Flashcards in deck_4811418 Deck (33)
how long is the generation time in flies?
what are the advantages of using flies as a model organism?
- easy and cheap tp rear- no recombination in males so tracking mutations is easy - only 4 chromosomes which are easily visualised- the exoskeleton of flies is good to look at patterning development of the embryo - has many homologues in humans- twice as many than c.elegans
what is the problem with screening for mutations in genes that have many different effects at different times?
- if you produce a mutant, only its first role will be disrupted- you cant get past this stage without using a conditional mutation. At example is Wg in flies. This is involved in development and in the adult
what is the problem with screening for pehnotypes of offspring that express lethal maternal effect genes?
the phenotype wil lonly be expressed in the offspring once the maternal source has run out- so you can see the role that it has before this point.
how did scientists try to address the problem of onlybeing able to screen for the role of the first role of a gene that is expressed at different time sin development?
They looked at mutations which were homozygous from large deletions of the genome. They then let the embryo develop until it died. They then noted that the gene that was deleted from the space which was the earliest acting from that which were deleted, would show its phenotype. They then repeated this with different regions there were abe to identify all the regions and genes which held the early acting genes. they found that there were only 7 early acting zygotic (non maternal genes)
give an example of using a dominant enhancer screen and why they are good
Loss-of-function mutations in almost all genes are recessive, which indicates that 50% of the wild-type level of a protein is sufficient for normal development. When a particular process is already par- tially disrupted by another mutation, however, this amount might no longer suffice, and mutations in the genes that are involved in the pathway can therefore be identified as dominant enhancers or suppressors in this sensitized genetic background.- because this relies on a heterozygote mutation (dominant) you can screen for these in the F1.
what are three good things about using sensitise backgrounds for screens: enhancers?
- you can screen in the F1 for dominant enhancers - if the sensitized background only effects one part of the body (such as the eye) which isnt required to live then you can see the effect of mutations to otherwise lethal genes that function as many stages in development as the sensiitzed background only affects the eye - Third, the whole genome can be screened at once, because there is no need to use balancers to make particular chromosomes homozygous
what are 2 reasons why sometimes looking for dominant enhancers or suppressors isnt good?
- not all components of a pathway will necessarily be dosage sensitive- the levels are not crucial to functioning sometimes- many mutations may turn out to be homozygous viable and have no phenotype when removed from the sensitive background- no guarantee that a gene has an essential role in the process.
why is the eye a good target for enhancer and suppressor screens?
- it is not essential for viability or fertility ad is easy to score. It is particularly good for modifier screens because it is composed fo 800 regulalry packed ommatidia and numerous defects in cell-fate determination and differentiation give a rough eye phenotype, the severity of which reflects the number of ommatidia affected. It is even possible to carry out screens for modifiers of genes the functions of which have not been characterized in the eye, if the eye-spe- cific expression of a wild-type or mutant construct of the gene produces a rough-eye phenotype
explain how the Flp/FRT can be used to generate mitotic clones
In mitosis one paternal and one maternal copy is inherited by each daughter cell. Flp is a recombinase which causes recombination between sites with a FRT site. Therefore, when activated and present with a transgenic line that has FRT sites on the same site on the matenral and paternal chromosomes (inserted via homologs recombination), then flp will cause recombination between the two and one paternal chromosome will the mutant so the daughter cell will get two copies and be homozygous.
when can the Flp/Frt be used?
- it can be used to study the difference in growth rates between cancerous cells and normal cells. This can be done by also putting a marker that is linked to the mutation- e.g red vs white eye s
explain how the Flp/Frt system can be used to screen for maternal effect genes in the F2.
1. cross a male which expresses the dominant ovoD sterility gene in its germ line and a balancer, with a female that expresses a mutant and a balancer. These both have an FRT below the mutant position. The mother will also express a heat sensitive Flp allele so that after fertilisation it can be triggered2. The F1 female is produced that has a genoptype of the maternal expressing ovoD and the paternal expressing the mutation. It also expressed Flp which is heat sensitive. 3. Flp can be activated and the germ line cells can undergo recombination when they divide. This will result in either no recombination which will not be layed due to the ovoD, a double ovoD which also wont be laid and a double mutant which will be laid 4. the F2 can be screened and the male siblings used to keep the mutation.
describe how tissue specific Flp/FRT can be designed in such a way to distinguish between each cellular outcome of the FRT/Flp
1. You can introduce a "Minute" muttation (M) onto the non mutation atm that cause cell death when homozygous. Therefore, twin spot cells die and the non recombined hets have inhibited growth.This means that the twin-spots die and the non recombined are out competed by the recombined homo mut which has normal growth. By having a mutant white eye allele next tot the minute mutation, the different cell types can be viewed. This is good because you can basically erradicate all of the non non-clonal cells in order to look at the effects of your homo mutation in one part of the organism 2. another way is to put a mutated White eye allele in the arm that has a mutation on the other chromsome. This means that the twin spots are dark red, the hets (non-recombined) are red, and the homo mutatnts are white. However, this doesnt get rid of the other cell types. This can be used to look at cancerous mutation's growth relative to a wild type cell.
why is using the FRT/Flp system useful? (2)
- it allows you circumvent the problems that arise with looking at the effects of mutations which are affective at different times of development. If you knock out a gene, you will only see the phneotype for the first time that it acts. So you can express genes specifically in different tissues at different times - it is good for comparing cancerous mutation as you can compare the growth of the different cell types
why are drosophila good model organisms in terms of compromise for human studies?
they are very easy to work with and easy to do genetics in but they also share many conserved genes with humans
name 2 famous examples of pathways in flies being transferable to vertebrates?
patterning of the D/V axis: Dpp; BMP4 and sig;chordin
are flies good for forward genetic screens?
what is the easiest way to mutagenise a fly?
feeding them EMS which induces point mutations
why are flies particularly good for developmental investigation?
their exoskeleton (cuticles) provide an exquisite read out of the patterning of the embryo. Secondly, the mother provides her eggs with most of RNA required for embryogenesis so very few mutantionf or embryonic lethal and very few block embryonic development at early stages- this means screens were ver efficient at identifying the transcription factors and signalling molecules that generate positional information in the embryo.
what does a saturated screen mean?
Saturation screens are used to uncover all genes involved in a particular phenotype of an organism or species. The screen is carried out by mapping mutants of a biological process until no new genes/gene mutations can be found.
what is a general problem with using screens for genes that are recessive lethal?
you can only observe their first essential role, as after this point the embryo is dead so can not longer be screened for future roles of the gene
how did Wieschaus identify the first early acting genes in drosophila?
they made flies that were homozygous for large deletions. They then were able to work out the regions which contained the essential early acting genes- they found that 7 were required
what is the general premise of enhancer and suppressor screens?
Loss-of-function mutations in almost all genes are recessive, which indicates that 50% of the wild-type level of a protein is sufficient for normal development. When a particular process is already par- tially disrupted by another mutation, however, this amount might no longer suffice, and mutations in the genes that are involved in the pathway can therefore be identified as dominant enhancers or suppressors (mutations) in this sensitized genetic background.
what are the advantages of performing an enhancer of suppressor screen?
First, the progeny of mutagenized flies can be screened directly (an F1 screen), because the mutations do not need to be made homozygous, which means that an order of magnitude more flies can be screened than in an F3 screen. Second, lethal mutations in essen- tial genes that function at many stages of development can be isolated, because the sensitized background only affects the eye, which is not required for viability ( so basically the identified gene may be involved in early processes so you can't make a homozygous mutant but you can have a sensitised background in an area that will not cause lethality when perturbed). Third, the whole genome can be screened at once, because there is no need to use balancers to make particular chromosomes homozygous.
what do you do if you have used a sensitised background to carryout a screen but you can only identify the pathway so far before the components become less sensitive to the sensitised background, the more downstream they are. and what if the most downstream gene you have identified is an early essential gene so you can't produce a sensitised background using it? (fly)
you can express the component which you want to know the downstream components of in the eye, if this gives a particular phenotype, you can then look for mutations which perturb this phenotype- having the ey in the drosophila which you can perturb without causing lethality is a very useful trick
what are the drawbacks of sensitised screens?
not all pathways are dosage sensitive. many of the mutations turned out to be homozygous viable and have no phenotype when removed from the sensitized background. Suppression or enhancement of the phenotype of interest is therefore no guarantee that a gene has an essential role in the process. It may also be necessary to carry out screens in a variety of different sensitised backgrounds to uncover all of the components of the pathway.
where is the bets place to carry out sensitised screens in the fly and fly?
In the fly eye, Furthermore, it is particularly suited to modifier screens because it is composed of ~800 regularly packed OMMATIDIA, and numerous defects in cell-fate determination and differentiation give a rough-eye phenotype, the severity of which reflects the number of ommatidia affected. It is even possible to carry out screens for modifiers of genes the functions of which have not been characterized in the eye, if the eye-spe- cific expression of a wild-type or mutant construct of the gene produces a rough-eye phenotype
what are clonal screens in flies?
screens in which only the area of interest expresses homozygous alleles for the mutation.
why are fly clonal screens useful?
they can be used to study pleiotropic lethal mutant genes