DMAP1 complementation tests

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.)

Comp test ex

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….)ex comp full shade b&yNotice 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.

ex comp half shade b&y

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.

Df map detail 02

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:

Df comp y&b

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.

Ratio tableCrosses and ratiosAnd now here are the actual data (Figure 7):

DMAP1 full interse y&b

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?

KEEP&CHUCK

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!

Drosophila Lamin C mutations: Preamble

I have friends who pick complicated men. I pick complicated genes. As a PhD student, I worked on genes stuck in heterochromatin that tended to be HUGE (hundreds of thousands of base pairs, mostly introns, mostly repetitive DNA).  My last publication (eons ago in 2012) was about a gene that went and quintuplicated itself. For my post doc, I worked on Lamin C, an essential gene nested inside the intron of another essential gene called tout velu. What fun!

Lamin C is a nuclear envelope protein. In multicellular animals, the nucleus has a mesh-like structure that lines the inner surface of the nuclear envelope, made out of filamentous proteins called lamins. Humans have two main types of lamins, A types and B types, and the division of labour between them is still not all that clear, but B types seem to have a predominantly structural role while A type lamins do other things, some of which are very mysterious indeed. Long story short, A type lamins – which in humans are further subdivided into Lamin A and Lamin C – seem to be required inside the nucleus, (as well as the periphery), where they probably play a role controlling nuclear organization and gene expression – very interesting epigenetic stuff. Mutations in A type lamins lead to a very wide range of rare diseases that affect multiple tissues – muscular dystrophies, neuropathies, bone disease – perhaps because when mutant, these lamins upset developmental gene expression programs that commit cells to specific tissues or organs. Or perhaps the tissue is rendered less mechanically sound – a lot of the diseases are muscle disorders, and cardiomyopathies, which are tissues under mechanical stress. Jury is still out, but by far the most unusual disease caused by defects in A type lamins is a premature aging disorder called Hutchinson Gilford Progeria, where children age very rapidly, usually dying before they are teenagers. HBO broadcast a very compelling documentary about the life of one of these unfortunate kids called “Life according to Sam” – although the kid was so amazingly positive and bright that “unfortunate” almost doesn’t seem to apply…

The mechanism of this progeria is still not at all clear. How does a mutant nuclear envelope protein cause rapid and system-wide aging? Do the stem cells fail to replenish themselves? Is differential gene expression messed up? I don’t think any one knows.

So lamins are interesting. They are also very highly conserved  – in animals anyway. Plants seem to have something similar but via a separate evolutionary pathway – a kind of convergent evolution. Among animals, invertebrates tend to have only the B-type lamins, while vertebrates have the A and B types. EXCEPT that Drosophila has two types. There is the B type, in flies called Lamin Dm0 (strange name deriving from its initial discovery involving different forms in the cell cycle) and the A-type called, confusingly, Lamin C. Why Lamin C and not Lamin A? Has to do with something called a prenyl group, and the mechanics of lamin incorporation into the nuclear envelope. It is horrible to explain and I distinctly recall my colleagues’ eyes running and noses bleeding whenever I tried so I won’t go on about it just yet here. Suffice to say that lamins are either prenylated or not, which means they either have a pointy-branchy sticky carbohydrate membrane anchor attached to their C-terminus, or not, because sometimes it gets clipped off post-translationally, which is what happens to human Lamin A. Human Lamin C turns out to be an alternatively spliced version that skips the bit of the gene that encodes the signal to put the anchor on, so it is never prenylated. Drosophila Lamin C is like that too, the gene does not encode a prenyl anchor. And it is true that when you compare the physical location of Lamin C and Lamin Dm0 using confocal microscopy, Lamin C tends to be both at the periphery and in the interior; Lamin Dm0 is more concentrated at the envelope (see figure – sorry no scale bar but the (polytene) nuclei are each about 15-20 microns across – 2 different mags).Lam C & Dm0 confocal

So what is Drosophila Lamin C (dLamin C) doing inside the nucleus? Anything analagous to the human Lamin C?  Are fruit flies just more efficient than humans? No messy alternative splicing or post translational processing to remove the anchor. Turns out you don’t really need Lamin A at all…well, that is if you are a mouse (PMID:16511604).  Maybe the complicated and messy and disease-causing Lamin A is just an evolutionary boo boo that has to get mopped up post-translationally (potentially another example of what I like to call “unintelligent design”. )

Hence, my interest in Lamin C. Well, me and MANY other people. During my post doc I worked on generating a mutation in dLamin C (LamC) which turned out to be a bit tricky because it is nested inside this great big giant essential gene called tout velu (ttv: French for all “all fuzzy” – plays a role in Hedgehog diffusion and pattern formation…another story: PMID:9665133). So making a mutant meant generating a lesion in LamC without disrupting tout velu. Lam C gene regionHere is what it looks like in Flybase: the blue line ticks are 10kb apart so ttv  is a pretty enormous gene.

But that is all ancient history. We made nice mutations and characterized them and it was great fun. There are other mutants about but some of them are iffy – so I wanted to make a new one, using the same method – imprecise excision of a transposon hit. With mutants, the more, the merrier. Most importantly because if you want to use a mutant to study something biological, you have to worry about what else got buggered in the background when you made the mutant, and is that buggery causing the interesting biological effects you are seeing. So duplicating a given experiment with mutations in the same gene but from two different genetic backgrounds is really important.

First thing is to find a fly strain with a P element (transposon) inserted in the right place. There are lots, but finding one that affects LamC without affecting ttv is hard. I highlighted the two best choices below. G00158 is what we used all those years ago during my pos doc (PMID:15965247). They are both in LamC’s first intron, When I started here at WWU I chose EP(2)2199 which is no longer available (probably because it was unhappy and kept dying for reasons that will become clear). So that is where this story begins.

Lam C P hits