Genes and Chromomeres: A Puzzle in Three Dimensions

DROSOPHILA polytene chromosomes have been used for more than 60 years to explore eukaryotic chromosome organization and the nature of genes. T. S. PAINTER 1934 , noting the distinctive and constant chromomeric patterns of the synapsed multistranded chromosomal arms, wrote "... it was clear that we had within our grasp the material of which everyone had been dreaming. We found ourselves out of the woods and upon a plainly marked highway with bypaths stretching in every direction. It was clear that the highway led to the lair of the gene." From the banded patterns of these chromosomes, maps of the "highway" were constructed. Mutational and recombinational analyses used in conjunction with these maps have produced a remarkable series of advances in understanding the organization of genes in these chromosomes and have paved the way for molecular cloning and sequencing of individual genes and gene complexes.

Still unresolved, however, is what the chromomeres and interchromomeres represent relative to genes and the structure and organization of chromosomes. It's not that this question has not been addressed. H. J. MULLER in a series of papers throughout his career probed the nature of genes and their arrangement in the chromosome, using X-ray-induced chromosomal rearrangements charted on the polytene maps. He spoke directly to the question as early as 1935 in a paper with A. A. PROKOFYEVA entitled "The individual gene in relation to the chromomere and the chromosome." They selected seven inversion breaks, three of which appeared to be identical, in region 1B of the X chromosome that caused phenotypic changes of the tightly linked genes yellow, achaete, or scute. By obtaining crossovers between rearrangements with different breakpoints, Muller created small deletions and duplications of the genes in the region. The cytological analysis of the breakpoints and the phenotypic effects of left/right combinations (i.e., deletion or duplication) showed that these genes are arranged in a discontinuous linear order and that the nodes (chromomeres) contain genes. Muller concluded that some chromomeres in the 1B1–7 region appeared to contain clusters of genes, judging from the variety of scute and achaete effects exhibited by some rearrangement combinations. That should have settled the question of the gene-chromomere relationship right there, but it did not. What Muller had opened is an early analysis of what is now known as the achaete-scute complex (ASC). More about how this and selected other genes fit within the chromomere-gene concept is discussed below.

Muller and Prokofyeva used the relationships they observed between the numbers of genes and chromomeres in this region of the X chromosome to estimate that the number of genes in the genome of Drosophila melanogaster is between 5 and 10 thousand. CALVIN BRIDGES' (1935) inference that faint chromomeres contain 1 gene while the heavy-walled doublets with an interspersed faint band might contain 3 supported this estimate. The detailed polytene chromosome maps (C. B. BRIDGES 1938 ; P. N. BRIDGES 1942 ) showed 5072 distinct bands in the four chromosomes. That number has been increased insignificantly through the use of the electron microscope to about 5100 bands (SORSA 1988 ).

WOLFGANG BEERMANN's (1961, 1962) studies of the puffing patterns in the giant polytene chromosomes of the dipteran Chironomus led him to propose that each puff originates from a single band. His demonstration that a particular secretion granule generated in a lobe of the salivary gland is correlated with the puffing of a specific band supported the concept that the information necessary for production of that protein resides in a single band. It was unresolved, however, whether there was just one or several functions encoded in the DNA of a chromomere.

	In search of a eukaryotic operon

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I was aware of most of these pioneering studies relating genes and chromomeres, but I did not at first set out to address that problem. In 1961, I presented a seminar to the genetics group at the University of Texas about the work of MADELINE GANS 1953 . It concerned the intriguing manner in which the mutation zeste (z¹) repressed the wild-type function of the white (w) locus in Drosophila females but had no effect on w⁺ expression in males. I was studying white locus structure at that time, and I wanted to discover more about the interaction between these two tightly linked X chromosome genes. The question that interested me then was whether the unique interaction between these two loci is influenced by other elements in the region.

The operon model of gene organization and regulation in bacteria, developed by FRANCOIS JACOB and JACQUES MONOD (1961), was at that time a major advancement in understanding prokaryotic gene organization and expression. The question that intrigued me was whether there is a eukaryotic counterpart to the operon. The far-fetched idea that zeste and white might be elements of a eukaryotic unit similar to the bacterial operon was a throwaway notion that I raised at the close of the seminar. Later in a discussion of Gans' work with Wilson Stone, the idea that discovering what types of genes occupy the region between z and w might be worth pursuing developed. That region, according to then-current cytological and recombination maps, consisted of about a dozen bands on the polytene chromosome map and about 0.5 cM linkage units. At that time no genes that definitely mapped to the region were known.

	The zeste-white region

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I had been analyzing the products of recombination events that generated reciprocal duplication and deficiency products in the regions within or flanking the white locus (JUDD 1961 ). These were later demonstrated to be due to recombination involving ectopic pairing and crossingover between interspersed transposable elements (DAVIS et al. 1987 ). One of the deficiencies, designated Df(1) w^rJ1, removes the 16-band chromosome segment between 3A1 and 3C2,3 of the polytene chromosome map. The deletion includes the zeste locus at 3A3 and the white locus at 3C2. I decided to use this deficiency in a genetic screen to saturate the zeste-white region with mutations.

The first experiment was designed to generate and recover mutations in all of the indispensable genetic elements located between these tightly linked loci and then to relate their function to zeste and white. The first series of X-ray experiments was carried out at Harvard University in the summer of 1962. George Lefevre had invited me to spend the summer there working in his lab. That was an exciting time as the first mutants were recovered: 16 lethal mutations composing five complementation groups localized between z and w (JUDD 1962 ). That the number of complementation groups was so large surprised me, and George too, as I recall. Early tests failed to show any discernible interactions of the lethals (as heterozygotes of course) with the zeste-white system. The questions then became whether there were indeed an even larger number of loci in that small region of the X chromosome and what would be required to identify all of them. Most important, Did any of them relate directly to the interaction of zeste and white? Answering those questions took on a life of its own as the number of lethals and semilethals identified by the mutation screen grew.

Margaret Shen joined the lab in 1964 and undertook the task of mapping the complementation groups, first into subsets defined by complementation tests against a battery of deletions and duplications. This was followed by the very tedious work of placing each mutation of a subset into an allelic group, which required a very large number of inter se complementation tests. The linear order of the genes within and between groups (cistrons) then had to be determined by recombination. This latter operation required the scoring of rare recombinants from many crosses that generated very large numbers of flies. It was Margaret's endless patience and meticulous work that kept this project going.

As the recombination map was worked out, we matched the position of each complementation group to its placement on the polytene chromosome map. This was determined by using overlapping deletions and duplications of the region in complementation tests with each group of lethals. The placement became more precise as the number of available rearrangement breakpoints increased. By the time Thom Kaufman came to the lab in 1967, we had identified and mapped 12 complementation groups flanked by zeste at 3A3 and white at 3C2. A comparison of the cytological and recombination maps strongly suggested that there was a one-to-one correspondence between the order and position of chromomeres and complementation groups. Judging from the number of allele at each identified complementation group, we calculated that we were approaching saturation of the region. However, since all of the early mutants were X-ray induced, there was concern that we might be failing to mutate some loci with X rays.

Thom began a mutation project to induce mutations with the chemical mutagens nitrosoguanidine and ethyl methanesulfonate. He recovered a large number of mutants but identified no new loci between z and w. However, his experiments extended the search to include two loci distal to zeste, one of which turned out to be the rediscovery of a previously described locus for which the mutants no longer existed. When examining putative lethal cultures, Thom noted one in which some of the third instar larvae that appeared later than normal were very large in size. He thought that possibly the culture had been contaminated with Drosophila virilis or D. hydei, both rather large flies kept in the Texas collection. Instead of discarding the culture, Thom kept on with his observations and determined that he had induced a semi-lethal mutation in the giant locus.

Thom also explored the region proximal to white by an analysis of the mutant phenotypes created by a series of deletions extending into that region. Now with the increased number of mutations, saturation for lethal loci in the z-w region appeared to be very close indeed. I was becoming convinced that we were not going to find any genes that interacted with zeste and white, a feeling that was bolstered by early results from the developmental analysis of the mutation groups by Mary Shannon, a post-doc in the lab, and Thom (SHANNON et al. 1972 ). The gene-chromomere question now was much more interesting than it had been and soon became a primary focus of the experiments.

Thom made two very interesting discoveries in addition to giant that are indirectly related to the gene-chromomere question and illustrate the value of saturation studies. First, while mapping the X chromosome breakpoints of the inversion In(1)e(bx), he discovered that the distal break created a mutation of zeste that behaved like the null allele, z^a (KAUFMAN et al. 1973 ). That inversion was induced and described as an enhancer of bithorax years earlier by ED LEWIS 1959 . That zeste might play a role in the function of the bithorax complex was surprising and exhilarating. Thom's observation was the beginning chapter of zeste as a major factor in the phenomenon of transvection. But that's another story altogether (for review, see ASHBURNER 1989 ).

Second, Thom found some semi-lethal alleles of a locus just distal to zeste, the survivors of which exhibited a remarkable behavioral phenotype. When the culture vial was struck sharply on the table, all of the flies convulsed and fell to the bottom of the vial. After a short while they revived and moved about quite normally. We named the locus tko. The tko gene has subsequently been cloned and sequenced by Vince Pirrotta and his collaborators (ROYDEN et al. 1987 ) and shown to encode a protein similar to a mitochondrial ribosomal protein, S12.

	Nonlethal loci

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The discovery of the tko phenotype pointed up an important weakness in a part of our search for all of the loci in the z-w region. We were relying on mutations that caused either lethality or observable changes in the morphology of the fly. How many genes might there be that are dispensable to flies raised in the lab or that, when mutated, give no discernible phenotypic change? The work of Seymour Benzer, who had designed a screen for behavioral mutations, was very much on our minds. We had to decide how we could extend our mutation screen to include other categories of changes. Plans for additional experiments were begun as Michael Young came to the lab. He decided to work on a screen to detect mutations that were viable but caused sterility and to detect changes that cause significant reduction in the rate of development, resulting in delayed emergence of the adults.

Rather than delay and wait for Mike's results, we decided that it was time to try to publish the data on the lethal and semi-lethal loci. Seven years had gone into these studies since that first summer at Harvard (1 year of which I spent pushing papers in the Biology Division of the Atomic Energy Commission). We had already published abstracts of the progress of the work, and the results had attracted quite a bit of attention, particularly among the molecular biologists. We also knew that Ben Hochman was well along with an analysis of the genes in the small chromosome 4 of Drosophila. That analysis would give him an estimate of the number of vital loci in the entire chromosome. His masterful account (HOCHMAN 1971 ) reported 40 recognized loci in the chromosome distributed among about 50 chromomeres. His positioning of the genes in the chromosome depended entirely on deletion mapping, since there is essentially no recombination in that small chromosome. Nonetheless, his estimate that there were probably 50 or fewer vital loci in chromosome 4 fit well with our data.

We reported (JUDD et al. 1972 ) finding 16 complementation groups associated with 15 adjacent chromomeres in the 3A1–3C2 segment. The order of the complementation groups, determined by recombination, matched with the position determined by deletion mapping, with the exception of 3 complementation groups in region 3B, where breakpoints separating them were not available. These groups were mapped by recombination, which by our analysis placed 6 complementation groups in region 3B, where we could distinguish only 5 chromomeres. However, HANS BERENDES 1970 had described six bands in 3B from an electron microscope study of the region. If we accepted his map, there was an equal number of genes and chromomeres, and the recombination and deletion maps showed essentially 1 complementation group per chromomere.

As our work progressed, Drosophila workers from other labs sent mutants and rearrangements to be used and tested in the z-w screen. Particularly valuable contributions came from George Lefevre, who sent lethals and a number of deletions and duplications that were crucial to assigning complementation groups to chromomeres. Johng Lim, Mary Louise Alexander, Seymour Abrahamson, Ben Hochman, Bill Welshons, and Raphael Falk also contributed to the pool of lethals.

	A concept in flux

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Johng Lim played a rather enigmatic role in the rise and fall of the one gene:one chromomere concept. We thought that we had discovered all of the indispensable loci in 3A1-3C2, until LIM and SNYDER 1974 carried out complementation tests on more than 100 lethals they induced in the region with alkylating agents. All but three of their mutations mapped at already known loci. Those three, however, failed to complement with each other but complemented all of the z-w loci we had discovered. Here then was a new lethal locus that mapped in section 3A. Lim and Snyder pointed out that electron microscope chromosome maps by Sorsa and Sorsa (in BEERMANN 1972 ) showed nine bands in region 3A, one more than we or Bridges had detected. Johng's cytological observations confirmed the existence of the band; furthermore, we discovered by recombination tests that the new locus mapped precisely where the additional band was located.

Was the one gene:one chromomere concept saved? For 3A maybe, but overall, no. During the time that we were working with Johng Lim to position the new locus, we had the results from Mike Young's experiments. The mutations causing delayed adult emergence all proved to be allelic to previously described loci, but Mike also found six mutations that produce female sterility. They formed two tightly linked complementation groups, fs(1)Y^a and fs(1)Y^b, that mapped to region 3B and were not allelic to any of our known loci. Try as we might to fit them into their own chromomeres, even using Hans Berendes' map, we could not.

In Benzer's lab at Cal Tech, Ron Konopka had discovered a mutant, per, that exhibited an abnormal diurnal activity rhythm. KONOPKA and BENZER 1971 had done preliminary mapping showing that per was a candidate for inclusion in the z-w region. Mike obtained three alleles of per from the Benzer lab. With deletion mapping of the mutant activity patterns, he placed the locus between two lethal loci in 3B. We had to conclude that the relationship of loci and bands, although very close to one-to-one, certainly did not hold as a general rule (JUDD and YOUNG 1973 ; YOUNG and JUDD 1978 ). However, a much more important question had emerged as we came closer to discovering all of the genes in the z-w region: Are genes in Drosophila really as big as these data suggest?

	Drosophila genes are large

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If our estimate of the total number of genes was accurate, we could calculate the average gene size from measurements of the amount of DNA in this region of the X chromosome. GEORGE RUDKIN 1965 had measured the amount of DNA in the polytene X chromosome and calculated that in each haploid strand, the average chromomere contains about 3 x 10⁴ nucleotide pairs. If the ratio of genes to chromomeres is about 1:1, a gene of average size in the z-w region consists of approximately 20 to 30 kb of DNA. About the same average value is obtained for all Drosophila genes by dividing the amount of DNA in the haploid genome by the total number of chromomeres. There are about 5100 chromomeres, and Charles Laird, then a colleague at the University of Texas, had determined that about 80% of the approximately 1.4 x 10⁸ bp of DNA in the haploid genome are unique copy sequences (LAIRD 1971 ). On that basis, the size of an average gene comes out to be about 22 kb. Considering that a typical polypeptide chain could be encoded by about 1.2 kb, there is, by that measure, sufficient DNA in the Drosophila genome to encode more than 100,000 different polypeptide chains.

What was then known about the reassociation kinetics and processing of RNA molecules further complicated the picture. It was evident that very-large-molecular-weight heterogeneous nuclear RNAs (hnRNAs) were transcribed in the nucleus, the sizes of which suggested that most of the DNA of a chromomere might be transcribed. However, most of that hnRNA turned over in the nucleus. The poly(A)-containing messenger RNAs (mRNAs) found associated with polysomes for translation in the cytoplasm were very much smaller and appeared to be derived from the 3' ends of hnRNAs by a series of processing steps. We were not molecular biologists, but clearly those observations caused us to question the possibility that there could be 100,000 different mRNAs generated from Drosophila melanogaster DNA sequences.

Considering our cytogenetic data on gene numbers and the molecular studies of the complexity of DNA sequences and the dynamics of RNA metabolism, Mike Young and I (JUDD and YOUNG 1973 ) proposed the following:

1. In the most general case, the chromosomal subunit, the chromomere, and the operational unit, the cistron, are coextensive. (2) The majority of the DNA of a chromomere is transcribed. (3) The transcript contains only one or a few structural gene sequences at the 3' end, whereas the remainder contains regulatory information. (4) Adequate processing of the transcription product depends on the integrity of all or most of the regulatory information elements. As a result, mutations derived therein act in a cis-dominant fashion. (5) Some of the 5'-end sequences may be released during processing of the transcript and activate other cistrons of a biosynthetic or developmental pathway. Mutations in these elements may act in pleiotropic fashion.

Speculation, yes, and it caused us to catch a lot of flack. To many molecular biologists of that time, our data suggesting that genes are very large, with only a small fraction of the DNA encoding protein, were hard to accept. Some notable exceptions, however, were Francis Crick and also Roy Britten and Eric Davidson. CRICK 1971 , having considered many of the data I have outlined above, including one of our abstracts (SHANNON et al. 1970 ), proposed a general model for chromosomes of higher organisms. He theorized that most of the DNA does not encode protein. His view was that in Drosophila a genetic complementation group usually is contained in a band plus an interband of the polytene chromosomes. He envisioned chromomeres to be globular, unpaired DNA used for gene control. The protein-coding sequences were proposed to be found in the much smaller fraction of fibrous DNA characterizing the interbands. Crick had been stimulated in his considerations by ROY BRITTEN and ERIC DAVIDSON (1969), who earlier in their seminal theory of gene regulation had suggested that large amounts of DNA are devoted to regulatory functions. They proposed five different types of genetic elements, only one class of which, the producer genes, encoded protein. The other classes were envisioned to be sensors, activating RNAs, integrators, or receptors, all of which acted in arrays of varying complexities to regulate the developmental expression of producer genes.

	Other genomic regions compared

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Despite these theoretical models concordant with our observations, the question concerning whether the density of genes in the z-w region might not be representative of the genome as a whole was raised. LIFSCHYTZ and FALK 1968 , LIFSCHYTZ and FALK 1969 had examined a small region encompassing 5 to 10% of the proximal X chromosome within which they identified 34 functional groups from the analysis of 105 X-ray or EMS-induced lethals. Those studies did not attempt to correlate directly bands with genes, but those data, along with Hochman's, were compatible with our studies. The gene-chromomere relationships and the question of gene size remained open.

In the next few years, however, there were a number of studies of other regions of the Drosophila genome. Several came from George Lefevre, who was a major contributor to the polytene chromosome organization studies. Possibly his most memorable contribution is the outstanding "dream nucleus": a representation of the polytene chromosomes that he compiled as a montage from a number of photographs. That today serves as the standard light microscope photographic map of the Drosophila melanogaster genome (LEFEVRE 1976 ). George approached problems in a variety of interesting and important ways, combining expert cytological analysis with studies of recombination frequency and mutation induction. He and Abraham Schalet published an extensive cytogenetic analysis of the proximal region of the X chromosome (SCHALET and LEFEVRE 1976 ). This region includes genes located in euchromatic and heterochromatic regions where the cytology is very difficult. Earlier, George studied the frequency of crossing over between two genes relative to the banding pattern of the region to determine whether recombination frequency depends not on the numbers of intervening chromomeres but on their relative sizes, i.e., DNA content (LEFEVRE 1971 ).

Michael Ashburner also has dealt with polytene chromosomes almost as much as anyone. Examining developmentally programmed puffing patterns, some hormone or heat shock induced, brought him face to face with the issue of chromomeres and the products encoded therein (ASHBURNER 1972 ). It also made him an early adventurer into chromosome pairing and gene expression, i.e., transvection. Again, that is another story. Michael's major contributions directly to the study of the gene-chromomere relationships come from an exhaustive analysis of the region surrounding the gene for alcohol dehydrogenase (Adh). Michael and his colleagues published at least seven papers about that region. In the first two, he and Ron Woodruff (WOODRUFF and ASHBURNER 1979A , WOODRUFF and ASHBURNER 1979B ) reported finding 21 lethal and 9 visible complementation groups in section 34D-35C of chromosome 2. Their calculations indicated that a total of about 34 complementation groups are located within that approximately 34-band segment. A complete molecular analysis of Adh and surrounding regions, undertaken by Michael and Gerry Rubin and their collaborators, is now almost ready for publication. More about that below.

Janos Gausz and associates, one of whom was Michael Ashburner (GAUSZ et al. 1979 , GAUSZ et al. 1981 ), examined the 86F-87C region of chromosome 3 and found that, with the exception of 87C1-3 where no genes mapped and 87C4-5 containing 1, there was apparently 1 complementation group per chromomere. In region 87D-87E,F in 23 to 24 bands, ARTHUR HILLIKER et al. 1980 in Art Chovnick's lab found 21 complementation groups. Igor Zhimulev and colleagues (ZHIMULEV et al. 1981 ) analyzed band 10A1-2 and adjoining regions. There also the ratio of genes to bands was near 1:1. Ted Wright and associates (WRIGHT et al. 1981 ) found 12, possible 13, genes in a 7-band segment in 37B10-37C4, clearly not a 1:1 relationship. Further, only 1 gene was found in 37C5-7 and none in 37D1. These cytogenetic analyses showed that the z-w region is quite typical and reinforced the concept that chromomeres and genes, although similar in number, could not be considered strictly coextensive. Most important, the number of genes per unit DNA still indicated that gene size averages about 20 kb.

	A large fraction of DNA does not encode protein

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What do all these cytogenetic data translate to at the molecular level? That genes contain exons interspersed with noncoding introns, which are spliced out during transcript processing, was not known in the early 1970s. Also, although enhancer and suppressor mutations were known, transcription enhancer and silencer elements that can act at a distance to regulate promoter elements had not yet been described. As molecular techniques for cloning and labeling were developed, the polytene chromosome maps became an important tool in facilitating the analysis of specific genes and chromosome regions. An early attempt to examine the arrangement of genes and their transcripts relative to polytene chromosomes came in 1983 from Pierre Spierer and collaborators (HALL et al. 1983 ). They mapped the transcripts from a 315K-bp segment from 87D5-6 to 87E5-6, which contains about 14 bands. Twenty discrete poly(A) RNA species collected at various stages of development, transcribed from nonrepetitive DNA, were defined. At least 12 complementation groups were recognized in the region. There was good correlation between the position of transcription units, chromomeric units, and complementation groups. Except for two large bands, E1-2 and E5-6, each band contains more than one transcription unit. No detectable transcripts were found from the large E1-2 band. The gene/chromomere number predicted by this analysis does not alter that shown by the cytogenetic studies more than twofold, and it is possible that several transcripts could be related to a single complementation group. Most important, it made clear that much of the DNA does not code for protein. What, then, is the function, if any, of the noncoding DNA?

	Lesions with nonmutant effects

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Ten years earlier George Lefevre had opened an interesting window to that question when he carried to another level the type of analysis used by Muller. George analyzed cytogenetically both mutant and nonmutant chromosome rearrangement breakpoints to find what proportion of breakpoints cause lethal or detectable morphological changes in the fly. That was a gargantuan undertaking because it required making salivary gland chromosome smears from essentially every individual culture, both mutant and normal, generated from offspring of mutagenized parents. A series of experiments identified almost 150 nonmutant breakpoints, from which George estimated that about 50% of X-ray-induced euchromatic breaks are not associated with lethal or other mutant effects (LEFEVRE 1973 ).

A similar result, but from a different approach, has now been added by George Miklos and associates (MALESZKA et al. 1998 ). Their molecular analysis of the 6-band 19F region surrounding the flightless locus shows 12 genes encoded by just 67 kb of genomic DNA. Seventy-five percent of the 67 kb contributes to 12 transcription units. At least three of the transcripts are alternatively spliced to specify two protein products. Notable, however, is the observation that the simultaneous deletion or disruption of most of the 12 loci results in unobtrusive morphological changes, changes that in all likelihood would not be detected in the usual genetic screens for mutations. They interpret these results to indicate that about 30% of Drosophila loci can mutate to a lethal state, and approximately 20% can produce morphological or behavioral changes. That means that about 50% of loci do not contribute strongly to the morphological phenotype.

Still another approach to the question of gene organization and chromomere structure is to ask whether a gene occupies all or only a part of a band. GEORGE LEFEVRE 1969 analyzed a series of breaks in and around the vermilion locus in bands 10A1-2 and showed that breaking a band known to house a specific gene need not always result in a mutation of that gene. This point was also addressed for the white locus by SORSA et al. 1973 , who reached the conclusion that w occupied only part of a band. A much more definitive answer comes from the lab of John Sedat (RYKOWSKI et al. 1988 ). These investigators hybridized labeled segments of Notch (N) locus DNA to stretched preparations of the polytene chromosomes. Using high-resolution, computer-aided optic microscopy, they aligned the molecular map of the gene with the cytological features of band 3C7, the position of Notch. This showed that band 3C7 is 34–37 kb in length, within which is found all or most of the 37-kb Notch transcription unit. The coding portions and introns are all contained within the band while the segment positioned 5' to the transcription start site lies in the open chromatin conformation of the interband between 3C6 and 3C7. Thus, the regulatory sequences are in an interband; Crick's model comes to mind, but with structural and regulatory elements reversed.

Sedat's observations of polytene chromosome structure with computer-assisted analysis of light microscope images give some support to the idea that some of the bands defined by Bridges may consist of smaller sub-bands separated by very small interbands. This had been suggested previously by several investigators (KEPPY and WELSHONS 1980 ; LEFEVRE and WATKINS 1986 ) to account for cytological changes seen at some chromosome breakpoints. This is yet another variable in defining the gene chromomere relationships.

	The cut locus

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Cut (ct) presents an interesting and possibly extreme view of how a single gene obviously extends across chromomere boundaries. The locus is huge, with mutations mapping over almost 200 kb of genomic DNA in 7B1-2 of the polytene map (JACK 1985 ). It is a highly mutable locus in which mutations comprise three distinct phenotypic classes. The kinked femur phenotype is characterized by small body size, kinked femurs, and small opaque wings that seldom expand. The cut-wing phenotype consists of incised wing margins of several patterns and some head capsule abnormalities, particularly antennae. The third phenotype consists of three distinct lethal classes. The complementation pattern is complex (LIU et al. 1991 ). All lethal classes fail to complement each other and the cut phenotype mutant class. However, one lethal class complements kinked femur, but the other two do not. Kinked femur and cut mutations all complement each other.

For a locus this big with a complex complementation map, what does the molecular structure look like? In 200 kb of genomic DNA, kinked femur lesions map most distally and appear to occupy up to 50 kb. This is difficult to determine because some mutations are deletions that encroach, possibly from outside the locus. The cut phenotype class is caused by changes, many of which are insertions of transposable elements, in about 25 kb just distal to the center of the locus. The lethal classes form three rather clustered groups of lesions positioned over about 70 kb proximal. A cDNA of 8217 bp derived from this 70 kb of genomic DNA has been sequenced. It contains an open reading frame encoding 2175 amino acids in which there is a 60-amino-acid homeodomain. The protein product is required in embryonic and adult organs, regulating sensory organ identities in the wings and various other body parts (JACK et al. 1991 ).

Back in 1979, Terry Johnson and I mapped the various classes of cut mutations (JOHNSON and JUDD 1979 ). On the basis of the complementation patterns, we rather brashly suggested that a very major portion of the locus, where the kinked femur and cut mutations map, is all regulatory in function and that the structural gene(s) was likely to be found in the proximal region where the lethals map. The molecular work pretty much bears that out. JO JACK and YVONNE DELOTTO (1995) have determined that cis-acting regulatory sequences of cut are spread over the distal and central region more than 85 kb upstream of the transcription start site. Lesions such as retrotransposon insertions in this region block expression of cut in subsets of tissues where normally it would be active. The effect is polar, with lesions farthest from the promoter affecting the fewest tissues.

	The achaete-scute complex

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I want to return to examining the ASC to relate what has been learned about its structure and expression to what Muller and Prokofyeva discovered about chromomeres and genes more than 60 years ago. Almost all of the ASC complex has now been cloned, restriction mapped, and sequenced. Extensive study has correlated the developmental functions of genes in the complex with transcription units. ANTONIO GARCIA-BELLIDO 1979 subdivided the ASC complex into four components from the sites of lesions causing mutant phenotypes. A fifth component was added by DAMBLY-CHAUDIERE and GHYSEN 1987 . Nine different transcription units have been identified in the seven-band 1B1-1B7 segment. Some lesions in the region, associated with at least one of the transcripts, affect sex determination and are thus excluded as members of the ASC complex. Four of the transcripts encode helix-loop-helix proteins that can bind as dimers to DNA. These products account for the neurogenic functions attributed to achaete, scute, lethal(1)scute, and asense of the ASC. Three of those transcripts share a 15-nucleotide sequence encoding an acidic amino acid domain on the 3' end. Genetic analysis of the ASC mutations shows that different components specify different but sometimes overlapping subsets of cellular effects. For example, the ac and sc genes have similar patterns of expression in imaginal discs. Juan Modolell and collaborators (GOMEZ-SKARMETA et al. 1995 ) have shown that this coexpression is accomplished by shared, position-specific, enhancer-like elements distributed along most of the approximately 90 kb ASC. Several enhancers located either upstream or downstream of sc drive the expression of the ac and sc promoters. The patterns of ac and sc expression observed when the ac and sc regions are separated, as by the rearrangements selected by Muller and Prokofyeva, show that the enhancer elements with similar specificities are not found in both regions. Also breaks such as the sc⁴ inversion they studied, which has a break inside one of the enhancer elements that disconnect the long downstream region from sc, actually remove expression of both ac and sc from the proneural cell clusters of the imaginal discs.

Muller noted that the rearrangements that appeared to have identical breakpoints in the scute region, but with different proximal breaks in the X chromosome, produced slightly different phenotypic effects. He reasoned this to be due to position effects brought on by placing different neighboring elements adjacent to the sc locus (RAFFEL and MULLER 1940 ). However, he anticipated the gene complex by noting: "The question is raised of to what extent the breakage in the region of scute in this and other cases where they seem identical in position are really identical, or may be separated by parts of a `gene-complex' (or `gene,' according to definition) which, although able to reproduce separately, function to produce bristles only, or mainly, when in proximity to one another, and function normally only when in proper arrangement."

Do the molecular studies significantly change how we view the relationship between genes and chromomeres, or is it confirmation of the cytogenetic data discussed above? Answers to this question may soon be forthcoming. Michael Ashburner and Gerry Rubin and their collaborators, in not-yet-published work, are now analyzing the sequence of about 2.7 mb of DNA surrounding the Adh locus on chromosome arm 2L (34D-36A). The genetic analysis had identified 72 genes, about 50 of which have lethal alleles in these 69 polytene chromosome bands. The computational analysis of the sequence, however, has identified about 109 protein-coding genes. I'm not sure what this means, but it will be very interesting to see how all of this unfolds. It is clear that only about one-third of these genes are detected by even the most thorough genetic tests, and only one-fourth can mutate to lethality. There is clear evidence confirming the cytogenetic data that gene density is far from uniform. Clusters of genes are found in some regions, while long, seemingly empty stretches characterize other parts of the genomic universe.

	A summary

TOP In search of a... The zeste-white region Nonlethal loci A concept in flux Drosophila genes are large Other genomic regions compared A large fraction of... Lesions with nonmutant effects The cut locus The achaete-scute complex A summary The chromomere LITERATURE CITED

The survey I have presented of selected genes, complexes, and extended regions of chromosomes, in my opinion, covers most of the spectrum of gene-chromomere arrangements. What emerges is that the relationship is not a simple one. Clearly, there is no apparent direct correlation between the chromomeric pattern and the numbers and arrangements of genes. In fact, judging from the cytogenetic and molecular maps and evidence from rearrangement breakpoints, genes do not respect chromomeric boundaries. In some regions, clusters of genes are found tightly packed into few bands, and on the other hand there are long stretches of DNA that seem devoid of recognizable genetic elements. Further, even with this nonuniform gene density, it is clear that, overall, a large fraction of the genomic DNA does not encode protein products and that much of the remainder is utilized in the regulation of gene expression. The maps, along with genetic dissection, molecular cloning, and sequencing, have shown us that Drosophila melanogaster genes on average are about an order of magnitude larger than is necessary to encode the polypeptides they specify.

Now, can we estimate how many genes it takes to make a fly? Maybe not with any great accuracy. I say that not just because of the points I have raised about undiscovered genes and bands or how regulatory elements and other apparently noncoding regions fit into the picture, but also because the c-value paradox looms in my mind. The paradox is that there is no direct correlation between the genomic DNA content of an organism and its developmental complexity. That takes the edge off estimating the number of required genes therein. Drosophila virilis has about 60% more DNA in its genome than D. melanogaster, and the mosquito (Aedes) has six times as much. Maybe here I should also mention, without further comment, that there are many examples of genes that encode proteins that perform two quite different functions. Evolution works, but in mysterious ways.

In the face of uncertainties about the numbers of chromomeres and the probability that many genes remain to be discovered, I am still impressed with just how close the association of genes with chromomeres remains. What has emerged is that the chromomeric map has proven to be one of the most powerful tools available to geneticists, cytologists, and molecular biologists. Genes can be located precisely by these maps, greatly facilitating genetic and evolutionary studies. Chromosome aberrations can be analyzed in great detail and correlated with their effects on genes at or near the breakpoints. Determining the effects of rearrangements in crossover suppression and aberrant chromosome transmission would have been extremely difficult without polytene chromosomes.

	The chromomere

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I would still like to know what a chromomeric unit is. I surmise that it is an inherent aspect of chromosomal three-dimensional structure, created by winding or folding parallel strands of DNA into tightly packed units, alternating with less dense interchromomeric stretches. The measurements done by John Sedat (RYKOWSKI et al. 1988 ) support the concept that in polytene chromosome chromomeres, the 10-nm nucleosome fiber is wound regularly into a higher-order structure. Band 3C7, where Notch is positioned, is not condensed above the level of the 30-nm fiber. As for interbands, their data are consistent with the suggestion by ANANIEV and BARSKY 1985 that interband DNA exists in the 10-nm nucleosome fiber form.

It is clear that breaks in chromosomes can fracture bands and create two condensed segments. From this I conclude that the winding or folding is not unidirectionally determined by sequence information localized at a band margin. With all of the rather extensive sequence data now emerging from analyses of large stretches of Drosophila DNA, there is not yet a clue about band/interband boundary junctions. Somewhere in that one-dimensional nucleotide sequence there certainly resides the necessary three-dimensional information.

I once heard Francis Crick comment after a seminar on transvection that genetic analysis has difficulty dealing with three-dimensional problems. The molecular data do not yet make this any easier.

	FOOTNOTES

I dedicate this essay to the memory of GEORGE LEFEVRE, who had a strong influence on my scientific career and who was a major figure in advancing the genetics and cytology of Drosophila.

	ACKNOWLEDGMENTS

I am very grateful for the efforts and many stimulating discussions of those who worked in my lab on various aspects of this problem, especially THOM KAUFMAN, MIKE YOUNG, and JO JACK. Also, very special thanks are due to JOHNG LIM for his contributions and discussions and to CATHY LAURIE, who stimulated the genesis of this essay, for her very useful comments. I gratefully acknowledge MICHAEL ASHBURNER's extensive and very helpful comments, and to him and GERRY RUBIN and their collaborators, special thanks for permission to cite the results of work in progress.

	LITERATURE CITED

TOP In search of a... The zeste-white region Nonlethal loci A concept in flux Drosophila genes are large Other genomic regions compared A large fraction of... Lesions with nonmutant effects The cut locus The achaete-scute complex A summary The chromomere LITERATURE CITED

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