Screening for DMAP1 deletions

Here is a description of the male recombination screen used to target DMAP1  which my colleague Dr. Kathleen Fitzpatrick (Simon Fraser University (SFU) in Burnaby BC Canada) and I wrote together. Dr. Fitzpatrick used this screen as a part of her undergraduate genetics lab teaching program, and so this work is discussed largely by her (I made the pretty pictures). An application of the Awesome Power of Undergrads in the service of a very tricky screen!

In our class (Bisc 302W) at SFU, we spent several semesters screening for a directed deletion of the gene DMAP1 in Drosophila melanogaster. We used a P transposable element located between DMAP1 and its near neighbour CG33785, on chromosome 2. The P element is called GS10389,  but I called it P* in the class, so I’ll refer to it that way here as well. Note here that P* is closer to the gene CG33785 than it is to DMAP1.

There are two main requirements for P elements to move in the genome: first: an enzyme called transposase and second, special DNA sequences on either end of the P element called inverted terminal repeats. The transposase enzyme recognizes those repeats, and can cut the P element out of the original position and insert it into a new position somewhere else in the genome. P elements tend to have insertion preferences: places where they are more likely to insert and places where they won’t insert. But there is no way to predict exactly where a P element will go. In this sort of work, you mobilize a P element – allow it to move – and then you have to select for the result you want. You can’t just make it do exactly what you want.

The majority of transpositions are precise – we call them clean (or perfect) excisions. Sometimes transposition is not neat and tidy; sometimes it’s a bit messy – the element is not cut out and moved discretely, but some DNA to one side or another of the element is cut out too. This is referred to as imprecise excision. For our purposes, we are very interested in those specific imprecise excision events that also include a crossover event with a homologous sequence, because in this way, we might be able to select the direction in which the deletion takes place  (and remember, P* is closer to a gene we do NOT want to disrupt). In 1996, Preston et al (PMID: 8978050) published the results of their analysis of crossovers (recombination events) induced by P elements. They found a crossover rate of about 1% of the chromosomes tested (though it varied with temperature) and – here’s the important thing – deletions were generated from the insertion’s original site to one side or the other of the P element in some of the crossover chromosomes. The direction of the crossover reflected the direction of the deletion. Thus, if you induce a P element to move, and the element starts out near a gene you are interested in, careful selection any recombinants that result may net you some deletions of your gene. The mechanism is kind of complicated – the P element sequences located on sister chromatids try to pair with each other, forming a complex structure, the resolution of which may generate the deletion we need. Take a look at Movie 1 (and many thanks to Dr. Bill Engel for letting us use it).


So, here is a strange thing about fruit flies that turns out to be very useful in this context. Everybody knows that during meiosis, homologous chromosomes pair and undergo crossing over during Prophase I. This is one of those very important generators of variability upon which our evolutionary history depends. But, in male fruit flies, for reasons I think are still not well understood, there is NO meiotic recombination. Very strange. In fact, reduced rates of recombination associated with heterogametic organisms – those with different sex chromosomes like X and Y – have been observed since the early part of last century – here’s a recent paper using Zebrafish that describes it well (PMID: 11861568). Male fruit flies represent an extreme example with no meiotic recombination at all, but that turns out to be very helpful to us, because if we do our directed deletion experiment with male flies, any crossovers that result are almost certainly due to the P element “attempting” to move and making a useful mistake (for us), as opposed to a general recombination event we can’t control.

But how do we set this up so that we can select for those aberrant male recombination events that move in the direction of DMAP1? We really want to make a deletion in DMAP1 and only DMAP1. How can we distinguish between deletions in the left or right direction?

The trick is to use other genetic markers – mutations in other genes, to the left and to the right of DMAP1 that can be recombined onto the chromosome at the same time, during the same recombination event in the male germline that generates the deletion when the P element is mobilized. So this is what we did in our class (Figure 1): we crossed flies with the P element beside DMAP1 (P*) to flies with eye colour mutations on chromosome 2, and a source of transposase enzyme on chromosome 3. We collected male offspring that combined P* with the transposase source. Then we crossed those males to females with the recessive eye colour mutations cinnabar (cn) and brown (bw) on both homologues of chromosome 2.  Here’s the important thing: these eye colour genes are located on either side of DMAP1 and P*, so ultimately, we can select for deletions in one direction or the other by selecting for one eye colour phenotype or the other (this is also depicted in Movie 1). The males were heterozygous for the recessive eye colour mutations, and therefore had wild type (red) eye colour, and the females were homozygous for the mutant forms (which combine to yield a white eye colour – a subject for a later post; just accept this curiosity for now). Therefore any crossover in the males could be detected in the offspring as either mutant for cinnabar but normal for brown (bright orange eyes) or mutant for brown and normal for cinnabar (brown eyes).

DMAP1 male RC 04

Most of the progeny will have either normal red eyes (one wild type allele of each gene) or white eyes (mutant alleles of both genes). Hopefully Figure 1 will help you work through the chromosome shuffling that’s going on. Figure 2 is what it looks like when the crosses are written out in fly language:

Cross scheme

Since we wanted deletions in the direction of DMAP1 (and not CG33785) we kept any brown-eyed males and crossed them individually to a stock of flies containing the SM6a balancer chromosome (having the dominant Curly (Cy) mutation and the recessive cn mutation, along with a bunch of rearrangements that prevent further recombination, thus allowing us to maintain potentially useful deletions). Brown eyed males have a recombinant chromosome that is likely to have a deletion towards, hopefully into, possibly all the way through, the DMAP1 gene. We had to cross each brown-eyed male individually because theoretically, each male arose from the union of a paternal gamete (sperm) in which the desired recombination event took place, and an egg from the white eyed, cn,bw mother. Every F2 male, in other words, represents a gamete from the F1 father, in which the P element was induced to recombine.

Preston et al. found that about 1/3 of the recombinant chromosomes they tested had deletions. The deletions ranged in size from just a few base pairs to more than 100,000 base pairs, but the majority fell within about 2000 base pairs. This is good because the DMAP1 gene is about 1800 base pairs long, and we hope to delete most or all of the DMAP1 gene without deleting additional genes. I set up the cross to make the F1 males, and gave those males to groups of students in several successive Genetics Lab semesters. Students crossed those males en masse to cn, bw females, and then sorted through the F2 progeny for brown eyed flies. Those males, when found, were crossed INDIVIDUALLY to females with the multiply rearranged balancer for the second chromosome that was also marked with cn, and in the next generation, the F3, the recombinant chromosome bearing the brown allele and P* was recovered and balanced as a genetically uniform population of flies, or a stock. If the induced recombination event caused a lethal mutation, then the stock should remain balanced, all flies showing curly wings, with no brown-eyed homozygotes present. If the event produced a mutation that was not lethal, then brown-eyed homozygotes should show up in the stock.

There’s a lot to keep track of in this screen. Lots of different markers and mutations, understanding what balancers are and what they are good for, why some matings are en masse (lots of males and females together in a bottle or vial) and why others have to involve single males. After several semesters, we generated seven stocks carrying independently induced recombinant events that hopefully have deleted into DMAP1. Table 1At first glance (Table 1), we see that some stocks are lethal, and some are not, which could mean that DMAP1 encodes an essential function, and we have possibly generated an allelic series, ranging from complete lethality due to a large deletion that removes most or all of the coding region (a null), to perhaps a small change that simply reduces the expression of the gene (a hypomorph). But lots of other, less appealing results are also possible – the generation of an off-site hit in some other gene (that happens to be essential), or a complex rearrangement that will be awful to try an analyze. We have lots of work to do…so we had better get started!



One comment on “Screening for DMAP1 deletions

  1. Pingback: Scholar’s Week Spring 2016 | SmallScienceWorks

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