DMAP1 Fertility Tests

So my colleague Kathleen and her inexhaustable supply of undergrads at Simon Fraser University in B.C., Canada, have generated seven potential DMAP1 lesions, using the male recombination screen described in the last post. Recall that in this screen, a P transposable element very close to the upstream gene CG33785 was induced to transpose in the male germline, hopefully generating a deletion in the direction of DMAP1, and thereby recombining a recessive brown eye colour mutation onto the same chromosome. Note here that four out of seven potential lesions produce viable brown-eyed (and therefore homozygous) flies. They look normal but are they really? In Figure 1, each potential mutant that produces homozygous progeny is tested for fertility. For each strain, four crosses, with virgin male and female parents, 3-4 of each sex (i.e, as close to identical conditions as possible). First, balanced males and females are crossed to each other (grey and black representing Cy males and females respectively – remember Cy is the dominant curly wing mutation that marks the SM6 balancer). Then, balanced males are crossed to homozygous females (which are dark blue in my colour scheme) – this tests the homozygous female fertility. Then the other way around: homozygous males (light blue) crossed to balanced females – this tests the homozygous males’ fertility. Finally, homozygous males and females to each other.

Fertility tests

The total number of flies for each cross exceeded 100, but I expressed the proportions of each genotype as a percentage of 100 in this graph. For balanced parents crossed to each other, the ratio of balanced to non balanced progeny (or blue shades to grey shades) should be 0.5.  (Think A/a X A/a, where A is Cy, and Cy/Cy is lethal. Half as many a/a to A/a, or 1/3 : 2/3). Note that doesn’t really happen except for 12-14 (and that ratio turns out to be 0.48).  For the two middle crosses, heterozygous males to homozygous females and the other way around (think A/a X a/a) half the progeny should be homozygous, and half should be balanced, or 1:1.  So you should see as much grey/black and light/dark blue for those crosses. Do you? Of course, homozygotes crossed to each other are depicted solely in the blue shades.

Clearly two of the potential mutants show sterility defects: 20-13 is male AND female sterile (no progeny at all for the second, third and fourth crosses), and 12-14 looks male sterile. Incidentally, in the very first post about this project, I yanked the ovaries out of 20-13/20-13 females, (look here), and asserted the testes didn’t look quite right either, so that’s consistent so far. (Could still be something in the background, but relish it for now!)

As for 20-11 and K2-13, the second and third crosses don’t produce quite the right ratios.  In addition, 20-11/20-11 parents produced approximately 70% fewer progeny than 20-11/SM6 parents, and for K2-13 that reduction was around 50%. So there might be viability and fertility issues for both lines.

In summary, 20-13 is homozygous male and female sterile, 12-14 is male sterile, 20-11 and K2-13 homozygotes may be semi fertile and perhaps even semi viable (particularly 20-11). But are these all mutations in the same gene? Stay tuned.

Fifty shades of Red: the Superhot history of PEV

Preamble to the preamble: this preamble consists of a rather leisurely perambulation through history. If historical context bores you, skip ahead to all of the amazing discoveries which at the time of writing this post I have yet to make. But if, like me, you like to try and climb inside the heads of the scientists who made these seminal discoveries, to try and see things through their eyes, with their knowledge, then read on! Most of the papers are publically available now, and the language is quite convoluted, but worth the effort. These (mostly) dead white males were mental contortionists…imagine how they built models of what was going on in what we now call epigenetics without the resolution our microscopes have today, let alone the knowledge of DNA and molecular biology. These folks are my heroes.

Position Effect Variegation. It just ROLLS off the tongue, doesn’t it? Haven’t you always dreamed of being able to expound knowledgeably about fluffy subjects like this at cocktail parties? Would you not become the life of the party? Doesn’t everyone want to know this stuff?

OK it is probably true that you won’t be pinned wriggling to the wall by people who want to know about position effect variegation (NOTE: they will more likely want to know about CLONING (the Dolly kind), GMO food, climate change and strange sexual practices in the animal kingdom. So make sure you have answers at the ready. Hmm. Subject for later blog.) But, understanding Position Effect Variegation, or PEV, is a very useful way of learning how chromatin structure affects gene expression, part of the revolution in epigenetics, which term has been bandied about of late in the press. PEV is a term coined way back in the second or third decade of last century, when almost anybody reading this (including yours truly, just FYI) were not even embryos; indeed, not even conceptions, real or imagined. Here are a couple of nice reviews of PEV with all the punchlines that include historical context: PMID: 18282501, PMID: 2127578.

Flemming and meIt all began with moose. That is, with German moose, which is to say, moss, a paper published in 1928 by the German Botanist Emil Heitz, who used Moose as a model organism for his cytological studies of the nucleus (for the teutonic among you: In I Jahrb Wiss Botanik, Vol. 69, 762 – 818  Das Heterochromatin der Moose). Cytology then was like molecular biology today – the really good cytologists were rock stars and stud muffins, and Heitz developed clever staining techniques to study the morphological transmogrifications that went on inside the nucleus during the cell cycle. It is remarkable, when you think about it, just what kind of dramatic contortions have to happen when a cell commits to mitosis (or meiosis). Heitz was particularly interested in the interphase nucleus – before all the drama of mitosis begins. Remember, this is 1928 – the chromosome theory of inheritance has been established (Sutton 1903: Biol. Bull 4:231-251) but what ARE chromosomes? Some ideas were bandied about (even DNA on the QT) but their behavior was mesmerizing, as it still is (sometimes I squash onion root tips for fun and stare at them for hours under the scope (Figure 1 – which is my image and which is Flemmings? Walther, that is, not Alexander of Penicillin. For more pretty pictures see here).

Among the most puzzling aspect of chromosomes was their constant appearance and disappearance during the cell cycle – from rod like structures that are easy to count and draw, to an amorphous mushy blur – even today my students don’t always believe me when I tell them the chromosomes are STILL THERE during interphase. (Thank goodness for the candy color chromosome painting techniques…take a look at the stunning 3D imagery in this paper PMID: 15839726). What Heitz noticed, especially looking at chromosomes during early prophase, or later in telophase, was that some portions of the nucleus, in fact the chromosomes themselves, when they began to be visible during early prophase, were diffuse and stained less intensely – he called these regions collectively euchromatic, whereas other portions were stubbornly dark – heterochromatic. One drawing from his 1928 paper is shown in Figure 2. Heitz chromosHeitz has colored the heterochromatin black; note that “m” is perpetually dark – a sex chromosome? He examined chromosomes from multiple plant species, and then insects as well, various species of Drosophila, including D. melanogaster, and his results were both reproducible and independent of artifact.

And so begins a flourishing analysis of the “longitudinal differentiation of chromosomes” – which is to say, how exactly are chromosomes constructed? If you were to dissect a chromosome out of the nucleus, and lay it shimmering in a petri dish, what would you see? Well, nothing of course, because that kind of dissection is actually impossible (almost – here’s a paper about microdissecting fly chromosomes: PMID: 2587252, and human chromosomes: PMID: 1282430) but if you COULD lay out a chromosome in a dish, what would you see? Would the compaction – the degree of coiling – be equal throughout the chromosome or unequal? Unequal, was Heitz’ guess and of course he was right. Chromosomes are highly compacted, compressed in certain regions – the heterochromatic regions around the centromeres and telomeres – and more open and loosely coiled in between – euchromatin. Other remarkable conclusions – Heitz distinguished between constitutive heterochromatin – that which remains condensed apparently throughout the cell cycle – and facultative heterochromatin, that which appears and disappears under different circumstances – notably one of the sex chromosomes (already a heated topic of debate a decade earlier including at least one dead white female – Nettie Stevens – among many dead white males – another story) and the nucleolar region (a prominent nuclear component already observed some seventy years earlier – yet another story: Montgomery 1898).

Heitz was well aware of Thomas Hunt Morgan’s work on Drosophila genetics (in the United States – the beginnings of Drosophila as a genetic model….rare to find any fly people today who aren’t geneticists deep down) and thus could compare his cytological findings with the genetic maps and linkage groups that were being churned out of Morgan’s lab by his army of insufferably prodigious students (Nettie Stevens, Calvin Bridges, Alfred Sturtevant among them). Heterochromatin was presumed to be gene-poor, euchromatin, gene rich, at least for flies. Euchromatin is gene rich across taxa, certainly, but as for heterochromatin, I know from personal experience (my Ph.D. in fact) that heterochromatin is not a hospitable place for genes to be, as we shall see.

And heterochromatin was soon discovered to be responsible for a “group of distinctly peculiar variants” in Drosophila, that variegated, in other words, gave a variable phenotype over a particular tissue. The most dramatic phenotypes involved eye color: an otherwise wild type red eye color gene would manifest as a mottled combination of red and white ommatidia (the individual photoreceptor units in the fruitfly’s compound eye). These mutations appeared among an amazing panoply of mutations induced by radiation – a discovery made by Herman Joseph Muller (author of the above “distinctly peculiar” quote), another of Morgan’s insufferably prodigious former students. Muller generated more mutated strains of flies in a matter of a few weeks using X rays than had been made since research using Drosophila had begun two decades earlier. He published a landmark paper in 1930, cataloging and describing the mutations he found. This paper is special because the peculiar variants had to be viewed in color. No fancy schmanzy microscope cameras in those days so instead, the specimens were drawn, and in the case of Figure 3 and 4, painted, with great accuracy and detail (from J. Genet. July 1930, Vol. 22, Issue 3). Muller's eversportsNote the fly in figure 3 has notched wings – the strain from which she arose originally had wild type (red) eyes, but Muller thought that perhaps the notch mutation resulted from an X-ray induced deficiency that removed both the notch gene (yielding the characteristic dominant wing notching) AND the neighbouring recessive red eye color gene as well (there had been precedents for this). So he crossed flies from this strain with another strain that was mutant for the red eye color gene (had white eyes) and expected to see white-eyed notched-winged progeny, but instead he got this fly – notched wings, but a mottling of red and white ommatidia in the compound eye. Go figure. In Muller’s words: “To the great surprise of the writer, the Notch winged offspring of this cross had neither white nor normal red eyes nor even eyes of any uniform intermediate colour (spelled English style HAH). They had mottled eyes, and exhibited various grades and sizes of lighter and darker areas…” Figure 4 shows details of various mottled eye colour phenotypes – including the more subtle and headache-inducing (I know) variegations of red on red.

Variegated phenotypes were at once stable and unstable. They were stable in the sense that once established, they did not revert back to wild type. This contrasted with the findings of other mottled phenotypes in other model organisms at the time, notably, the “mutable loci in Maize” observed as early as 1917 (Emerson PMID: 17245873) and so elegantly dissected by Barbara McClintock in the 1940’s and 50’s (PMID: 15430309). These unstable phenotypes involved transposable elements: pieces of DNA that could move from place to place in the genome. But Morgan’s mottled phenotypes behaved differently. He described them as “eversporting” (PMC1085688), a curious terminological throwback to the history of, well, natural history – even Charles Darwin used this term. “Sports” are varieties, usually very obvious strange variants, described by farmers, gardeners, pigeon fanciers and natural historians, before the genetic basis got hooked up with the term “mutation.” Muller’s “eversporting” mottled variants were unstable within the tissues they affected – so the pattern of mottling varied between individuals carrying the same mutation – but were stable once established in a strain, essentially forever.

Muller made two important observations: these ever sporting variants were recessive to the wild type allele (but dominant to a loss of function allele) and they all correlated with chromosome rearrangements, arising from treatment with X rays (hence he called them “eversporting displacements”). Muller’s expressive efforts to explain how the eye color gene could function then not function as the eye developed in an individual with this phenotype are fascinating – particularly in light of how chromosomes were thought to be constructed – remember – no DNA, no proteins, no sense of how this substance was organized and propagated.

Eversporting mutations were very puzzling to geneticists, which is to say, fascinating to the point of obsession, and I like this conclusion from a paper by Jack Schultz (no relation) and Theodosius Dobzhansky (he of the nothing-in-biology-makes-sense-except-in-the-light-of-evolution adage): “The data presented are consistent with the hypothesis that the behavior of genes is a function of their position in the chromosome, that is, depends upon their relation to other genes.” (PMID: 17246728). So this is what position effect variegation means – a gene’s expression (its phenotype) variegates (varies) depending on its position in the chromosome. Note that this is an epigenetic (“above genetic”) effect: only the location of the gene is different: in modern terms, the sequence of the variegating allele is unchanged.

Thus was born the genetic analysis of chromatin structure. The first reports that the mottling might be in some way connected with heterochromatin came in 1933 (PMC1085890) and then later in 1936 (PMC1076699). The first paper (Gowen and Gay 1933) has a very athletic title: “Eversporting as a function of the Y chromosome in Drosophila”.  Flies are heterogametic – which means they produce gametes with different sex chromosomes (Y-bearing, or X-bearing) just like we do. And like us also, (and indeed many heterogametic species) the Y chromosome is largely heterochromatic. Unlike us, it is also LARGE – almost the same size as the X (the Y chromosome in humans is much smaller than the X) Also very much UNLIKE us, whereas a Y chromosome determines maleness in humans, it is the number of autosomes to sex chromosomes that determines sex in flies. Thus, XY is male in humans and flies (in humans because of the Y, in flies because for any given autosome pair, there is only one X). XX is female in both flies and humans, but XO is female in humans (Turner syndrome), but male in flies (still one X per any given autosome pair), and XXY is FEMALE in flies (2 X’s for any given autosome pair) but male in humans (Kleinfelter Syndrome, in fact). This digression about sex determination is necessary to understand the findings in Gowen and Gay’s 1933 paper.

So here is a quote from Gowen and Gay paper: “On cytological examination of oogonial divisions, mottled-2 females are found to have typical chromosome figures, but every figure from red-eyed females contains an extra chromosome, which looks like a Y” (my emphasis). First, cytological inspection is usually how the kinds of rearrangements producing the eversporting variants were characterized. Second, mottled-2 is one of Muller’s eversporting red and white-eyed mosaic strains. And third, when mottle-eyed female fruitflies are genetically manipulated so they receive a Y chromosome (while retaining their two X’s), their eyes are no longer mottled, but red, like wild type. These females would have genotype w/w[m2]/Y, where w = a loss of function allele in the red eye color gene, w[m2] the rearranged chromosome bearing the eversporting red eye color variant which is wildtype for the red eye gene, but relocated by rearrangement, and Y = the Y chromosome (remember, these flies are female).  Yet these flies looked like they had perfectly normal (wild type) red eye color genes. In other words, the presence of a Y chromosome SUPRESSES the mottled phenotype, making it look MORE wild type (more red eye color pigment). It is almost as if there is something about the Y chromosome that sucks out (in proper parlance – titrates) the variegating substance, whatever it is, away from the red eyed gene locus. But I am getting ahead of myself.

The effect of supernumery Y chromosomes was demonstrated for several genes that variegated, and in 1936 Jack Schultz (no relation) attempted to extend the argument to other so-called “inert” regions of the genome. General understanding of a mechanism was still wanting: did the loss of eye color gene expression result from a loss of the gene itself, or a suppression of the gene’s function? Who knew? Here’s a quote: “…the proportion of “mutant” to wild type tissue, depends upon a quantitative relation between active and “inert” chromosome regions. In addition, however, the “inert” regions are involved in the rearrangements themselves. Thus both the chromosome structure associated with variegation and the extent of variegation are related to the “inert” chromosome regions.” Schultz correctly postulated that variegating genes were interacting in some way with blocks of heterochromatin into which or near which they had been relocated – he and others found that these genes had to be close to a chromosome rearrangement breakpoint that brought them into proximity to the “inert” regions. Also, that there had to be a balance between the active vs inert portions in the nucleus, and that variegation occurs when that balance is upset. This is reminiscent of a mass action model proposed much later (PMID: 3146523). He also proposes, in a footnote, to adopt Heitz’ terminology: “The terminology is obviously outworn: “inert” is certainly a misnomer. Heitz’s use of “heterochromatin” and “euchromatin'”‘ for “inert” and active regions is probably better.” And so it came to pass.

MetropoleThe interwar period – 1920’s and 30’s – is fascinating. Devastating, apocalyptic, hedonistic…some of the greatest literature, art, architecture, music came from this period. (The Lempicka image is a photo of an enormous poster I acquired after attending one of the best exhibits in my life – Montreal 1991 – each room exhibiting art, architecture, design from different major cities during this period). But for the health care, I think I would enjoy a visit to this time (so long as I was provided with enormous wealth).

All the work I’ve described took place during this period, and by the time WW2 had begun, Morgan and his (now grown up) prodigies were investigating background and environmental effects that modified the eversporting phenotypes. PEV became a tool to dissect “…the developmental influences that operate in the development of the compound eye” or any other tissue susceptible to variegation (there were lots – wing margins, bristle patterns etc.) Extra heterochromatin, in the form of Y chromosomes for example, we’ve discussed. But a pronounced temperature effect was also observed – higher temperatures suppressed the mottled phenotype (more wild type, more red), lower temps enhanced (more mottled, less red). It is very interesting reading this series of papers – all found in the Carnegie Institution Yearbooks for this period, beautifully digitized here.  Herein, the hypothesis that mottled tissue portions showing the mutant phenotype represent somatic loss of the relevant genes during development was addressed (not the case). So, too, the stoichiometric balance of heterochromatic vs euchromatic factors. Jack Schultz (no relation) undertook a screen for “modifiers of the grade of variegation” reasoning that there might be so-called “interstitial” heterochromatin – pockets of heterochromatin interspersed in the euchromatic arms of the chromosomes, deletion of which would modify variegation as loss and gain of Y chromosomes did. He was right about the interstitial heterochromatin (now called intercalary heterochromatin PMID: 11862456) but more than likely his secondary mutations (14 suppressors and 58 enhancers) that modified PEV were hits in genes we now know encode proteins important in establishing and maintaining heterochromatin. Of especial interest is Schultz’s analysis of the light gene – a mutation that yields a pale brownish red colored eye (as opposed to the normal bright brick red color – we fly pushers can distinguish considerably more than fifty shades of red). This gene is located in heterochromatin, and Schultz observed that it appeared to behave in the opposite way when compared with a euchromatic gene like the wild type red eye-color gene. Schultz discovered a weird phenomenon we still have not adequately explained today, which is that those few unfortunate genes stuck in heterochromatin seem to have adapted to the compressed, transcriptionally repressive environment, indeed they NEED it to function properly, and so what might suppress variegation in euchromatin enhances variegation in heterochromatin, and v.v. (I am reliving prenatal traumas here because this is what I studied for my Ph.D. Here is a classic review: PMID:8825487) There are many tantalizing findings in these Carnegie Institution papers: in addition to environmental effects on PEV and weirdo heterochromatic genes, the authors pondered parent-of-origin effects on PEV (today we may ask: PEV somehow connected to imprinting? PMID: 10101173 – and try this blog for an interesting overview the genetics of imprinting) the nucleic acid vs. protein nature of chromosomes (is heterochromatin maybe connected to RNA? NOT so far from the truth as we shall see) and so on.

But I am getting ahead of myself again. So now let’s skip over the war (such a boon for physicists – less so for Drosophila geneticists) and the paradigm shifting discoveries of DNA structure and the genetic code, and hit the 1960’s, man, where PEV got its groove back. Janice Spofford (a LIVE white female) published the first detailed genetic analysis of PEV (PMID: 6082620, PMID: 17248449) and a classic review (in a book – email me if you would like the reference) that is still used in publications today. Spofford picked up where Schultz and co. left off, mapped and characterized in some detail a third chromosome Suppressor of variegation ( which she called a “Suvar”). The main difference by this point is the preponderance of fabulously useful rearrangements that allowed her to maintain mutant lines, duplicate regions of the genome, move bits of heterochromatin into euchromatin etc. A panoply of what started out as X-ray induced peculiarities now serviceable genetic tools.PEV fig After Spofford’s work, a bit of a hiatus, and then in the 1980’s and 90’s a flurry of enhancer-suppressor screens using chemical and transposable element mutagens (PMID: 6795427, PMID: 2501647, PMID: 8382174 and others).These kinds of screens are very popular with geneticists – the idea being that there are probably secondary mutations that can be induced in the background of a variegating allele that can ENHANCE the mottled phenotype (ie. make it more extreme, more mutant white, less normal red eye pigment) or SUPPRESS the mottled phenotype (more red, less white). Map and identify the secondary mutations, identify the genes involved, and build a more detailed mechanistic picture of what is going on. Figure 5 is my artistic attempt to diagram the effects of these screens, using Muller’s white[mottled 4] rearrangement that moves the normally distal (telomeric) position of the red eye color gene proximally right next to heterochromatin. In this position, as the individual cells differentiate during development in the embryonic eye, the red eye color gene might be shut off by encroaching heterochromatin, (leading to white ommatidia) or escape silencing from the heterochromatin (leading to various distinct shades of red in the adult eye). The result is a mosiac of red(s) and white eye colour. Note this model suggests the heterochromatic border acts something like a tidal front, that ebbs and flows in development. Here are a couple of useful reviews: PMID: 9610404, PMID: 12524009.

By the 1990’s somewhere between 50 and 150 enhancers and suppressors of variegation had been mapped (big range because definitions varied but the point is, MANY genetic changes can modify PEV). Also by this time, the revolution in recombinant DNA technology had taken place, a HUGE tectonic shift for all biological science I am just skipping over here (really important, look here). This revolution meant that it was now possible to isolate the sequences of the genes responsible for modifying PEV, purify the proteins, test for interactions and so on. Huge discoveries, but best of all, these modifiers turn out to be highly conserved throughout taxa. Thus, the obscure eversporting spotty eyes of fruit flies end up providing the groundwork for understanding the epigenetic regulation of gene expression in all eukaryotic organisms, plants and animals.

The field has truly exploded. What I have written to this point, in this very long blog entry, represents the trunk of a massively branching tree of new information. Probably impossible at this point to summarize, but try reading this paragraph out loud in one breath, you might get an idea of how enormous this field has become, and how rapidly it has progressed. So for example: Suvar2-5 encodes a protein called Heterochromatin Protein 1 (PMID: 3099166), that binds to a particular residue on the N-terminal tail of Histone H3 that has been methylated (PMID: 11242053). The methyl group is put there by an enzyme called Suvar3-9, after a histone deacetylase encoded by another Suvar removes an acetyl group from the same amino acid residue, and since HP1 interacts with itself, this chain of events sets up a spreading tendency that compresses chromatin into a heterochromatic state (PMID: 11283354). In fact methylation is one of several post-translational modifications of specific residues in the N-terminal tails of histones that seem to create a kind of chromatin Braille that protein complexes can “read”, compressing the chromatin fiber for silencing (by reducing access to DNA sequences to which transcriptional activators bind) or opening the fiber up, loosening chromatin structure (to facilitate gene expression). These post translational modifications have been collectively dubbed a “histone code”(PMID: 11498575), the interpretation of which helps to explain things like developmental gene expression (how are genes turned on and off in the highly concerted manner required for the differentiation of cells and tissues), transcriptional memory (how does a genome remember which genes are on or off for a given tissue, through the contortions of mitosis), imprinting (parent of origin effects), centromere identity and chromosome segregation, even the early musings that RNA was playing a role have been borne out (though the original hypotheses could not have envisioned RNA interference targeting protein complexes to heterochromatin PMID: 12215653), and all this, the proteins and the mechanisms, all this is conserved – PMID: 11242053, PMID: 10202156, PMID: 8505380, PMID: 10581035 honestly I could go on for several pages with a list of PMIDs but here is a not too ancient review instead: PMID: 17173056.

These and other things I would love to go on about: the behavior of modifiers has shown that the heterochromatin at telomeres is somehow different from the blocks that surround the centromeres, a phenomenon known as telomeric position effect or TEV (PMID: 11293791, PMID: 12663532). There are modifers of PEV that have led to the discovery of boundary elements in the genome – sequences and protein complexes that control the spread of heterochromatin and effectively divide the genome into different transcriptional domains (PMID: 18082602, PMID: 11700282). There is a dominant PEV allele caused by a chunk of heterochromatin inserted right next to the brown gene (another eye color gene) that exerts its dominant effect by sticking to the wild type brown allele on the other homolog and dragging it into a repressive nuclear compartment (PMID: 8646782, PMID: 7672573). Indeed, another Suvar appears to organize chromosomes in the interphase nucleus (PMID: 11390354), and I am not sure we yet have any idea about those weirdo genes trapped in heterochromatin that require an apparently repressive environment to function (PMID: 16690158). All in all, the arcane intricacies of position effect variegation have unlocked some of the deepest mysteries of epigenetics – how a gene’s position in a chromosome, and that chromosome’s place in the nucleus, and the structure of the nucleus itself work together to orchestrate that magnificent generation of complexity from embryonic protoplam, a story of genetic and epigenetic regulation that continues to unfold today,even as you read this blog.

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