This term all the lab groups will contribute to a single project- Using male recombination (MR) to create directed deletions. The aim is to delete a gene called DMAP1 (a methylated DNA binding protein gene) for future work
In this project, we will use a strain of fruit flies that has a P element inserted between two genes: CG33786 and DMAP1. We are interested in DMAP1 because it encodes a protein that is part of a complex that binds to DNA and we suspect it may be involved in gene regulation. I will post some papers about this gene in the “resources” module. There is not very much known about it in flies. Most of the work involves mammalian cell culture.
When P elements attempt to move, breaks occur in the DNA on either side of them, and then the element moves, most often to a nearby location. Sometimes, a P element does not move quite correctly, breaks are made in the wrong positions and a crossover event takes place as a result (I will talk about this in lecture Tuesday Jan 21; there will be posted slides to look at in advance and some narrated power points to introduce you to P elements and how they are used in research). About 1/3 of the time a crossover event occurs, the DNA between the original P element position and the new position is deleted. The deletions made are called directed deletions because they all start at the original P element position and extend varying lengths away from it. If we set up our crosses correctly, we can tell which direction the deletion extends based on the genotype of the flies that result. Please note that the exact mechanism of this is not understood and we can’t make the P element make a mistake in transposing so that we can get a deletion. We simply have to create the right circumstances in which the P element will transpose, and when a mistake occurs, we will recognize it because crossover will happen at the same time. So we collect the flies that have a crossover chromosome, and then we’ll use molecular techniques (at the end of the semester) to see if there is a deletion in DMAP1 or not.
GENES WE ARE USING.
The P element we are interested in is on chromosome two. We will be using a mutant strain that has two “markers” on chromosome two: cn bw/cn bw. The combination of the cinnabar (cn) and brown (bw) mutations makes the eyes of the flies white (i.e. flies that are cn bw/cn bw have white eyes. Please note the way this genotype is written – what do you know about the two genes, based on this?). cn/cn alone makes the eyes a brilliant orange colour and bw/bw alone makes the eyes a dull brown colour. Flies with brown eyes also lack the pseudopupil, an area of darker pigmentation in the eye. (see below)
We will also be using a strain that has Sb, 2.3/TM3 Ser; Sb is the Stubble mutation; it is dominant and causes the bristles on the back of the fly to be about ½ the normal length. 2.3 is a wings clipped P element, which we use as a transposase source (see P element powerpoints in the week three module). Ser is the dominant Serrate mutation, which causes “bites” out of the wing margin. TM3 is the name of a balancer chromosome for chromosome 3. The P element stock has a P insertion between two genes, one of which is DMAP1 (see the powerpoint slides for more information). I am just calling it P*. The P element chromosome is homozygous viable so there is no mutant phenotype associated with it. The flies are mutant for the white gene, so they should have white eyes, but the P element has the white+ cDNA on it (white+ is the most commonly used reporter gene), so the eyes are pigmented. But the gene isn’t fully expressed, so the eyes are not quite wild type. They are more of a pinky-orange colour. The X chromosome in this stock also has a mutant yellow gene, so the flies are yellow in colour instead of the usual tan. You will have a chance to see all of these mutant phenotypes in the lab next week.
A note about…