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Signaling molecules of the transforming growth factor beta (TGF-beta) family contribute to numerous developmental processes in a variety of organisms. However, our understanding of the mechanisms which regulate the activity of and mediate the response to TGF-beta family members remains incomplete. The product of the Drosophila decapentaplegic (dpp) locus is a well-characterized member of this family. We have taken a genetic approach to identify factors required for TGF-beta function in Drosophila by testing for genetic interactions between mutant alleles of dpp and a collection of chromosomal deficiencies. Our survey identified two deficiencies that act as maternal enhancers of recessive embryonic lethal alleles of dpp. The enhanced individuals die with weakly ventralized phenotypes. These phenotypes are consistent with a mechanism whereby the deficiencies deplete two maternally provided factors required for dpp's role in embryonic dorsal-ventral pattern formation. One of these deficiencies also appears to delete a factor required for dpp function in wing vein formation. These deficiencies remove material from the 54F-55A and 66B-66C polytene chromosomal regions, respectively. As neither of these regions has been previously implicated in dpp function, we propose that each of the deficiencies removes a novel factor or factors required for dpp function.

Using an elaborate set of cis-regulatory sequences, the decapentaplegic (dpp) gene displays a dynamic pattern of gene expression during development. The C-terminal portion of the DPP protein is processed to generate a secreted signaling molecule belonging to the transforming growth factor-beta (TGF-beta) family. This signal, the DPP ligand, is able to influence the developmental fates of responsive cells in a concentration-dependent fashion. Here we examine the sequence level organization of a significant portion of the dpp locus in Drosophila melanogaster and use interspecific comparisons with D. simulans, D. pseudoobscura and D.virilis to explore the molecular evolution of the gene. Our interspecific analysis identified significant selective constraint on both the nucleotide and amino acid sequences. As expected, interspecific comparison of protein coding sequences shows that the C-terminal ligand region is highly conserved. However, the central portion of the protein is also conserved, while the N-terminal third is quite variable. Comparison of noncoding regions reveals significant stretches of nucleotide identity in the 3' untranslated portion of exon 3 and in the intron between exons 2 and 3. An examination of cDNA sequences representing five classes of dpp transcripts indicates that these transcripts encode the same polypeptide.

We have identified the molecular lesions associated with six point mutations in the Drosophila TGF-beta homologue decapentaplegic (dpp). The sites of these mutations define residues within both the pro and ligand regions that are essential for dpp function in vivo. While all of these mutations affect residues that are highly conserved among TGF-beta superfamily members, the phenotypic consequences of the different alleles are quite distinct. Through an analysis of these mutant phenotypes, both in cuticle preparations and with molecular probes, we have assessed the functional significance of specific residues that are conserved among the different members of the superfamily. In addition, we have tested for conditional genetic interactions between the different alleles. We show that two of the alleles are temperature sensitive for the embryonic functions of dpp, such that these alleles are not only embryonic viable as homozygotes but also partially complement other dpp hypomorphs at low temperatures. Our results are discussed with regard to in vitro mutagenesis data on other TGF-beta-like molecules, as well as with regard to the regulation of dpp cell signaling in Drosophila.

A convenient method is described for the intracistronic mapping of genetic sites responsible for electrophoretic variation of a specific protein in Drosophila melanogaster. A number of wild-type isoalleles of the rosy locus have been isolated which are associated with the production of electrophoretically distinguishable xanthine dehydrogenases. Large-scale recombination experiments were carried out involving null enzyme mutants induced on electrophoretically distinct wild-type isoalleles, the genetic basis for which is followed as a nonselective marker in the cross. Additionally, a large-scale recombination experiment was carried out involving null enzyme rosy mutants induced on the same wild-type isoallele. Examination of the electrophoretic character of crossover and convertant products recovered from the latter experiment revealed that all exhibited the same parental electrophoretic character. In addition to documenting the stability of the xanthine dehydrogenase electrophoretic character, this observation argues against a special mutagenesis hypothesis to explain conversions resulting from allele recombination studies.

Experiments expanding the array of mutants affecting the xanthine dehydrogenase (XDH) structural element in Drosophila melanogaster are described. These include rosy eye color mutants which exhibit interallelic complementation, and mutants with normal eye color but lowered levels of XDH. Evidence is presented which argues that these are structural alterations in the enzyme. Recombination experiments were performed using these mutants as well as some electrophoretic variants. The two ends of the rosy locus are marked with mutant sites which are clearly structural in nature; the XDH structural element and the rosy null mutant map are completely concordant. A possible procedure to recover control element mutants is described.

This report describes the genetic analysis of a region of the third chromosome of Drosophila melanogaster extending from 87D2-4 to 87E12-F1, an interval of 23 or 24 polytene chromosome bands. This region includes the rosy (ry, 3-52.0) locus, carrying the structural information for xanthine dehydrogenase (XDH). We have, in recent years, focused attention on the genetic regulation of the rosy locus and, therefore, wished to ascertain in detail the immediate genetic environment of this locus. Specifically, we question if rosy is a solitary genetic unit or part of a large complex genetic unit encompassing adjacent genes. Our data also provide opportunity to examine further the relationship between euchromatic gene distribution and polytene chromosome structure.----The results of our genetic dissection of the rosy micro-region substantiate the conclusion drawn earlier (SCHALET, KERNAGHAN and CHOVNICK 1964) that the rosy locus is the only gene in this region concerned with XDH activity and that all adjacent genetic units are functionally, as well as spatially, distinct from the rosy gene. Within the rosy micro-region, we observed a close correspondence between the number of complementation groups (21) and the number of polytene chromosome bands (23 or 24). Consideration of this latter observation in conjunction with those of similar studies of other chhromosomal regions supports the hypothesis that each polytene chromosome band corresponds to a single genetic unit.

Three gene systems have been shown to exhibit proximity-dependent phenotypes in Drosophila melanogaster: bithorax (BX-C), decapentaplegic (DPP-C) and white (w). In structurally homozygous genotypes, specific allelic combinations at these loci exhibit one phenotype, while in certain rearrangement heterozygotes the same allelic combinations exhibit dramatically different phenotypes. These observations have led to the suggestion that, through the process of somatic chromosome pairing, such loci are brought into sufficient proximity to permit effective passage of molecular information between homologues; rearrangement heterozygosity would then displace the homologues relative to one another such that this trans-communication is obviated. The genetic properties of the proximity-dependent allelic complementation (termed transvection effects) at the BX-C and DPP-C, are quite similar. Chromosomal rearrangements which disrupt transvection possess a breakpoint in a particular segment of the chromosome arm bearing the transvection-sensitive gene (arm 2L for the DDP-C and 3R for the BX-C); this segment of each arm has been termed the critical region by Lewis (1954). As determined by cytogenetic analysis of transvection-disrupting rearrangements, the critical regions for the BX-C and DDP-C transvection effects extend proximally from these loci for several hundred polytene chromosome bands (Lewis 1954; Gelbart 1982). The interaction between the zeste and white loci appears to depend upon the proximity of the two w+ alleles. By use of insertional duplications, displacement of w+ homologues has been shown to interfere with the zeste-white interaction. In contrast to transvection at bithorax and decapentaplegic, however, only breakpoints in the immediate vicinity of the white locus can disrupt the zeste-white interaction (Gans 1953; Green 1967; Gelbart 1971; this report). In this report, we investigate the basis for the difference in the size of the BX-C and DPP-C critical regions from that of white. We test and eliminate the possibility that the difference is due to the presence near the white locus of a site which mediates somatic chromosome pairing. Rather, our evidence strongly suggests that the zeste-white interaction is, at the phenotypic level, much less sensitive to displacement of the homologous genes than is transvection at either the BX-C or DPP-C. We also show that many of the breakpoints in the vicinity of the white locus do not behave as if they are disrupting a critical region for somatic chromosome pairing. Given these results, we suggest that the zeste-white interaction and transvection are two different proximity-dependent phenomena.

Zeste (1-1.0; 3A3) mutations have been known to modify the expression of two gene complexes: white (1-1.5; 3C1.5) and bithorax (3-58.8; 89E1-4) in Drosophila melanogaster. Certain mutations of these complexes have been shown to behave in a synapsis-dependent fashion. That is, certain bithorax and white genotypes exhibit one level of expression when the two copies of these loci are able to synapse in somatic tissues and another level when heterozygosity for chromosomal rearrangements interferes with their ability to pair. Such phenomena are termed transvection effects by LEWIS (1954). In the case of the white locus, asynapsis leads to a more normal state, whereas at bithorax, asynapsis leads to a more mutant phenotype. Recently, a third case of transvection was described at the decapentaplegic (2-4.0; 22F1-3) gene complex (GELBART 1982); phenomenologically, it is very similar to transvection at bithorax. In this report, we demonstrate that zeste mutations can also interact with those decapentaplegic mutations that exhibit transvection effects. In addition, we present more information on the zeste interactions with white and bithorax. Interactions with zeste may be diagnostic of loci that can exhibit transvection effects. However, different groups of zeste alleles interact with each complex. z1 interacts with white, za alleles interact with bithorax and all tested zeste mutants interact with decapentaplegic. These differential effects of zeste mutations may be a reflection of the neomorphic nature of the z1 allele.

A new mutant, mit (mitotic loss inducer), is described. The mutant is recessive and maternal in action, producing gynandromorphs and haplo-4 mosaics among the progeny of homozygous mit females. Mosaic loss of maternal or paternal chromosomes can occur. The probabilities of either maternal or paternal X chromosome loss are equal. mit has been mapped to approximately 57 on the standard X chromosome map.-Using gyandromorphs generated by mit, a morphogenetic fate map, placing the origins of 40 cuticular structures on the blastoderm surface, has been constructed. This fate map is consistent with embryological data and with the two other fate maps generated in different ways.

Our laboratory has been concerned with the structure and function of the decapentaplegic gene complex (DPP-C) in Drosophila melanogaster. To define the boundaries of the complex, we have studied the genetics of mutations allelic to a previously discovered mutation shortvein (shv), known to reside near decapentaplegic. We found that shortvein resides distal to Hin-d and dpp within the same polytene chromosome doublet, 22F1-2. Lesions in shv can affect not only the formation of the wing veins but also can interfere with normal development of parts of the adult and/or be lethal. Like those of dpp mutants, the shv-associated adult abnormalities affect distal epidermal structures. Some shv lesions cause a larval lethal syndrome which is associated with an unusually long larval stage (ca. five to six times its normal duration). Lesions in shv exhibit an involved pattern of complementation with dpp mutations, indicating that both shv and dpp are parts of a single gene complex. A subset of the array of mutant phenotypes displayed by shv/dpp trans-heterozygotes appear to be dpp-specific phenotypes; we interpret these as reflecting an inactivation effect of certain shv alleles on dpp functions. The other abnormalities displayed by these trans-heterozygotes appear to be shv-specific defects; we view these as indicating an inactivation effect of certain dpp mutations on shv functions. Furthermore, embryonic lethal (EL) mutations within the DPP-C exhibit allelic interactions with all shv mutations. We conclude that the shortvein region represents a newly identified integrated portion of the DPP-C.

Experiments are described that provide an opportunity to estimate the genetic limits of the structural (amino acid coding) portion of the rosy locus (3:52.0) in Drosophila melanogaster, which controls the enzyme, xanthine dehydrogenase (XDH). This is accomplished by mapping experiments which localize sites responsible for electrophoretic variation in the enzyme on the known genetic map of null-XDH rosy mutants. Electrophoretic sites are distributed along a large portion of the null mutant map. A cis-trans test involving electrophoretic variants in the left- and right-hand portions of the map leads to the conclusion that the entire region between these variants is also structural. Hence most, if not all, of the null mutant map of the rosy locus contains structural information for the amino acid sequence of the XDH polypeptide. Consideration is given to the significance of the present results for the general problem of gene organization in higher eukaryotes.

The Ace locus in Drosophila melanogaster is known to be the structural gene for acetylcholinesterase. Ace is located in a region of chromosome arm 3R which has been subjected to intensive genetic and molecular analysis. Previous deletion mapping studies have identified a 40-kb region within which the Ace gene resides. This report focuses on the further localization of Ace within this 40-kb interval. Within this region, selective fine structure recombinational analysis was employed to localize three recessive Ace lethals relative to unselected restriction site variations. These three mutations fall into a segment of 7 kb within the Ace interval. Fine structure recombinational analysis was also used to confirm that the Ace phenotype of one deletion, Df(3R)AceHD1, co-segregated with the molecular deletion. This deletion does not fully remove Ace activity, but it behaves as a recessive Ace lethal. Df(3R)AceHD1 is the most distal Ace lesion identified and indicates that the Ace locus must extend at least 16 kb. Several poly(A)transcripts are detectable in the region defined by the Ace lesions. The position and extent of the Ace locus, as well as the types of transcripts found, is consistent with the recent findings which identified Torpedo-AChE homologous cDNA sequences in this region.

A selective system for recovery of exchanges between trans mutations at adjacent loci, l(3)S12 and rosy, is described. In addition to the expected crossover and conversion classes, two exceptional types of offspring were recovered. Triploid offspring arose as 0.01% of all zygotes; the diploid chromosome set was apparently of maternal origin. Nine tandem duplications derived from unequal exchange between nonsister homologues were recovered among 2.25 x 10(6) zygotes screened. From considerations of the proportion of the genome that was assayed in this system, and on the assumption that the rate of unequal exchange observed is typical for the genome as a whole, it appears that one unequal exchange occurs per 500 female meioses in Drosophila.