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1

what is a forward genetic screen

this involves using a screen to identify a gene involved in a process in an unbias and from an uninformed background.

2

what is a reverse genetic screen?

a bias approach in which candidate genes are screened for their involvement in a pathway or process

3

what is the general approach to performing a screen in drosophila?

You use a DTS (temperature sensitive dominant lethal mutation) and a balancer female. You have a wild type male which you expose to a mutagen. You then cross these to produce the F1. You are given DTS/balancer gets and mutagenised chromosome/ balancer. You then grow these at 29 degrees so you kill the DTS/balancer. then you are left with only must/bal. You cross these and get a ball/bal which is lethal, must and ball (which will have the balancer marker) and a homo mutagenised . You can observe these for a phenotype

4

how is a screen generally carried out in c.elegans?

A population of wild-type hermaphrodites is exposed to a mutagen and genes are randomly mutated in the germ cells (mutated germ cells are indicated in red). For example, one sperm could be mutated for the gene unc-32, which is required for the correct functioning of the nervous system. Fertilization of an egg by this sperm will result in a heterozygous F1 individual. Because this animal is a self-fertilizing hermaphrodite, it will produce eggs and sperm that bear this mutated gene; one-quarter of its F2 progeny will be homozygous for the mutation and result in a coiled phenotype (shown in b). Such an animal can be transferred to a plate, and in three days, its F3 progeny can be inspected to determine whether the mutant phenotype breeds true.

5

how do you carry out a screen in zebrafish?

mutagenise a malecross with a femaleeach F1 offpsring will make a family So cross with a wild type to make a family which contains heterozygous F2 fish when these two hets cross you get 25% homozygous in the F3

6

what type of mutations so UV and x-ray normally induce?

large deletions

7

what type of mutations do chemical mutagens normally make?

single point mutations

8

describe how EMS works

it is an alkylating agent, it induces G-c pairings to change to A-T. it does this because it methylates guanine which makes it pair with T so when the DNA replicates, an A-T pairing is formed

9

describe how ENU works?

- most toxic but most well used mutagen- it can change G-C to A-T and vice versa.

10

how do optogenetics mutations work and in what organism are they used in?

Caenorhabditis elegans expressing His-mSOG in the germline behave and reproduce normally, without photoinduction. Following exposure to blue light, the His-mSOG animals produce progeny with a wide range of heritable phenotypes. We show that optogenetic mutagenesis by His-mSOG induces a broad spectrum of mutations including single-nucleotide variants (SNVs), chromosomal deletions, as well as integration of extrachromosomal transgenes, which complements those derived from traditional chemical or radiation mutagenesis.

11

what are the 4 type of mutation phenotypes that can be generated following a genetic screen?

- no signalling (null)- less signalling (loss of function)- increased signalling (gain of function) - increased signalling (over expression)

12

can some loss of function mutations be temperature sensitive?

yes

13

how can the order of a genetic pathway be achieved by using epistasis? what are the conditions when making double mutants and how can this issue be circumvented?

You have candidates for a pathway that you wish to order. You make single mutation mutants of each the candidate genes and take note of them. You then make double mutants of all of the possible pairings. The downstream gene will have the same mutant in the single and in the double mutants. The double mutants must have opposite phenotypes - you can use gain of function mutations for one if they both have the same phenotype

14

what is good about ordering genetic pathways in organisms such as the c.elegans? give an example

they are very easy to carry out genetics in, once the pathway has been order it can be investigated in other more complex organisms. For example, the TGF0beta pathway homologues (?) are found in c,elegant, drosophila and verterbates and are all involved growth patterning

15

describe a GFP screen carried out in c.elegans and cite it.

flowers and poole et al., 2010: They sought to find gene involved in the patterning os the ASEL neurons in c.elegans. they used GFP labelling by driving the expression of ASER and ASEL specific markers in the worms. They then did an RNAi screen and looked form probes which called defects in the left/right patterning. They identified lsy-22 and lsy-5. When they were both knocked down there were no ASER reporter expression so they are involved in ASER patterning. They did epistasis analysis by using lsy-6 mutants which express the opposite phenotype (2 ASERs) and found they were both epistatic to lsy-6 but act upstream of die-1 which is after lsy-6. they found that they were both alleles of grouch which is a transcriptional repressor and work in conjunction with cog-1 to help repress die-1 which stimulates right fate. They did this by looking at binding domains.