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1

Describe a normal F2 screen

Because c.elegans can be hermphrodites, screens can be carried out on the F2. 1. a mutagen such as EMS is applied to the herm 2. one of the germ cells will be heterozygous and so will create offspring which are heterozygous for a mutation. Each F2 can create its own family. 3. these heterozygous F1s will then self-fertilise and produce 25% double mutants.

2

what did Brenners screens aim to isolate?

visible mutants such as large small/ fast moving etc

3

what is the simplest way to identify genes that are all involved in one pathway?

Look for mutants that all have the same phenotype and then do complementation tests to see if the mutations are occurring at the same loci. If not they you have identified other genes

4

what is a suppressor screen and give an example

suppressor screens involve looking for a mutation in a known mutant screen which reverse the mutational phenotype. For examlple a gene called Lin-15 was identified which caused constitutively active RAS pathway signalling (it is a negative regulator). However, another mutation of downstream targets of the pathway reverse the phenotype as it couldnt signal any longer.

5

what types of pathways are supressor screens particularly good for?

negative regulatory hierarchies because loss-of -function mutations can cause const activation of a pathway and another mutation can suppress this pathway

6

describe two clever epistatic screen which involved the sex pathway

you want to find a mutation which changes a male to a female but how do you spot transformed female among lots of normaly female? You can carry out a screen in a him-5; dpy-1 background. Him-5 causes lots of males to occur and dpy-1 causes any XX organisms to be dumpy. So a mutation which trusn XO males into XO herm which cause a non-dumpy herm to present - XOL-1 is a protein which specifies males and is involved in increasing the doage of X genes to compensate for the XO genotype in males. him-5; XOl-1 produce numerous XO animals because of him-5 but these die because of the xol-1 mutation. Finding a mutation which restores this phneotype to viable males will allow other genes in the pathway to found.

7

give an example of a " screen from heaven" involving drugs and explain why it is good

An ideal screen is one which eradicates all of the organisms not of interest. For example screening for resistance to a drug. For example Aldicarb cause ach to build up in the synaptic cleft and cause death by overexcition. However, some mutants develop resistance as they cant release ach from the synaptic cleft- these are still alive

8

what makes a screen from heaven?

a screen in which the relevant mutants can be easily identified- for example, offspring which arent of interest die. Or selection for a suppressor mutant in which a mutation restores viability

9

what are the 3 general advanatges of being able to perform such large scale screens in c.elegans

1. saturation screens: saturation of genes that can be mutated to give a specific phenotype can identify many or all of the components in a particular phenotype. The presence of many alleles in all complementation groups groups shows that a screen is saturated 2. can identify unusual mutations that arise less frequently than null alleles. For example null mutations for most of the components of the RAS signalling pathway but some mutations are partial. Another example is finding allele specific suppressor mutations- this can be found in proteins that interact with each other- a mutation may cause one component to compensate for the loss of function of another and rescue the phenotype- specifically in receptors or cofactors. 3. allows structure and function analysis- different mutations will effect different domains within the gene which can have different functions

10

what are examples of screens from hell?

- phenotypes that cant be screened in the F2- germ line ageing- can take occur over 10 generations and can look for mutations that affect this- microscope screens that require extreme precision - laser ablations

11

why are the standard protocol for lethal screens not applicable?

normally you would screen the F2's, however for lethal homozygotes there are no F2s! SO you need to think of a way of screening so that you can still maintain a source of mutant chromosomes to use for lines

12

when are lethal genes normally implicated temporally?

in development normally

13

explain a cool way of screening for mutant effect genes?

you can use mutant strains that lin-2 which prevent eggs from being laid. This allows the 25% arrested progeny to be examined easily. You can then keep the F1's knowing that they are het for a mutant.

14

how do you do a maternal effect screen and why do they pose such as difficulty?

The problem with maternal effect is that the F2 mutants are viable because they receive the maternal effects proteins/mRNA from their het mother. As with lethal mutants, you can use a genetic background which has lin-2 mutant preventing egg laying. So you mutagenise worms and then plate each F1 mother and observe her F2 offspring. F2 mothers that are homozygous for a mutation cant give their embryos the right proteins so they die and stay in the mother as a bad of eggs rather than a bag of worms. you can then use the siblings of the F2 mothers which are het so rescue the mutation for lines. (

15

what are the problems with investigating lethal mutations and how can this be circumvented?

It is hard to determine the pathways that are involved in the lethal- you could try a suppression screen. or you can anaylse development very closely

16

explain how you carry out an ENU and include the numbers and logic for each step.

After mutagenesis, about ten worms (P0 generation) are transferred to large Petri plates and allowed to lay ~100 F1 progeny. The P0s are then removed and the F1s grow to adulthood. After one day of egg laying, the F1 parents and any hatched F2 progeny are washed off. However, the F2 eggs stick on the plate, leaving a 14-h cohort of F2 progeny to survey. So, a fraction (~20–30 individuals) of the 250 potential progeny from any particular F1 are sampled. One-quarter of these 20 progeny will be homozygous for the mutant chromosome, and so about five individuals that are homozygous for any mutation that occurred in the germ line of the P0 are expected. Because each plate of worms descends from 100 F1s, 200 copies of a particular locus can be assayed for mutations. Using typical concentrations of a mutagen (for EMS (ethyl methane sulphate) this is 50 mM), 2,000 copies of a gene need to be screened to find a mutation. So, about ten plates of worms need to be inspected to find a mutation in the gene of interest. See also REF. 105. The F2 progeny are inspected for the phenotype of interest and candidate mutants are removed to a fresh plate to evaluate if the character breeds true.

17

describe a suppressor screen and an example

Screens that are suppressor screens as well as selections are particularly powerful. In some cases, the initial genotype can be engineered into the strain by expressing proteins that constitutively acti- vate a pathway. For example, the trimeric G protein Gsα can be locked in the active form by mutating the GTPase domain. The expression of activated Gs under the control of a heat-shock promoter30 or a neuron- specific promoter31 causes neuronal degeneration, which induces death or paralysis in the animal. In the- ory, mutations that suppress this phenotype, by restor- ing viability or movement to these animals, could be found. Specifically, mutations in downstream compo- nents in the pathway — that is, in genes that encode proteins required for neuronal death — were identified as second-site mutations that restored cell viability. Suppressors of neuronal degeneration showed that necrotic cell death acts through cyclic-AMP signals because adenylyl cyclase is required for normal levels of Gs-mediated cell death.

18

there is a problem with lethal mutants in that it is not a very specific phenotype, how can you screen for the specific cause of your mutant lethal gene?

Pierre Gönczy and colleagues screened chromo- some III for all maternal-effect-lethal mutations, by using an inverted chromosome to balance and main- tain the induced mutations. By re-screening these strains under high magnification for defects in cell division in the one-cell-stage embryo, these researchers identified 34 loci that are required for mitosis and cytokinesis in the first cell division. Another successful strategy for identifying interest- ing lethal-mutant phenotypes has been to manipulate worms physically and thereby predict the relevant phe- notype for which to screen. For example, because it has an invariant lineage, C. elegans was originally character- ized as a simple developmental system that depended on the segregation of determinants into specific lineages — quite different from the development of higher organisms. However, Jim Priess removed a single cell in the four-cell-stage embryo and discovered that the development of a neighbouring cell, called ABa, was dis- rupted. ABa made epidermis and neurons, as it did usually, but failed to produce pharyngeal muscles. By screening for mutants in which ABa failed to produce pharyngeal cells, Priess and colleagues identified three genes that are involved in the Notch signalling path- way. These studies and others revealed that the stereoptypical lineage of C. elegans relies on invariant cell–cell interactions more than it does on the segrega- tion of determinants into specific lineages.

19

what is genetic redundancy and how does it occur, and how can you identify redundant genes?

In the simplest situation, functional redundancy arises because a gene has been duplicated and two or more closely related genes exist, any of which can carry out a particular task. This phenomenon is called homologous redundancy. The synergism between two redundant genes can be discovered because a deletion exists that removes both genes. Interestingly, the C. elegans genome contains many small duplications that carry pairs of potentially redundant genes. Mutagens that generate deletions might therefore be useful for the design of screens that circumvent redundancy- can also do sensitised screens - or double RNAi