdown

mutant alleles are heritable, knockdown usually isn’t (unless the silencing construct is expressed as a transgene)

mutation usually affects a single gene, knockdowns can target multiple genes with shared sequences, uncovering redundancy

Question 18

arabidopsis - basics on it’s genome?

Answer 18

5 chromosomes - no sex chromosome

Encodes about 27,000 genes. Similar number of genes to humans but arabidopsis genome is 135 Mb long, which is smaller than humans, so is more condensed

Each gene = ~ 2 kb with 4 introns
Note - this is a relatively small genome compared to other plants like e.g. wheat (17,000 Mb, 96,000 genes)

Question 19

arabidopsis is a good model for what?

Answer 19

Evolution and adaptation (can grow in different places, so you can compare)

Population genetics

Development for more complex plants (maize/wheat etc…)

Environmental interactions (light, water, disease etc… they can’t run away like animals can)

Plant genomics

Gene regulation

Question 20

what makes arabidopsis a good plant model?

Answer 20

Short life cycle (6 weeks seed-to-seed)

Large numbers of progeny (you need lots of organisms to see rare events)

Can be grown in restrictive conditions

Can be efficiently transformed using Agrobacterium tumefaciens (inserts tDNA into the plant genome)

Genome sequence available (since 2000*)

Large collection of mutant stocks, natural ecotypes (plant collected from a particular place) and genomic resources

Question 21

briefly, what is the life cycle of arabidopsis like?

Answer 21

germination and seedling establishment, vegetative growth, bolting, flowering, senescence (plant dies, releases it’s seeds)

Question 22

do plants have a germline?
what do their cells do - mitosis/meiosis?

Answer 22

Plants do not have a germ line, any cell could technically become a germ cell
Both diploid and haploid cells undergo mitosis

Diploid = sporophyte, they can undergo meiosis to form ‘haploid spores’

Haploid spores undergo mitosis = forms adult gametophyte (still haploid)

Gametophytes become gametes that fuse with other gametes to form a zygote

In higher plants (including Arabidopsis), the haploid phase (or gametophyte) is reduced to just a few divisions in the ovule (egg) or pollen (male gamete)
Ancient plants (ferns and mosses) haploid phase is bigger and sometimes dominant

Question 23

arabidopsis - how does it reproduce?

Answer 23

Most of the time you get self-fertilisation -
you have the pollen on the anthers, which is transferred onto the stigma in the centre, travels down and fertilises the ovule

But you can cross them in the lab -
Choose your maternal parent, remove the sepals, petals and anthers before it flowers (to remove the pollen and prevent self fertilisation)
Mark it with a bit of cotton so you know which plant you’re using (idk do it for both)
For the paternal parent, remove the whole anther, touch to the maternal stigma to pollinate it/fertilise the ovum

Question 24

why is it useful that arabidopsis can be selfed, and crossed?

Answer 24

Homozygous lines can be stably maintained by self fertilisation
Crosses can be performed when required (e.g. mapping genes, backcrossing to parental lines after mutagenesis, looking at the interaction between genes)

Question 25

what are ecotypes/accessions?

Answer 25

Strains collected from different places are generally homozygous (or loci) and are called ecotypes or accessions

two famous ones are Columbia (USA) and Landsburg erecta strains

Question 26

what is the 1001 genome project and its aims?

Answer 26

sequenced genomes of 1001 accessions, which are naturally inbred lines, that are products of natural selection under diverse ecological conditions, enabling a research program that links genotypes and phenotypes to fitness effects (available from the stock centre to everybody)

Question 27

what are the steps in making transgenic arabidopsis?

Answer 27

Bacteria Agrobacterium tumefaciens has a T-plasmid containing T-DNA (transfer DNA)
T-DNA is transferred from the plasmid to a random spot in the nuclear genome of the plant

Let your plant grow and bolt…

Dip infiltration of T0 - dip in solution of: your bacteria with gene of choice, detergent to break cell membranes and help bacteria get in, plus some sucrose
Wrap in clingfilm and later remove

Let it recover, then grow seeds on some ms -minimal salts - media…

The media also has kanamycin in, and the T-DNA has a kanamycin resistance gene (so only the plants that have taken in the T-DNA will survive and grow

Question 28

what is the result of the transgenic process in arabidopsis, and what extra step is needed?

Answer 28

the result -
Each seedling has an independent T-DNA insertion (independent because the T-DNA is inserted randomly/in different places)
When the insertion occurs in an actual gene it likely knocks out the gene entirely - you’ve shoved a gene inside of another one (it’s not like the smaller changes you’ll get with EMS)

Unlike in yeast (homologous recombination), you need to find out where your T-DNA is -
Backcross your lines to get a line with just one T-DNA insertion (multiple insertions in different genes mess up the process…)

Flanking DNA - sequence the gene you’ve inserted, ‘sequence out’ to see what’s surrounding your gene/locate it

Note - large collections are already available

Question 29

arabidopsis has a lot of resources - name some

Answer 29

Mutant collections
Stock Centres
Genome information
Epigenome information - whole genome has been sequenced via its DNA methylation status in response to stressors
Expression profiling - looking at the expression of 1000s of genes at once

Specifically - Nottingham arabidopsis stock centre
ABRC (arabidopsis biological resource centre), like yeastgenome.org
1001 genome project
BAR - expression of genes throughout development

Question 30

what three big discoveries were made using arabidopsis?

Answer 30

Flower development, homeotic mutants and the ABC model*

Vernalization and epigenetic silencing

RNA silencing

Question 31

what is vernilisation?

Answer 31

Vernalization is flowering after prolonged cold. Wants to flower in spring - but how does it tell this apart from autumn? Can’t use day length as 12 hr days occur in both march and September

Epigenetic control of the floral repressor gene is the solution

Question 32

briefly explain epigenetic control of the floral repressor gene in vernalisation

Answer 32

Plant won’t flower in autumn because FLC (flowering locus C, a floral repressor gene) is high
Then it gets cold, slowly reducing FLC expression throughout winter (requires a prolonged cold, if its e.g. a week FLC will go back up)
It gets to spring and FLC stays low allowing the plant to flower
COOLAIR silences FLC
Seed development - FLC turned back on again

Vernalisation requirement - plants in different places require different periods of cold before they flower

Question 33

explain the process of RNA silencing

Answer 33

Originally identified as a response to viral infection (in arabidopsis) but has been shown to be a vital part of development

An RNA dependent RNA polymerase generates double stranded RNA from a single stranded precursor. The double stranded RNA is cleaved into 21-25nt fragments by nucleases of the Dicer family.

These small interfering (si)RNAs are double-stranded RNA molecules, and have two unpaired bases at the 3’ ends of each strand

Once separated, one of the two strands becomes bound to an Argonaute nuclease, or Slicers. This RNA then forms base paired duplex structures with longer, target mRNAs (the SiRNA is complementary to the mRNA target) , so the siRNA is what guides the Argonaute ‘slicer’ to its target.
RNA silencing = combination of Dicing and Slicing

Question 34

explain the steps in a typical forward genetic screen when using arabidopsis

Answer 34

1. Perform a forward genetic mutant screen (apply a mutagen, look for phenotype of ‘I can’t properly do the process you’re interested in’)
2. Cross back to the parental strain, to check if results are 3:1, to ensure the problem here is due to a single gene inheritance
3. Identify the gene responsible by mapping
4. Verify that we have the correct gene by ordering a null mutant from a stock centre and looking for the phenotype we observed in our screen/should match
5. Find out more about the new gene, and plan our next experiment using information from publicly available databases

Question 35

use slides for tables comparing model organisms