C. B. Bridges' Repeat Hypothesis and the Nature of the Gene

IN producing the definitive maps of the giant salivary gland chromosomes of Drosophila melanogaster, C. B. BRIDGES 1935 interpreted certain structures as tandem gene duplications that had become established in the species. He wrote:

In my first report on duplications at the 1918 meeting of the A.A.A.S., I emphasized the point that the main interest in duplications lay in their offering a method for evolutionary increase in lengths of chromosomes with identical genes which could subsequently mutate separately and diversify their effects. The present demonstration that certain sections of normal chromosomes have actually been built up in blocks through such "repeats" goes far toward explaining species initiation (p. 64).

I will call this Bridges' repeat hypothesis, and in this article I show how Bridges had hoped to test it. Although he was unsuccessful, it led others to discover (1) recombination within the gene and (2) the existence of gene complexes, or clusters of closely linked and functionally related genes.

Bridges had, in fact, provided a cytological basis for challenging the concept of multiple allelism that had dominated research on the nature of the gene for nearly 40 years. A. H. STURTEVANT 1965 , who defined the concept, succinctly describes the difficulties it began to present:

Multiple alleles had been supposed to represent changes in a single original gene, and there were two criteria for their recognition: they occupy the same locus in the chromosome and were not separable by crossing over; and their heterozygote (trans type) was mutant with respect to their common recessive phenotype, since neither carried the wild-type allele of the other (p. 89).

Sturtevant points out that these two criteria failed to agree when, in the 1940s, crossing over was shown to occur within what had been considered single genes, namely Star (S) (LEWIS 1942 , LEWIS 1945 ) and lozenge (lz) (OLIVER 1940 ; GREEN and GREEN 1949 ). Both cases cited Bridges' repeat hypothesis.

In preparing a talk on the immense indebtedness of the genetics community to C. B. Bridges, I had occasion to examine the vast collection of cards¹ on which he recorded the data from his experiments. They show that, as early as 1938, he had begun a deliberate attempt to subject another multiple allelic series to a recombination analysis, namely that of bithorax (bx), whose prototypic allele, bx, he had discovered in 1915. That was also the year in which he was awarded the Ph.D. degree at Columbia University. His doctoral thesis, "Non-disjunction as proof of the chromosome theory of heredity," became a classic and appeared the next year as the first article in the first volume of GENETICS (BRIDGES 1916 ).

In 1919, Bridges discovered another mutation, bithoraxoid (bxd), which fully complemented bx.² These mutations cause partial transformation of the third thoracic segment of the fly into the second. As such, they are examples of homeosis, a phenomenon that Bateson had described in 1898 (see reviews in LEWIS 1994 ; MCGINNIS 1994 ). Bridges' bx and bxd mutants were the first examples, at least in animals, of homeosis that were the results of gene mutation, as opposed to being accidents of development.

Bridges' renewed interest in bx mutations was prompted by W. F. Hollander's discovery of two new bx mutations in the summer of 1934. At the time, Hollander was a graduate student of L. J. Cole at the University of Wisconsin, and, as an assistant in the laboratory course in genetics, Hollander maintained stocks of Drosophila.

Dr. Hollander informed me that he had found the mutations in a single fly and was able to set up stocks of them that he then sent to the stock center at the Cold Spring Harbor Laboratory, probably in 1935. Bridges was at the time a research associate of the Carnegie Institution of Washington at Caltech, but he spent summers at Cold Spring Harbor (CSH), where, along with Demerec, he helped maintain the CSH stock center and thus would have become aware of Hollander's stocks.

Hollander has kindly shared with me handwritten letters that Bridges sent him, concerning these mutations. In the first letter, dated March 8, 1935, Bridges indicates that he is sending Hollander stocks of bx, bx^34e, an allele found by J. Schultz (BRIDGES and BREHME 1944 ), and bxd. Hollander followed Bridges' advice and tested all possible combinations of these mutations with his two new mutations. Hollander named one of the mutations bithorax-Wisconsin (bx^w). In a letter dated August 10, 1937, Bridges suggested that the other mutation be named bithorax-Dominant (bx^D), since bx^D/+ flies have slightly larger halteres than wild-type flies.

Clearly Bridges was intrigued with bx^D, since it behaved as allelic to both bx and bxd, even though all previously known bx mutations fully complemented bxd. In the same letter (in which "allel" was the old abbreviation for allelomorph), Bridges wrote: "Whether it really is an allel nevertheless is one of those unsettled questions; I should say not an allel by preference until proved otherwise."

At Bridges' suggestion, HOLLANDER 1937 described the phenotypes of all the possible trans-heterozygotes involving his mutations and those that Bridges had sent him. Bridges also advised Hollander to examine the salivary gland chromosomes of bx^D; however, Hollander was awarded the Ph.D. degree in 1937 and had to give up work with Drosophila. He has pursued a long career studying the genetics of pigeons, a field in which he was one of the pioneers and in which he is still actively engaged. For a history of pigeon genetics at Wisconsin, in which Hollander participated, see OWEN 1989 .

In early 1938, Bridges began a genetic and cytological analysis of Hollander's mutants. I believe he saw it as a way to test his repeat hypothesis. One of the data cards (undated) has a diagram that shows that Bridges had repeated, and essentially confirmed, all of the interactions that HOLLANDER 1937 had described, namely, that trans-heterozygotes involving bx^D and either bx or bxd have, in every case, a more extreme mutant phenotype than that of bx^D/+, whereas trans-heterozygotes involving a given bx mutation and bxd are in every case wild type.

Bridges recorded, on a card dated December 25, 1937, "no aberration" associated with bx^D in the salivary gland chromosomes. Thus, he ruled out at least a gross deficiency or chromosomal rearrangement as an explanation for the failure of bx^D to complement bx or bxd in trans. A card dated March 9, 1938 (Fig 1) shows that he had already completed the scoring of progeny from matings of Sb bx^D/bx^w females to a homozygous bx^w male. Stubble (Sb), located at 58.3, provided a closely linked flanking marker to the left of the locus of bx^D at 58.8. Among 1169 progeny, Bridges reports an Sb fly that was wild type for bx. He bred the fly, confirmed its genotype, and on another card (not shown) labeled it a "revertant." Bridges' diagram of the maternal genotype (Fig 1) shows that he also considered it to be a possible crossover between bx^w and bx^D, provided that bx^w lies to the left of bx^D. It is interesting that the diagram allows for the possibility that bx^w lies to the right of bx^D. The reason must have been that Bridges recorded on a raw data card (Fig 2) a possible double-mutant fly that he designates Sb bx^D bx^w/bx^w. On the reverse side of the card, he sketched the fly (Fig 3). Since it expressed the Sb marker, to be a double-mutant crossover, bx^w would have had to lie to the right of bx^D. Although bx^w is now lost, all other existing bx mutations lie to the left of bx^D. It seems more likely, therefore, that the fly was the result of a new mutation, either bx or an enhancer.

View larger version (0K): In this window In a new window Download PPT slide   Figure 1. (Top) Bridges' 3 x 5-inch data card showing progeny of a mating of an Sb bxD (e4)/bxw female(s) to bxw males that gave a single Sb fly (column 6). Column 1 is dated March 9, 1938 (38c9) and shows culture numbers that contain the raw data on which the summary is based. Columns 2–7, inclusive, show the number of progeny having the phenotype at the head of the column (with the paternally derived bxw omitted). Inserted between column 6 and 7 in the header is the genotype of a possible double mutant from culture 21705 (see Fig 2). Flies were not scored for ebony-4 (e4) since not all parental females carried it. (Bottom) Interpretation of the maternal genotype and frequency of the wild-type recombinant (1 in 1169 offspring). See text for description of other mutant symbols.

View larger version (0K): In this window In a new window Download PPT slide   Figure 2. Bridges' card showing progeny scored in March 1938 (38c) from three cultures: 21704; 21705, which produced the possible double-mutant fly of genotype Sb bxD bxw/bxw; and 21706, which produced an Sb fly, a possible revertant or wild-type crossover between re and y. A large delete sign is Bridges' indication that the data were copied to a summary card.

View larger version (0K): In this window In a new window Download PPT slide   Figure 3. The reverse side of the card in Fig 2 with a sketch of the possible double mutant. Bridges' comments are "died no offspring"; "bigger thorax & wings than bxw/bxw"; "seen struggling on pupa case & hauled out"; "bxw stock had been seen (38b) to have sc6 wa in it." The last comment suggests that the stock had been contaminated at some point.

A frequency of one revertant in only 1169 offspring must have encouraged Bridges to repeat the experiment. He did so rather quickly and added stripe (sr), at 62.0, as a flanking marker to the right of bx^D. A summary card dated June 14, 1938 (Fig 4) records his failure to obtain any wild-type or double-mutant crossovers with respect to bx from a mating of Sb bx^D/bx^w sr females to homozygous bx^w sr males. There is no record that Bridges recorded the total progeny of this mating. I have therefore calculated from the total progeny listed on each of the 37 summary cards a grand total of 6753 progeny. The result must have been very discouraging.³ Bridges became ill at about that time and on December 27, 1938, he died of a heart infection at the age of 49.

View larger version (0K): In this window In a new window Download PPT slide   Figure 4. (Top) The first of 37 summary cards showing F1 progeny (cumulatively totaled) from a mating of trans-heterozygous females backcrossed to bxw sr es males. (Bottom) Interpretation of the parental genotype. Numbers inserted on the line are the C-o intervals. No crossovers were recorded in interval 2. es, ebony-sooty.

In spite of Bridges' failure to repeat his success in obtaining a possible recombinant between two bx mutations, his repeat hypothesis became the catalyst that motivated subsequent successful attempts to use crossing over as a tool to dissect "multiple allelic" series. Those attempts have been reviewed in three Perspectives: by DUNCAN and MONTGOMERY 2002A , DUNCAN and MONTGOMERY 2002B for the Star and bithorax series and by GREEN 1990 for the lozenge series. Not only were wild-type crossovers between "alleles" obtained in those early studies, but the reciprocal double-mutant crossovers were derived as well. Without the latter, the possibility remained that the wild-type products were instead reverse mutations associated with crossing over (OLIVER 1940 ).

Recovery of the double mutant has usually proved difficult because of a striking position effect, or "cis-trans effect," in which the cis-heterozygote for recessive mutations is wild type, and the trans-heterozygote is mutant in phenotype. In the Star-asteroid case, the trans-heterozygote, S +/+ ast, is nearly eyeless, while the cis-heterozygote, S ast/+ +, has a nearly normal eye like that of S/+ (figured in LEWIS 1951 ). Similar strong cis-trans effects were found for heterozygotes involving bx^D and for each of the various bx mutations (bx^D and bxd). However, in this case, cis- and trans-heterozygotes for bx and bxd are both wild type (LEWIS 1951 ).⁴ Two other series of mutations were found to be quite similar in complementation and recombination properties to those of the bx series, namely the Notch series (WELSHONS 1958 ; WELSHONS and VON HALLE 1962 ) and the dumpy series (CARLSON 1959 ); however, critical tests for cis-trans effects were not carried out, because of failure to obtain the double-mutant recombinant class.

Subsequently, many multiple allelic series proved divisible by recombination not only in Drosophila, but also in microorganisms, phage, and even in maize (see review in LEWIS 1967 ). In an ingenious experiment, NELSON 1962 used iodine staining of maize pollen from a trans-heterozygote for two waxy (wx) mutations to obtain high yields of the non-wx (wild-type recombinant) pollen. Similar high yields of wild-type recombinants between alleles were readily obtained in many examples of multiple allelic series involving microorganisms and phage. However, recovery of the reciprocal double-mutant class was rarely achieved or even attempted.⁵ Instead, it came to be taken for granted that the cis-heterozygote would be wild type for two recessive mutations.

In Drosophila, several cases of recovery of the cis-heterozygote were made possible by the technique of half-tetrad analysis.⁶ Thus, from trans-heterozygous attached-X females that are mutant with respect to two sex-linked mutations, rare wild-type females can be recovered that carry the wild-type recombinant in one arm of that chromosome and the reciprocal, or double-mutant, recombinant in the other arm. In this way, the double mutants for two different white (w) mutations were obtained (LEWIS 1952 ),⁷ as were two different garnet (g) mutations (HEXTER 1958 ; CHOVNICK 1961 ). In both cases, there was the typical strong cis-trans effect; namely the trans-heterozygote was mutant and the cis-heterozygote was wild type, being in fact the basis on which it was selected. Many multiple-mutant combinations of bx mutations, with resultant cis-trans effects, were derived using attached right arms of the third chromosome (LEWIS 1967 ).

Working with the ascomycete, Aspergillus nidulans, PRITCHARD 1955 used a different type of half-tetrad analysis that involved mitotic recombination to derive the double mutant for two adenine (ad)-requiring recessive mutations. Pritchard was able to obtain the cis-heterozygote for two of the ad mutations and established a cis-trans effect. Thus, the trans-heterozygote required the addition of adenine to grow, whereas the cis-heterozygote was wild type and grew without addition of adenine.

Historically, Bridges' repeat hypothesis stimulated research that led to the discovery of recombination within a multiple allelic series at first in Drosophila and later in many other organisms. Similarly, it stimulated work that eventually led to the discovery of the tightly linked clusters of related genes known as the bithorax complex (BX-C) (LEWIS 1978 ) and the Antennapedia complex (ANT-C) (KAUFMAN et al. 1980 ). Finally, MCGINNIS et al. 1984 and, independently, SCOTT and WEINER 1984 discovered the homeobox (HOX), a DNA motif in the protein-coding region of the genes of these two complexes. In most animals the BX-C and ANT-C form one continuous complex known as the homeobox complex (HOX-C). The incredibly high degree of conservation of the HOX in all higher animals, including humans, points to the evolution of the HOX-C by a process of repeated gene duplication and diversification.

I believe Bridges would have liked that!

	FOOTNOTES

^* Author e-mail: lewise{at}its.caltech.edu
¹ Bridges' records are held at Caltech in their original filing cabinet and consist of thousands of 3 x 5-inch cards that are numbered consecutively, commencing with number 7662, ca. 1917 (since cards starting with 7700 are dated October 1917), and terminating with card number 22,002, dated June 1938. The fate of cards 1–7661 from the Columbia University period is not known. Cards 7662–22,002 contain mainly the raw data on which the linkage maps of D. melanogaster were based. Not every number is represented. Other cards, often 3 x 5-inch pieces of paper, provide summaries and raw data on which Bridges based the map locations for almost all mutants known at the time of his death.
² Unfortunately, the figure of the mutant in BRIDGES and BREHME 1944 and in later compendia is that of an early bx^b mutant allele, now lost, which apparently became confused with bx^d. A penciled drawing, labeled bxd by Lillian V. Morgan's artist, E. M. Wallace, was never finished. It showed a cup-shaped halter wing and a missing first abdominal segment. The latter was overlooked in the description of the mutant. Figures of the halteres of the bx and bxd mutants (LEWIS 1951 ) show that bx and bxd have mutually exclusive phenotypes. Thus, bx mutants have only the anterior half of the halter transformed toward an anterior-like wing, while bxd mutants have only the posterior half transformed to a posterior wing.
³ Bridges' failure to obtain any more recombinants in 6753 offspring is not unexpected, since the frequency of crossing over between bx and Ultrabithorax (Ubx) is estimated to be 0.01% on the basis of experiments (LEWIS 1954B ) in which the use of the method of STEINBERG 1936 resulted in an approximately threefold increase in crossing over in the bx–Ubx region.
⁴ bx^D was renamed Ultrabithorax (Ubx) when it was found to lie between bx and bxd (LEWIS 1954A ). The mutation is now known to be the insertion of a transposable element into the 5' exon of the Ubx transcription unit (BENDER et al. 1983 ). Bridges' bx and bxd mutations are now known to be insertions of such elements in large cis-regulatory regions that control where and when the protein coded by the Ubx gene is expressed during development. Intriguingly, though, as shown by LIPSHITZ et al. 1987 , the wild-type bxd region codes for a 26.5-kb transcript that results in a family of non-protein-coding RNAs of unknown function.
⁵ Another difficulty encountered with microorganisms was the phenomenon of gene conversion, which made it difficult to order the alleles even when there were flanking markers. Gene conversion, however, did not complicate the analysis of S, lz, and bx. The mutations, at least in the case of bx, are either deletions of many kilobases of DNA or insertions of transposable elements (BENDER et al. 1983 ). Apparently such large lesions cannot be readily corrected by gene conversion.
⁶ The technique was pioneered by Lillian V. MORGAN 1931 , who used it to obtain, simultaneously, the reciprocal products of unequal crossing over at the Bar locus.
⁷ Wild-type recombinants between two w mutations were reported by MACKENDRICK and PONTECORVO 1952 .

	ACKNOWLEDGMENTS

I thank Welcome Bender, Giuseppe Bertani, Allan Campbell, Andrew Dowsett, Robert Drewell, Ian Duncan, Willard Hollander, Norman Horowitz, Howard Lipshitz, Geoffrey Montgomery, David Perkins, and Allan Spradling for help in the preparation of this Perspectives.

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