And now time for Ye Olde Fashionede Geneticse. We have 7 lines of flies, each a potential mutation in DMAP1. Four produce homozygous flies with variable levels of viability and fertility and three are lethal (see here). Are they all lesions in the same gene? We expect so, perhaps even more than we might from a standard mutagenesis screen using a chemical mutagen like EMS, because we used a mutator P element so close to our target. But still, all pairwise combinations of crosses between all the lines are necessary, resulting in a grid that is symmetrical on either side of a line separating reciprocal crosses – that is, the same cross repeated with the contributing parents’ gender reversed.
Let’s consider an example of a simple complementation cross assuming five independent mutant lines that are all recessive lethal (so they are all balanced – remember the balancer carries a dominant visible marker that is recessive lethal). A PLUS sign indicates the cross produces transheterozygous progeny that are therefore not balanced: mutant 1/mutant 2. This means the mutations are in different genes, and so the progeny have wild type alleles for the two different genes, and so they complement each other. The genotype of the progeny is written as:
m1+ m2 / m1 m2+
or more simply (if obliquely):
+ m2 / m1 +
(If an allele is wild type we tend to make the symbol vanish and just leave the + sign, like the Cheshire cat’s smile).
If the alleles fail to complement, indicated by a MINUS sign, then only balanced progeny are obtained, arguing the independently isolated mutant alleles are in fact in the same gene. In Figure 1, note that mutant 1 fails to complement mutant 2, but complements all the other mutants. So mutant 1 and mutant 2 are in two different genes; mutant 2 has only one allele, and mutant 1 has 4 alleles (mutant 1, 3, 4 and 5.)
Another way to visualize this is by using colour. Yellow for failure to complement (alleles of the same gene) and blue for complementation (alleles of different genes) as shown in Figure 2. I chose these colors partly because I get to make what looks like a reverse Swedish flag, and partly to help those with red green colour blindness (but not people with blue yellow colour blindness which is far more rare – highly unlikely that the well over three people reading this blog are afflicted….)Notice the symmetry. Usually, only half the crosses are done, in one direction only, if no parent-of-origin effects are suspected. What are parent-of-origin effects? An example is imprinting, discussed in an earlier post. Something that causes the expression of otherwise identical alleles to change, depending on the sex of the parent from which they were inherited. If no parent-of-origin effects are suspected (often a rather rash conclusion), only half the cells in the table are provided with data, the undescribed cells on the flip side of the line of symmetry are assumed to be the same, as shown for our hypothetical example in Figure 3.
So what about our DMAP1 mutants? The first and most important crosses to carry out use a different genetic background with a known lesion in the DMAP1 region. Why? Look here for an explanation of the importance of genetic backgrounds.
My colleague Kathleen Fitzpatrick ordered in some fly stocks with deficiencies in the region – molecularly or cytologically defined deletions of genetic material. Since these deficiencies remove many essential genes, they are usually recessive lethal, and so are balanced. Kathleen ordered two different deficiencies (two different genetic backgrounds) that remove DMAP1, and one that does not, but which is located very near DMAP1. Take a look at this map from Flybase: the regions DELETED by the deficiencies are indicated by red bars.
The three relevant deficiencies (indicated with asterisks) are Df(404) (DMAP1 still present), Df(403) and Df(701), (both of which delete DMAP1, but were generated from different screens and so have different genetic backgrounds). I crossed all the putative DMAP1 lethals to each other; Kathleen introduced the deficiencies. Figure 5:
So here is the first surprise (and inevitable disappointment). Remember that YELLOW means failure to complement, and BLUE complements. Yellow all down the diagonal as we expect – all the stocks involved in these crosses have recessive lethal mutations or deletions. First look at the inter se crosses between the putative DMAP lesions. Notice that 10-14 complements the other two putative DMAP1 lesions – 22-13 and K-14 – which fail to complement each other, so there must be hits in at least two different complementation groups, or genes, here. Already we have information showing that at least a subset of our putative deletions are unlikely to be in DMAP1. Buggah.
The cross to the deficiencies tells the tale, however. All three putative DMAP1 lesions we have made complement the deficiency that does NOT remove DMAP1 (+DMAP1) but only 10-14 fails to complement the deficiencies that remove DMAP1 (-DMAP1). Notice I have blue hatching to indicate results with K-14. These crosses were not actually done, but since K-14 complements 10-14, and fails to complement 22-13, I suspect K-14 carries the same background lethal as 22-13 (which is odd, because they were isolated in different years, so are bound to be independent events). But it looks like only 10-14, of the three lethal putative DMAP1 male recombination mutants, is actually likely to be a lesion in DMAP1. Which is better than nothing.
What about parent-of-origin effects? DMAP1 stands for DNA methyltransferase 1-associated protein, and DNA methylation plays a crucial role in imprinting, which is an evolutionarily conserved parent-of-origin effect. So we developed a more complicated complementation grid – somewhat incomplete – that shows data from crosses in both directions. Also, since this test includes potential DMAP1 mutants that produce homozygous progeny, some with fertility issues, I have altered the colouring scheme to reveal potential hypomorphic effects, assuming a reduction in DMAP1 activity, as opposed to a complete loss on function – which we assume to be lethal (see here for argument). Remember that yellow means failure to complement, and blue means full complementation. Use Table 1 to follow how variations in these colours mean variations in viability. The cut-offs are arbitrary, simply based on my experience. For every cross, I scored (counted) at least 100 progeny flies. If you are unsure of where the ratios come from, study Figure 6.
So it looks like 10-14 is the closest thing we have to a lesion in DMAP1. The other lethals are suspect, and should be chucked. Since the original P hit in DMAP1, used for the male recombination scheme, was not itself a lethal, the lethal lines that complement the deficiencies which take out DMAP1 must have incurred a second hit during the male recombination scheme. That means that the lethal lines 22-13 and K-14 share the same newly induced background lethal. As I mentioned, and you might suspect from the naming system for these mutants, they were isolated in different years (2013 and 2014). So the same lethal was generated twice! I am not a big fan of coincidence, so something interesting (but annoying) happened here. But since DMAP1 wasn’t apparently involved, we really should abandon it. Buggah.
What about the non lethals? 20-11 and K2-13 sort of fail to complement each other. Moreover, 20-11 and 10-14 – our putative DMAP1 lethal – also sort of fail to complement, but only in one direction, when the lethal comes in from the male parent. So maybe we do have a hypomorph here (20-11) that supports an argument for a DMAP1 function in a parent-of-origin effect (like imprinting). To make things messier (why not), note that K2-13 also partly fails to complement 10-14, but in the other direction that we see for 20-11. (K2-13 also partly fails to complement 12-14, which itself pretty much complements 10-14. This is the sort of shop talk that drives normal people nuts). What does that mean? Dunno. I scored 100 flies total for each cross – the numbers (and therefore shading) might be slightly different if a larger number had been scored. Sample size, statistics etc.
So if I were in a chucking mood (which I rarely am, being something of a fly hoarder), which stocks should I chuck, and which should I keep?
Of course I should simply keep 10-14 and possibly 20-11. Alas, all the interesting sterility issues must now be revisited….that wonderful messed-up ovary picture I took was for 20-13, which steadfastly complements everything.
Well it was nice while it lasted.
Now what? We have pretty much reached the limits of classical genetics. We could cross our 10-14 lesion to increasing numbers of deficiencies to narrow down cytologically where our lesion is. We could do some cytology to look at chromosomes, to try to visualize the actual nature of the lesion. All these things we might have done twenty, thirty years ago. But at this point, we have to go molecular, which is great because we can drill down in much more detail, but also not so great because it costs money.
The best thing would be to do a Southern blot at this point. A Southern is a procedure where genomic DNA is fractionated by cutting it up in a defined manner (using a restriction enzyme that cuts at a specific DNA sequence) and separating out all the fragments by size in a gel. The gel is then blotted with a nylon membrane and the pattern of fragments is transferred to the nylon membrane by capillary action. The membrane is then washed with a labeled piece of DNA (a probe) that matches the region containing DMAP1 (for instance, the probe could be a DNA copy of the mRNA). The probe is labeled with something that can be visualized (colorimetric, radioactivity etc) and the pattern of bands analyzed. This method could tell us (1) if the region containing DMAP1 has been disrupted at the molecular level and (2) what the nature of that disruption might be. It could be a complete deletion of the coding sequence for DMAP1, or partial. Look here for a description from Ed Southern who developed the method in 1975. Initially he couldn’t get the work published, so scribbled it on an envelope for colleagues who really wanted to use it. No blogs then.
But Southerns are expensive. You need labeling materials, special nylon membranes, masses of chemicals etc. Fortunately, there is a cheaper alternative (though it does not give as much information) and that is PCR – polymerase chain reaction.
PCR is the one molecular acronym my freshman biology students usually recognize (outside of DNA), because it shows up in CSI etc. Yey, for science in crime shows (a great source of boo boo material for my exams) but it takes a solid understanding of how DNA replication works to fully appreciate the power of this highly efficient technique. It is also a method that doesn’t have to cost a ton of money. So that’s where we will head. Time to get down to the DNA, and characterize the nature of the 10-14 lesion in DMAP1!