Monday, 16 February 2009

Drosophila: Virgins, balancers and jump-outs

A wise man once said that the sooner you find your model organism, the better. Ignoring the fact that the wise man I am quoting is actually me, I think I generally agree with him. This year I am working on an exciting project which extensively uses the model organism Drosophila melanogaster, geneticists’ favourite little invertebrate. In the 15 short weeks I have been working with flies, I have learned a great deal from these wonderful creatures and experienced emotions from adoration to amusement to frustrated rage in my dealings with them.

Briefly, this is your average everyday “fruit fly” (even though a more appropriate name would be “vinegar fly”) with the complete package of a head, a thorax with 2 wings and 6 legs, an abdomen, and everything else that makes flies flies. I will spare you the tediously detailed anatomy and physiology lecture and will instead jump straight to the cool stuff that interests us geneticists and you readers of genetics blogs.

One of the most interesting things about Drosophila is in fact the people that use them in research. These people are commonly known as “fly pushers”, since they do in fact spend a lot of their time pushing flies around under a microscope using an old half-mangled paintbrush. One thing that you will inevitably hear about once you start hanging out with fly pushers is how interested they are in virgin females. Flies, that is. You see, since experiments in fly genetics involve crossing male flies from one line to female flies of another in order to obtain progeny of the required genotype, one must ensure that the females have not already been fertilised by males before picked out and crossed to a different line. Collecting virgins involves looking for flies that have a dark spot in their abdomen called the meconium, which is visible for 2-3 hours from eclosion (i.e. emergence of the adult from the pupa). Females will not be receptive to males for up to 8 hours from eclosion so any fly that has a meconium can be safely considered a virgin. The most efficient way to collect virgins is to empty a vial of eclosing flies in the morning, selecting females with visible meconium, and returning in the evening. Any females in that vial will be virgins even if the meconium isn’t visible, since less than 8 hours will have passed since your last collection. Much like the world we live in these days, virgins can be rather hard to find, so if you ever hear a scientist worrying about the fact that they need as many virgins as possible, you now have an idea of what they are talking about.

Now let’s delve into some actual genetics. One of the multitude of reasons Drosophila are an excellent test tube for genetic experimentation is something called a “balancer chromosome”. A line of flies carrying a recessive lethal mutation can be maintained by tracking the mutation using dominant markers, or by “balancing” it over a different lethal mutation on its homologous chromosome. This way each chromosome “rescues” the mutation the other is carrying. However, if recombination takes place between these chromosomes, it is likely that you will then have one chromosome carrying both mutations over a wild type chromosome, which will quickly take over the stock and the mutations will be lost. You could always outcross males every generation (there is no recombination in male Drosophila) but that would be very laborious. In order to get around this problem, fly geneticists initially discovered and subsequently intentionally generated balancer chromosomes. These are full chromosomes which contain multiple inversions (essentially all the genes are still there and intact, but their order is jumbled up) in order to suppress recombination with its homologous chromosome and make any gametes with recombined chromosomes non-viable due to aneuploidy. They also carry some dominant visible markers such as Cy (curly wings), Tb (tubby), Sb (stubble) and others, and some recessive lethal markers, meaning that they can never go homozygous.

So, to illustrate this, if you have a recessive lethal mutation on chromosome 2 (let’s now call that chromosome “Yorf”), in order to establish a line that will self-perpetuate and not need constant attention, all you need to do is cross it to a line that carries a balancer second chromosome, for example the chromosome CyO. You will now have a line whose flies will always have the same genotype, i.e. Yorf/CyO. As the Yorf/CyO flies interbreed, any Yorf/Yorf progeny will not survive because the mutation will be lethal, and any CyO/CyO flies will also not survive because of the recessive lethal marker that CyO carries. There will also never be any recombination between the Yorf and CyO chromosomes because of the multiple inversions in CyO that prevent homologous pairing. Wonderful, isn’t it?

Another great thing about Drosophila is insertional mutagenesis. P-elements are transposons, short stretches of DNA which, in the presence of transposase and absence of inhibitor, are excised from the genome and reinserted randomly into another site. Some P-elements are rendered inactive by having the transposase gene deleted. This temporarily immobilises the transposon, until that fly is crossed to a fly carrying an active transposase gene. In the progeny, the P-element will jump out, and if it inserts into the coding sequence of a gene, it can disrupt its function. A mutagenesis screen can then be carried out and the insertion locus can be mapped using standard complementation or cytological mapping. There is however an application of P-element mutagenesis that has accelerated Drosophila genetics in recent years.

The targeted gene knock-out system developed in mice is an excellent way to study the role of single genes, but the long generation time and ethical concerns make the mouse a rather cumbersome model organism. An analogous targeted gene disruption tool has been developed in Drosophila, taking advantage of a property of P-element transposition. When a P-element is transposed, its excision from the genome can sometimes be imprecise, taking some of its flanking DNA along with it, thus causing a deletion in that gene. Since an extensive library of mapped P-element inserts already exists (in over half the fly genome), a line carrying a P-element insert in a particular gene can be obtained and the P-element “jumped out” by crossing to a transposase line. In the cases where the jump-out has been imprecise, the result will effectively be a targeted gene knock-out.

This is, of course, only scratching the surface of the range of genetic tools that Drosophila offers. The best way to understand fly genetics is to try some crosses on paper yourself. Some quite amazing things can be achieved within a few weeks’ time simply by performing a series of crosses and studying the progeny. The waiting time is still a little too long for impatient people like myself, but careful time planning can ensure that you always have some flies to play with and are never stuck staring at a pupa, waiting for the damn fly to emerge.