My Drosophila Story (2021 corrections)
The science of Genetics, was a hot topic during my undergraduate years at NYU. It was the late 1950’s and DNA had only recently been proven to be the genetic material. Then to everyone’s amazement Watson and Crick had figured out the beautifully logical and functional structure of that key macromolecule.
My interest in genetics grew when I found a vibrant group at Brooklyn College that used the yeast, Saccharomyces cerevisiae, to analyze aspects of crossing over, gene expression etc. So under the guidance of a faculty member of that team, I used this yeast for my Masters thesis. We shed some light on the synthesis of purines in that unicellular organism, using several mutants blocked at different steps in this pathway. (These mutants could be grown on purine supplemented medium.) The accumulated intermediate products, made just before those blocks, were various shades of red, and could be easily visualized by paper chromatography. Since all living organisms ultimately have a common ancestor, a metabolic pathway in any organism is usually similar to the same path in many others species. I don’t know how widely applicable the exact pathway we then elucidated really is, but I know of a rare human genetic condition in which an accumulation of an intermediary of purine synthesis products causes a devastating syndrome. I now regret that I didn’t have the wisdom then to pursue this fact. But I was at that time excited about getting ready to move to California, in pursuit of a PhD.
When one wants to study genetic mechanisms the choice of the subject organism is crucial. For example, Gregor Mendel, who invented the field of Genetics, could not have succeeded if the pea plants he studied did not have distinct traits located on different linkage systems (which we now know to be chromosomes); and the T.H. Morgan lab at Columbia University could not have demonstrated fundamental phenomena, like crossing over and the effect of radiation on mutation rates, without the tiny, prolific, Drosophila melanogaster flies (1), which produce a new generation in weeks and can be housed in small milk bottles with a layer of porridge.
After the DNA breakthrough, the organism used post prominently in genetic research was the prokaryote, Escherichia coli. With this unicellular organism, geneticists (now called molecular biologists) cracked the genetic code (2), and cell differentiation started to be elucidated by studying the changes this bacterium makes when it is grown on various media. But the vast changes cells that multicellular organisms make, which appear to be more permanent throughout the organisms lifetime (3), i.e. changes that underlie the process of embryonic development cannot be studied in unicellular organisms.
Embryology is a field with a long history that had used ingenious experiments, manipulating eggs and embryos of echinoderms and as well as vertebrates. Drosophila, with its complex morphology, ease of cultivation and genetics already elegantly studied, seemed to be ideal for studying the genetic control of development.
Luckily, Dr. James Fristrom had joined the UC Berkeley faculty a couple of years before I arrived on that campus and he accepted me as a graduate student. He had devised a method of isolating en masse the embryonic anlage (imaginal discs, 4) within fly larvae, so a biochemical approach to the study of their development became feasible.
At the time when I joined the Fristrom lab, ecdysone, the steroid hormone that triggers metamorphosis (the change from a larva to flying adult) in insects could only be isolated from huge quantities of larvae, and only one, well-endowed lab at Harvard University was able to accomplish this. Consequently, getting even small amounts of the purified substance was difficult. I happened to read a paper by a Czech scientist, Dr. Herout, reporting that a molecule, almost identical to ecdysone (5), was found in a common fern plant, and that its isolation from that source was relatively easy. Because of my Czech heritage I did not hesitate to write to Dr. Herout, who graciously sent us a large quantity of the hormone, in a plain white envelope. I think the fact that Czechoslovakia was then in the grips of a repressive Communist government had something to do with his magnanimous gift.
Having a large source of isolated target tissues (imaginal discs) as well as a good quantity the trigger hormone (ecdysterone, 6), I set out to study changes in RNA synthesis associated with metamorphosis. I showed that bulk RNA synthesis increases in isolated imaginal discs exposed to ecdysterone, and that the optimal ecdysterone concentration for that in vitro effect is within physiological range. Bulk chemical measurements can be affected by various factors, including dynamics of product stability and precursor availability, and I eliminated these possible influences in my theses, and proved that actual new synthesis was taking place. However, I did not even approach study of the activation of specific genes.
When we moved to Hawaii (7), I went see Dr. Elmo Hardy, who had been recommended to me by my professors at Berkeley. Dr. Hardy was at that time studying the insects endemic to the Hawaiian Islands. He in turn graciously urged me to go see Dr. Hampton Carson, who was famous for many studies of genetics and evolution, and who serendipitously was just at that time moving to the University of Hawaii to head the “Hawaiian Drosophila Project”. Dr. Carson listened to my history and showed me the beautiful giant polytene chromosomes he was studying. I proposed looking whether these chromosomes, when freshly dissected from larvae, would react to exposure to ecdysterone, and Dr. Carson arranged for me to become a member of his team.
In interphase (i.e. non-dividing) cells, chromosomes cannot be seen under a light microscope because at that stage the chromosomes are expanded into thin threads. But during cell division (mitosis), they become contracted, or packed up, to facilitate their exact apportionment into daughter cells. (This is when chromosomal shapes and sizes we know as the karyotype are produced.) The polytene chromosomes are an exception to the light microscopic invisibility of expanded, thread-like interphase chromosomes. Consisting of fibrous chromosomes made by a thousandfold replications of expanded DNA threads, polytene chromosomes are found in specific tissues in various organisms, notably Diptera or true flies (8).
In Berkeley the genetics graduate students and professors sometimes held informal get togethers. I recall one of these that featured Dr. Curt Stern, an intellectual son of the founder of Drosophila genetics, T.H. Morgan (9). Dr. Stern, an emeritus lecturer in Berkeley at that time, said: “if you had asked me in the 1920’s what I want for Christmas, I would have said: giant chromosomes in which you can see genes.” And that is in fact what giant polytene chromosomes really are. The thick and thin banding visible in these chromosomes appear to represent genes, which moreover sometimes appear differently active in various stages of development.
Polytene chromosomes had been first seen in the salivary glands of another fly in 1881, but it was not until the 1930’s when they were demonstrated in Drosophila, where giant polytene chromosomes are found in non-dividing, larval salivary glands (8). A crucial factor is that all the DNA replicates in these chromosomes are annealed together, i.e. exactly lined up. In addition each pair of homologous chromosomes (10) are united within each giant polytene chromosome.
Many species (11) of the tiny fruit fly, Drosophila, are found, distributed all over the world, and the banding patterns of their polytene chromosomes have been used to track their evolution. Although each Drosophila species appear to be related, there are many obvious, “scramblings” of the banding of their polytene chromosomes, i.e. translocations (the movement of a section of a chromosome to another place in the same or different chromosome) and inversions (the sequence of bands is moved “head to foot” so to speak). Such scrambling does not occur and mainly does not survive very often (for it must not interfere with the smooth functioning of the genetic material), and the chromosomal changes that do persist can be a way of tracing how the various species of Drosophila are related.
Nearly two thirds of the 1500 Drosophila species known worldwide, are endemic to the relatively tiny Hawaiian Islands. This astounding statistic is a consequence of the fact that the Hawaiian Archipelago was formed by volcanic action in the middle of the Pacific Ocean. It is the most isolated land mass in the world. Consequently, for a small fruit fly to reach it (in the past before human migration) was very hard. In fact it has been calculated that maybe only one gravid female was blown into the islands by a huge storm about 26 million years ago. Then on those originally sterile islands, the progeny of that pioneer fly became adapted to various habitats, including feeding on different secretions or on different parts of various plants, and their radiating adaptation was unimpeded by competition from closely related insects. Twenty six million is only about half the time during which Drosophila have been evolving on all the world’s continents, and this amount of time appears not to have been long enough for the Hawaiian Drosophila to develop more than only a relatively few chromosomal translocations and inversions. So as a result of their single source origin, many of the Hawaiian Drosophila are homosequential (i.e. they cannot be distinguished by the banding patterns of their polytene chromosomes). Nevertheless, even these homosequential Hawaiian Drosophila are different species, since when interbred (as can be done by raising two species together in bottles on plenty of food) they do not produce fertile offspring. But their divergent morphology and habits in the wild must still be due to different gene action, which should be demonstrable by studying the visible gene activity in their polytene chromosome, known as puffing.
In the late nineteenth century it was reported that polytene chromosomes, produce different puffs at various stages of development. Much later these puffs were characterized as RNA, therefore indicating they are products of activated genes. In Hawaii in 1970 I proposed to study the effect of ecdysterone on the puffing activity of isolated polytene chromosomes of late third instar larvae, of different Hawaiian Drosophilae.
Although I managed to observe only the short end of the X chromosome I made some interesting findings: I showed that one identical region can be activated in two different species, even though in one of these species the chromosomal region involved was inverted. Thus the activation mechanism may not be dependent on an adjacent regulator.
Most significantly (in my opinion) I also showed that there can be a difference in whether a puff is induced in two different species. To prove that this difference was not an artifact of the developmental stage of the individual larvae I used, I made (with the help of Dr. Ken Kaneshiro, who was a graduate student at the time) a hybrid between the two species involved. (Please note that the resulting interspecific hybrid could not metamorphose, but this was enough because all I needed was the late stage larva.) I found the end to the polytene X chromosome in that interspecific larva was not annealed and only one half of it responded to my ecdysterone treatment by puffing.
Since I had to stop (12) my research on the puffing of Drosophila polytene chromosomes, many investigators have used this system. For example, It was shown by Ulrich Clever in 1973 that ecdysterone can induce sequential puffing in polytene chromosomes. Dr. Ulrich had already shown previously that the RNA product of one gene is required to induce an RNA product of another gene by using the inhibitor streptomycin D.
During the fifty years that followed, new techniques (including micro-manipulation of the puffing products) have confirmed that the puffs are products of activated and strategically needed genes. Many of the puffs are mapped and given code names. But no one seems to have addressed the questions about what makes chromosome homologs anneal and why changes in gene coding can prevent this annealing, as I had observed.
We still do not fully understand the manipulations of DNA that occur naturally within our chromosomes. For example, it defies thermodynamics that the DNA molecule can replicate so quickly in vivo, since replication logically requires unwinding of the DNA helix. The study of polytene (and perhaps also lampbrush chromosomes, found in amphibian eggs) may give further clues to how DNA is arranged, activated, regulated, and replicated.
1. The little fruit fly, Drosohila melanogaster, was used during the first part of the twentieth century by T.H. Morgan and his illustrious students at Columbia University to reveal several basically important genetic laws, most notably linkage on the microscopically visible chromosomes. Consequently a whole library of genes was characterized and mapped for this, easily-handled organism.
2. It is astounding that the triplet genetic code, elucidated in E. coli is found in all living organisms; I mean really all: bacteria, plants, invertebrates and vertebrates, which includes, of course, human beings.
3. Almost all cells within a multicellular organism, barring mutation, have basically identical genetic instructions. This is guaranteed by the accuracy of mitosis. Yet obviously different genes are active in different cells, with their widely different morphologies and functions. ( I say that almost all cells have identical basic genetic sequences, because it is clear that vertebrate white blood cells, which produce antibodies, have variable DNA regions that code for different specific antibodies.)
4. It was known from earlier studies, using meticulous dissections, that the imaginal discs were preprogramed to become specific parts of the adult (i.e. imago).
5. The fern, Microsorum scolopendria , produces ecdysone and ecdysterone. The latter differs from ecdysone because it has one hydrogen side group replaced by a hydroxyl, but like ecdysone it triggers insect metamorphosis. I speculate that the advantage for plants, in producing these chemicals may be that it upsets the hormonal balance of insect larvae that feed on these plants, and this probably stops the predator insects’ development. Ecdysterone is now commercially available. I’m not sure if it is still isolated from botanical sources, which I understand are commonplace.
6. The trigger for metamorphosis is actually a shift in the balance of two insect hormones, ecdysone and juvenile hormone. When juvenile hormone concentration is greater than ecdysone, the insect larva molts into a larger larva, but when ecdysone concentration is greater than that of juvenile hormone metamorphosis occurs.
Interestingly ecdysone (and ecdysterone) is a steroid, thus related to the many steroid hormones involved in vertebrate development, and juvenile hormone is a fatty acid derivative, identical in structure to vitamin A. Later during my “cancer research years”, I showed that feeding vitamin A to a strain of mice that normally had a high incidence of breast cancer, significantly reduced that incidence.
7. We moved to Hawaii because my husband, Bob, received a post doctoral grant to study the evolution of the Hawaiian Honeycreepers, birds endemic to the Hawaiian Islands.
8. Giant polytene chromosomes have been found in other places and in other organisms beside flies. They appear to arise in tissues whose function is to produce an unusually high abundance of a particular substance.
9. Dr. Curt Stern had been in the 1920’s a postdoctoral student at the famous Columbia Fly room, founded and run by Dr. T. H. Morgan.
10. Each diploid cell contains one chromosome set from the paternal and one from the maternal parent. So, for example, there are two homologous number one chromosomes, etc.
11. A species is defined as a group of individuals that can interbreed, i.e. form viable offspring.
12. After we moved to Pittsburgh I missed an opportunity to continue my study of Drosophila. The illustrious Carnegie Mellon University allowed me to give a seminar at their research institute, because I attempted to get a job there. In my seminar, I made the mistake of sticking to my rather dull (now in my opinion) studies on the dynamics of bulk RNA synthesis that I had made for my PhD thesis. I completely ignored my polytene chromosome findings, which were relatively preliminary, but perhaps more significant.