Yuichiro Hiraizumi and Forty Years of Segregation Distortion

Corresponding author: Barry Ganetzky

ONE cannot help but admire a gene as fiendishly clever as Segregation distorter (Sd). Not only has it figured out a way to cheat the system, but it is extraordinarily good at what it does. It violates the fundamental principle of Mendelian inheritance regularly and with remarkable potency by effectively eliminating the competition. And it has managed to keep its modus operandi a rather well-held secret for 40 years. With the recent identification of the Sd gene product as a mutated version of one of the key players in nuclear transport, the end game may at last be in sight (MERRILL et al. 1999 ). But it would be a mistake to underestimate this wily prey.

The first published report describing SD chromosomes appeared in the pages of GENETICS almost exactly 40 years ago (SANDLER et al. 1959 ). It was the first of a series of eight papers published over the next several years that represented the outcome of a very productive collaboration between Larry Sandler and Yuichiro Hiraizumi. There was much serendipity in the inception of these studies. In 1957 Sandler and Ed Novitski published a paper in which they coined the phrase meiotic drive (SANDLER and NOVITSKI 1957 ). In their usage, meiotic drive referred to any alteration of the normal meiotic process that resulted in the excess transmission of one allele over its alternative from a heterozygote. The article was largely a theoretical consideration of the evolutionary implications of meiotic drive in natural populations. Sandler had applied for and obtained a postdoctoral fellowship to work in the laboratory of James F. Crow. But initially he was not planning to work on meiotic drive. Instead, as far as I can tell from ancient correspondence retrieved from the files of Professor Crow, he was planning to pursue some arcane problem analyzing exchange in reversed acrocentric compound-X chromosomes.

Meanwhile, at about the same time that Sandler and Novitski published their article, Hiraizumi had just joined Crow's laboratory as a new graduate student. One of Crow's previous superstars, Motoo Kimura, had finished up his degree in 1956 and promised to find an appropriate successor upon his return to Japan. He found Hiraizumi. (This system of self-replacement has much to commend it. It seems most unfortunate to me that it has never operated like that in my own laboratory.) Hiraizumi was not thinking about meiotic drive either. He soon embarked on a thesis project aimed at determining whether "recessive" deleterious alleles in natural populations of Drosophila melanogaster are completely recessive, or whether they have sufficient dominance that selection acts primarily on heterozygotes (HIRAIZUMI and CROW 1960 ). The experiments consisted of extracting recessive lethals and semilethals from natural populations in Madison and measuring their heterozygous effects on preadult mortality, rate of development, longevity, and fertility. Numerous testcrosses of +/cn bw males to homozygous cn bw females were performed.

Among the several hundred chromosomes analyzed by Hiraizumi, six were rather remarkable in that heterozygous males produced red-eyed (+/cn bw) progeny almost exclusively. Very likely, most investigators would have decided that some error in the crosses had been made and discarded those few wayward cultures. Fortunately, Hiraizumi's penchant for detail was present from the beginning, and he found these chromosomes impossible to ignore. Thus, he discovered SD chromosomes. Another bit of serendipity was the use of the cn bw chromosome as the marked homolog in his experiments. The ease of scoring the white-eyed phenotype of cn bw homozygotes made this chromosome particularly useful for the studies Hiraizumi was undertaking. There is no way he could have known at the time that cn bw is rather sensitive to the action of SD (and is still the preferred tester chromosome in most crosses involving SD), whereas many other marked laboratory chromosomes are relatively insensitive. If he had used a differ-ent marked homolog, Hiraizumi might have missed SD chromosomes altogether. It is impossible to say how many other investigators before Hiraizumi failed to detect the presence of SD in natural populations either because they were using homologs insensitive to the action of SD or simply because they missed the grossly aberrant transmission ratios, but it seems very probable that this happened more than once.

In any case, the stage was now set for Hiraizumi and Sandler to embark on the detailed characterization of the first meiotic drive system to be identified in Drosophila melanogaster. The details of exactly how the team of Hiraizumi and Sandler operated in carrying out their collaboration have not survived, even anecdotally, among those who shared the same laboratory space. But it must have been an interesting combination—Sandler, large, brash, loud, and domineering and Hiraizumi, small, reserved, soft-spoken, and polite. And yet by all accounts, Hiraizumi had an iron will and refused to be intimidated by Sandler, holding his own in any disagreement about interpretation of the data and preferring to stick with his own views until the data could resolve the issue one way or another. Whatever the day-to-day operating procedure, there is no doubt that Hiraizumi's ability to focus intently hour after hour on counting the flies for one huge experiment after another was a vital component to the enormous amount of work that was accomplished over the next few years.

It is remarkable how much of the story Sandler and Hiraizumi (actually Sandler, Hiraizumi, and Iris Sandler) got right in the first article. They distinguished the two common types of SD chromosomes (SD-5 and SD-72) on the basis of the inversions they carried, showed that distortion happens in males but not in females, and demonstrated that distortion could occur with a variety of homologs other than the cn bw chromosome. From recombination experiments they showed that recombinants lacking inversions could still distort, indicating that segregation distortion was a genic meiotic drive system rather than a process dependent upon a gross structural property of the chromosomes. On the basis of a small number of recombinants, they also positioned SD to the left of cn at the base of the second chromosome "either in or very near the centromeric heterochromatin of the right arm of chromosome II." In retrospect, this conclusion was influenced by several additional factors. One of them, I suspect, was Sandler's lifelong fascination with heterochromatin. In addition, Hiraizumi and Sandler were unaware of the presence of a small pericentric inversion that limited the recovery of recombinants. Nor had they yet realized the multicomponent nature of SD chromosomes in which distorting elements and target elements must be present in the right combination to see distortion. A recombinational dissection of SD chromosomes demonstrating the existence of two discrete elements with distinct roles in distortion was first presented by Sandler and Hiraizumi in 1960, in the fifth article of their series (SANDLER and HIRAIZUMI 1960 ). But real clarification awaited the detailed analysis of HARTL 1974 , who was able to avoid many of the complexities that plagued the interpretation in earlier attempts. This work led to the clear identification of the Responder (Rsp) locus as the target of distortion. Subsequently, as a student in Sandler's laboratory, I found that the Sd locus itself is located on the left arm in polytene region 37D—not all that close to the heterochromatin (GANETZKY 1977 ). However, I also found that Rsp was located in the centric heterochromatin of 2R. But this concurrence with the conclusion of Sandler and Hiraizumi on the basis of their mapping data was entirely fortuitous. More recent molecular and cytogenetic analyses by WU et al. 1988 and by PIMPINELLI and DIMITRI 1989 have defined Rsp as consisting of an array of simple satellite repeats whose copy number on a given second chromosome is proportional to the sensitivity of that chromosome to distortion.

Another important conclusion drawn by Sandler and Hiraizumi was that distortion resulted from gametic dysfunction. This conclusion was based on egg hatch studies as well as on the results of a very cleverly designed cross involving a T(Y;2) that ruled out the possibility that distortion involved an extra replication of the SD-bearing chromosome with elimination of its homolog. Eventually, more definitive evidence for sperm dysfunction was obtained in tests of male fertility carried out by Hartl and Hiraizumi (HARTL et al. 1967 ) and by Nico-letti and his colleagues (NICOLETTI et al. 1967 ). The ultimate proof was provided by the compelling elec-tron micrographic analysis by TOKUYASU et al. 1972 , which revealed the failed chromatin condensation in half of the developing spermatid nuclei and led in turn to subsequent defects in sperm maturation. Incidentally, because meiosis progresses perfectly normally in heterozygous SD males (PEACOCK and ERICKSON 1965 ), segregation distortion is not an example of meiotic drive at all, according to the original definition of SANDLER and NOVITSKI 1957 . The definition has now been expanded to include any alterations in meiosis or gametogenesis that result in preferential transmission of a particular allele or chromosome.

It is not too surprising, given what we now know about the complexity of the SD system, that not all of the conclusions and interpretations of the 1959 paper were correct. Sandler and Hiraizumi noticed that, when SD was heterozygous with various balancer chromosomes, such as In(2LR)Cy, transmission of the balancer was normal. They concluded that distortion was suppressed in these cases and, therefore, that proper synapsis at least in the vicinity of SD is requisite for distortion to occur. What they did not realize at the time is that the balancer chromosomes they used carried an insensitive allele of Rsp. It was the presence of this allele rather than any effect on chromosome pairing that rendered the balancer immune to the action of SD (HARTL 1975 ). Evidence from more recent studies in which either Rsp or Sd is relocated to a different chromosome indicates that Sd acts with full potency on its target wherever it happens to be located irrespective of any pairing between the loci. Sandler and Hiraizumi also proposed a model involving targeted chromosome breakage to explain the dysfunction of the SD⁺-bearing spermatids. The hypothesis was rather clever, but no recent data provide support for any mechanism of distortion that involves chromosome breakage. It is clear from the closing paragraphs of the article that the authors were much influenced by the work of McClintock, then in recent vintage, and were trying to squeeze SD into an Ac-Ds-like model.

The subsequent history of the investigation of the SD system has been described elsewhere, and there is no need to review it again here (HARTL and HIRAIZUMI 1976 ). Most likely, given the strong start to the analysis of segregation distortion, Sandler and Hiraizumi never anticipated that 40 years later a handful of investigators would still be butting heads with SD, trying to make it yield to rational interpretation. With the identification of the Sd product and a plausible link to defects in nuclear function and chromatin condensation, we feel that we are now getting very close (MERRILL et al. 1999 ). But, 40 years ago, Sandler and Hiraizumi probably felt that way too.

A recent Perspectives article was a tribute to Larry Sandler (LINDSLEY 1999 ). But what about the other key player in the SD story, Yuichiro Hiraizumi? After leaving Wisconsin, he returned to Japan for several years before taking a faculty position in 1969 at the University of Texas in Austin, where he remained for the rest of his career and is still active as an emeritus professor. In addition to other projects, he continued working on SD through the years. His attention to detail and his penchant for counting large numbers of flies to detect small but significant differences are legendary. And he never farmed out these experiments to someone else, believing strongly that he should gather and analyze his own data so that subtle results would not be missed. Long hours at the microscope, seven days a week, were his norm, fueled primarily by Dr. Pepper and cigarettes—he continued to smoke even as he anesthetized his flies with ether!

He isolated an SD chromosome from a Japanese population, the first to be found in Asia (HIRAIZUMI and NAKAZIMA 1965 ). He was also the first to recognize the existence of supersensitive chromosomes. These chromosomes bear an Rsp allele (Rsp^ss) that renders them even more sensitive to distortion than the standard cn bw tester chromosome (MARTIN and HIRAIZUMI 1979 ; HIRAIZUMI et al. 1980 ). Molecular analysis has confirmed the genetic studies and demonstrated that Rsp^ss chromosomes have an elevated copy number of the short AT-rich satellite repeat that composes the Rsp locus (WU et al. 1988 ). These chromosomes continue to be extremely useful tools in many experiments involving measurements of distortion. He discovered another component of the SD system on the right arm of chromosome 2 that he named Modifier of SD [M(SD)]. The presence of this component is required for SD chromosomes to have strong distorting activity, and its absence in certain recombinant derivatives probably explains some of the unexpected properties of these derivatives (MARTIN and HIRAIZUMI 1979 ; HIRAIZUMI et al. 1980 ). Hiraizumi also discovered a strong X-linked suppressor of distortion (HIRAIZUMI and THOMAS 1984 ). These enhancers and suppressors of distortion remain tantalizingly attractive targets for molecular analysis. When they are finally nailed down, they will surely add additional clarification to the mechanism of distortion. Most perplexing of all, Hiraizumi discovered that under certain conditions some SD chromosomes manifest "negative distortion"—the chromosome bearing an insensitive allele of Rsp is recovered less often than its homolog bearing a sensitive allele (HIRAIZUMI 1989 , HIRAIZUMI 1990 ). No existing model of distortion can accommodate this result in any simple way, but Hiraizumi's data cannot be denied.

Hiraizumi searched natural populations in Austin for SD chromosomes, but he never found them there. As far as I know, this is the only population of D. melanogaster in which SD chromosomes seem to be lacking. But he did not come up completely empty-handed. He found chromosomes that promoted the occurrence of recombination in males (HIRAIZUMI 1971 ). This was one of the early indicators of the existence of the phenomenon of hybrid dysgenesis, a manifestation of the transposable P element—an Ac-Ds-like system at last! Even more remarkable than the discoveries themselves is the fact that on many of the article reporting them, Hiriaizumi is sole author.

Although work was his primary pleasure, Hiraizumi occasionally took time out for other activities. He was a Go champion in his section of Japan, but could not find anyone in Madison able to play at his level. An avid fisherman, what little vacation he took was usually spent fishing. He enjoyed his home life as well. He and his wife, Mitzuko, had two children, Kazuo and Midori (Mrs. Frank Campbell). Kazuo earned a Ph.D. in Genetics at North Carolina State University, working on Drosophila, and now teaches Biology at Gettysburg College. Mitzuko, an accomplished artist who painted in the classical style with dark ink on rice paper, died in 1993.

One final note of irony. In preparation for writing this article, I decided to have a look at Hiraizumi's thesis, but was puzzled when I could not find a copy in the library. It turns out he never received a Ph.D. from Wisconsin because he was unable to pass his language exams. Instead, Kimura arranged for him to receive the doctorate from Japan, where the degree is awarded on the basis of publications. Apparently, discovering a novel genetic system and authoring or coauthoring 10 papers, including several that were destined to become classics, were insufficient criteria for the Ph.D. at Wisconsin. Fortunately, our students are not held to that standard these days.

Among his colleagues and those who have had the pleasure of meeting him, Hiraizumi is known for his gentleness, patience, unfailing good humor, and sincere delight in interacting with students and associates. Never one to seek the limelight, Hiraizumi nonetheless has made lasting contributions to two of the most intriguing genetic systems in Drosophila. And because he is still at it, there is no telling what he might turn up next.

View larger version (91K): In this window In a new window Download PPT slide   Figure 1. Yuichiro and Mitzuko Hiraizumi in 1960. Photo by Leidner Studio, courtesy of Elaine Mange.

View larger version (99K): In this window In a new window Download PPT slide   Figure 2. Hiraizumi in the lab, 1999. Photo by Midori Hiraizumi Campbell.

	ACKNOWLEDGMENTS

I thank Yuichiro Hiraizumi's friends and colleagues, James F. Crow, Rayla Temin, Elaine Mange, Kathy Matthews, James Curtsinger, and Irene Eckstrand for sharing their reminiscences with me.

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