Genetic suppression of mutations in the Drosophila abl proto-oncogene homolog.

The Drosophila abelson (abl) gene encodes the homolog of the mammalian c-abl cytoplasmic tyrosine kinase and is an essential gene for the development of viable adult flies. Three second-site mutations that suppress the lethality caused by the absence of abl function have been isolated, and all three map to the gene enabled (ena). The mutations are recessive embryonic lethal mutations but act as dominant mutations to compensate for the neural defects of abl mutants. Thus, mutations in a specific gene can compensate for the absence of a tyrosine kinase.

Identification of a fibroblast growth factor-binding protein in Drosophila melanogaster.

As assessed by competitive binding and protein-crosslinking experiments, Drosophila melanogaster cells possess basic fibroblast growth factor (bFGF)-specific binding proteins that are similar to FGF receptors on vertebrate cells in molecular weight and binding affinity; these D. melanogaster cells, however, have no detectable binding proteins for acidic fibroblast growth factor (aFGF). Consistent with the presence of bFGF-specific binding proteins, D. melanogaster cells degrade bFGF but not aFGF. These results indicate the conservation of heparin-binding growth factors and receptors between vertebrates and D. melanogaster.

Molecular and developmental characterization of the heat shock cognate 4 gene of Drosophila melanogaster.

The Drosophila heat shock cognate gene 4 (hsc4), a member of the hsp70 gene family, encodes an abundant protein, hsc70, that is more similar to the constitutively expressed human protein than the Drosophila heat-inducible hsp70. Developmental expression revealed that hsc4 transcripts are enriched in cells active in endocytosis and those undergoing rapid growth and changes in shape.

Isolation and sequence of the Drosophila virilis 60 A gene, a transforming growth factor-beta superfamily member related to vertebrate bone morphogenetic proteins.

The complete protein-coding region of the Drosophila virilis 60 A gene, a member of the transforming growth factor-beta superfamily, was isolated and sequenced. The mature domain of the protein-coding region is 99% identical to the Drosophila melanogaster 60 A gene and 73% identical to human bone morphogenetic protein 5. In the pro-domain, a number of large blocks of amino acids are also highly conserved, indicating an important functional role for this portion of the protein as well. In the putative 5' and 3' untranslated regions, several short sequence motifs are conserved between D. virilis and D. melanogaster.

Differential mitotic stability of yeast disomes derived from triploid meiosis.

The frequencies of recovered disomy among the meiotic segregants of yeast (Saccharomyces cerevisiae) triploids were assessed under conditions in which all 17 yeast chromosomes were monitored simultaneously. The studies employed inbred triploids, in which all homologous centromeres were identical by descent, and single haploid testers carrying genetic markers for all 17 linkage groups. The principal results include: (1) Ascospores from triploid meiosis germinate at frequencies comparable to those from normal diploids, but most fail to produce visible colonies due to the growth-retarding effects of high multiple disomy. (2) The probability of disome formation during triploid meiosis is the same for all chromosomes; disomy for any given chromosome does not exclude simultaneous disomy for any other chromosome. (3) The 17 yeast chromosomes fall into three frequency classes in terms of disome recovery. The results support the idea that multiply disomic meiotic segregants of the triploid experience repeated, nonrandom, post-germination mitotic chromosome losses (N + 1 leads to N) and that the observed variations in individual disome recovery are wholly attributable to inherent differences in disome mitotic stability.

Characterization of cuticular proteins in the red flour beetle, Tribolium castaneum.

We are characterizing the cuticular proteins of Tribolium castaneum (Herbst) (Coleoptera:Tenebrionidae) to determine their role in the function of the exoskeleton. Based on qualitative analyses of cuticles, we focused on the sodium dodecyl sulfate (SDS)-extractable proteins. A small-scale cuticle "mini-prep" procedure was devised that yields preparations virtually free of contaminating cellular material compared to hand-dissected preparations, as assessed by fluorescent microscopy using DAPI to stain nuclei. Proteins extracted in 1% SDS from various developmental stages (last larval instar, pupal, adult) were analyzed by one-dimensional denaturing polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. The cuticular protein profiles show both similarities and differences among the stages examined. The amino acid composition, glycosylation, and partial amino acid sequence of several abundant cuticular proteins indicate similarity to cuticular proteins of other insects.

Sequence, biochemical characterization, and developmental expression of a new member of the TGF-beta superfamily in Drosophila melanogaster.

More than 20 members of the transforming growth factor-beta (TGF-beta) superfamily of growth and differentiation factors have been implicated in development. One member of the TGF-beta family has been previously reported from Drosophila, the decapentaplegic (dpp) gene which is involved in embryonic dorsal/ventral polarity, embryonic gut formation, and imaginal disk development. Using PCR methods, we have identified a second Drosophila gene in the TGF-beta family. It encodes a protein product that is more similar to the TGF-beta-related human bone morphogenetic proteins (BMPs) 5, 6, and 7 than it is to the Drosophila dpp gene product. Because of its localization on the polytene chromosome map, we refer to this gene as 60A. Expression of a 60A cDNA in Drosophila S2 cells was used to determine that 60A encodes a preproprotein that is processed to yield secreted amino- and carboxy-terminal polypeptides. The carboxy-terminal peptides are recovered as disulfide-linked homodimers. The 60A transcripts and protein are first detected at the onset of gastrulation, primarily in the mesoderm of the extending germ band. As the germ band retracts, and throughout later stages of embryonic development, the 60A transcript and protein are most readily detected in cells of the developing foregut and hindgut.

Drosophila transforming growth factor beta superfamily proteins induce endochondral bone formation in mammals.

Both decapentaplegic (dpp) protein and 60A protein have been implicated in pattern formation during Drosophila melanogaster embryogenesis. Within the C-terminal domain, dpp and 60A are similar to human bone morphogenetic protein 2 (75% identity) and human osteogenic protein 1 (70% identity), respectively. Both recombinant human bone morphogenetic protein 2 and recombinant human osteogenic protein 1 have been shown to induce bone formation in vivo and to restore large diaphyseal segmental defects in various animal models. We examined whether the Drosophila proteins, dpp and 60A, have the capacity to induce bone formation in mammals by using the rat subcutaneous bone induction model. Highly purified recombinant dpp and 60A induced the formation of cartilage, bone, and bone marrow in mammals, as determined by histological observations and by measurements of the specific activity of alkaline phosphatase and calcium content of the implants, thereby demonstrating that related proteins from phylogenetically distant species are capable of inducing bone formation in mammals when placed in sites where progenitor cells are available.

The Tribolium decapentaplegic gene is similar in sequence, structure, and expression to the Drosophila dpp gene.

We are characterizing members of the Transforming Growth Factor-ß (TGF-ß) superfamily in the red flour beetle, Tribolium castaneum, in order to examine the evolutionary conservation of the structure and function of TGF-ß-like genes during insect development. A decapentaplegic-like gene of the TGF-ß superfamily was isolated in Tribolium (Tc dpp) that is similar in sequence, organization, and expression to the Drosophila melanogaster dpp gene (Dm dpp). Conserved features include a high degree of sequence similarity in both the pro-domain and mature domains of the encoded polypeptide. In addition, the position of an intron within the protein-coding region is conserved in Tc dpp, Dm dpp, and two bone morphogenetic protein genes of the TGF-ß superfamily in humans, BMP2 and BMP4. Consensus binding sites for the dorsal transcription factor are found within this intron in Tc dpp similar to the intronic location of several dorsal binding sites in Dm dpp. During embryogenesis, Tc dpp is expressed in an anterior cap of serosa cells at the blastoderm stage, in the dorsal ectoderm at the lateral edges of the developing and extended germ band, and in the distal tips of developing embryonic appendages. Several aspects of embryonic expression, similar in both flies and beetles, suggest conserved roles for dpp in cellular communication during the development of these distantly related insects.