Sex determination in Flies Flashcards
(17 cards)
How is fly sex determined
X:A ratio. ratio of 1 = female, 0.5 = male and anything in between is intersex.
What are numerators and denominators and why are they important?
Numerators are X linked TF whilst denominators are autosomal TF. In the female they will each be able to form an equal number of homodimers and heterodimers. Homodimers are active TF.
In males as there are many more denominators then the numerators will be mopped up and so X TF will not be very active, if at all.
What is the first sex determining gene?
Sexlethal - mutants are always males. In females there is an inherent bias to skip the 3rd exon whihc includes a stop codon. So when the active TF bind to the early promotor there will be a surge in production of the sex lethal protein in females. In males if anything is transcribed it includes a stop codon and so doesn’t form any active product.
What is the function of Sxl?
Sxl protein binds RNA and influences splicing. It does this to its own RNA causing a skipping of the 3rd exon. So females with lots of sxl will skip the third exon in the late promter as well where as males who have non will still include it.
It also splices out a stop codon in the next gene - transformer.
What is transformer and how is it sex specific?
In females sxl splices out a stop codon from transformer and so females form an active transformer. In males as there is no sxl, no tra is formed.
Tra mutant females are pseudomales and sterile. Tra mutant males are completely normal. Tra binds to Tra2 which influences the splicing of the next gene double sex.
Tra2 mutants phenocopy tra except that the male mutants are sterile.
How is double sex sex specific?
DSX - M is the default pathway.
DSX - F comes about because of the incorporation of a exon 4 which has an early stop codon
Mutants for dsx are therefore intersex. The binding domain that dsx encodes is conserved in humans and naematodes.
Does dsx control all of sex specification?
No - dsx-m expression in females only changes body morphology. They still have the same behaviour and something called the MoL.
However tra-tra mutants do so tra must control mind whilst dsx controls the body. Note that dsx is capable of repression and activation of genes.
What is the gene intersex?
Co activator with dsx-f and the mutant only affects females.
Why might the X:A theory not be entirely accurate.
If correct we would expect X:A and XXX:AAA to be normal females. They are but their expression isn’t exactly the same. X:A expression of sxl resembles that of males and XXX:AAA cellularise early and are super females.
Appears that it is more to do with the counting of numerators than the ratio.
What dosage compensation occurs in the fly?
X chromosome in males has double the dose.
Are there any autosomal genes that when mutated cause males to be feminised?
Yes - male: maleless and male specific lethal (1-3).
All 4 associate with each other and H4 acetylation.
Do mle and msl bind to the X chromosome what prevents binding in females?
Yes staining for mle and msl-1 with antibodies covers the male polytene chromosomes. When msl-1 is mutated mle doesn’t bind and in sxl mutants the female x becomes stained. So does Sxl prevent binding?
Yes Sxl cases the retention of a stop codon at the 3’ end of msl-2 and binds to the 5’s end to stop translation (just in case)
Other than mle and msl does anything else cover the x chromosome?
Yes - Rox 1 and 2 which are both RNAs cover the X polytene chromosome. But if any of the DCC is mutated then rox isn’t transcribed. If both rox are mutated then DCC doesn’t work.
Mle (helicase) unwinds rox genes changing its shape and then triggers the compensasome to assemble.
Where does the compensasome bind to on the X?
MSL1 and MSL2 can bind to 30-40 sites on the X chromosome via CES which contain an MRE this includes the rox genes. There is spread of the complex up and down stream from these sites.
When there is a high ratio of msl:rox then there is clumping and when the ratio is normal there is spreading.
Used Chip-Seq to find these sequences. Cut away DNA around where msl-2 binds. Then release the fragment and put on a CHIP then test against genome. The MRE motifs are enriched on the X chromosomes - 150 sites in total all transcriptionally active
How does the DCC bind to the X chromosome and increase transcription?
Through the CES. If you attach a Rox promoter to a firefly luciferase gene with a CES on the end and insert into cells producing lots of the DCC genes then this will induce expression of the firefly luciferase. If you swap the rox promoter for an autosomal promoter with a CES then it is still transcribed.
If you mutate the MRE or the DNA flanking it within the CES then it alters the expression level.
Putting the CES on an autosomal chromsome causes the gene its next to and the surrounding genes to increase expression - the compensasome spreads.
Another protein in the DCC - MOF (males absent on the first) acetylates H4K16 to increase transcription.
How were Trojan horses and elephants used to fill the final piece of the puzzle for dosage compensation?
Trojan horse = sequence of DNA including 3 active genes and 2 inactive genes. Insert on different places in the X. Where ever it is places the active genes attract MSL even though they don’t contain the CES.
Do it on a bigger scale into barren regions of the X and yoou still see MSL attraction to the active genes.
Remove all the promoters and there is not attraction.
What is the final model for dosage compensation and what is one last problem yet to be solved?
- rox genes attract DCC
- DCC is then attracted to 150 CES
- DCC then spreads to any active genes
Dosage compensation starts before msl genes are expressed???
