Regulation of the expression of the sn-glycerol-3-phosphate dehydrogenase gene in Drosophila melanogaster

P element-mediated transformation has been used to investigate the regulation of expression of the sn-glycerol-3-phosphate dehydrogenase gene of Drosophila melanogaster. A 13-kb construct containing the eight exons and associated introns, 5 kb of the 5' region, and 3 kb downstream from the structural gene produced normal levels of enzyme activity and rescued the poor viability of flies lacking the enzyme. All the regulatory elements essential for normal enzyme expression were located in a fragment that included the exons and introns and 1-kb upstream noncoding sequence. Deletions of the 1.6-kb second intron reduced activity to 25%. Transformants with fusion constructs between the sn-glycerol-3-phosphate dehydrogenase gene and the beta-galactosidase gene from E. coli revealed three elements that affected expression. A (CT)9 repeat element at the 5' end of the second intron increased expression in both larvae and adults, particularly at emergence. A second regulatory element, which includes a (CT)7 repeat, was located 5' to the TATA box and had similar effects on the gene's expression. A third, undefined, enhancer was located in the second intron, between 0.5 and 1.8 kb downstream of the translation initiation codon. This element increases enzyme activity to a similar extent in larvae and adults but has little effect when the enhancer at the 5' end of the intron is present.     

A study of penetrance of Drosophila melanogaster forked gene

Group I intron endonuclease I-CreI is encoded by an open reading frame contained within a self-splicing intron in the Chlamydomonas reinhardtii chloroplast 23S rRNA gene. I-CreI initiates the lateral transfer or homing of this intron by specifically recognizing and cleaving a pseudopalindromic 19-24 bp homing site in chloroplast 23S rRNA genes that lack the intron. The gene encoding this enzyme has been subcloned, and the protein product has been purified and crystallized. The crystals belong to space group P321, with unit cell dimensions a = b = 78.2 A, c = 67.4 A. The crystal unit cell is consistent with an asymmetric unit consisting of the enzyme monomer. The specific volume of this unit cell is 3.3 A3/Da. The crystals diffract to at least 3.0 A resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector.     

Changes in gene expression during post-eclosional development in the olfactory system of Drosophila melanogaster

We have found that the expression of some genes in Drosophila melanogaster changes during the life of the adult fly. These changes can be illustrated by the use of enhancer trap lines which mark the expression of particular genes in the adult fly. Although the fly is considered able to perform most necessary adult functions within the first 72 h after eclosion from the pupal case, we find changes in expression over the first 10 days of life in the antennae of several of the genes we have examined. Some genes change by increasing from an initially low level of expression of the marked gene, while other lines, which we have termed 'late-onset' genes, show no expression of the marked gene until 4-5 days following eclosion. In contrast, some genes decrease their expression during the first 10 days of life. The changes in gene expression seen over the first 10 days of the fly's adult life provides molecular evidence of the many maturational changes occurring during the early life of the adult fly.     

Evolutionary history of the sex-peptide (Acp70A) gene region in Drosophila melanogaster

A phosphorothioate oligonucleotide (PS-ODN) with sequence identical to the repeat sequence of the mammalian telomere, 5'-d(TTAGGG)-3', was incubated with a Burkitt's lymphoma-derived (OMA-BL1) cell line. This hexanucleotide inhibits telomerase activity in cell lysates, lengthens cell doubling time, and induces apoptosis. Concatenated repeats (12-, 18-, and 24-mers) of the 5'-d(TTAGGG)-3' motif induce similar cellular responses. Scrambled sequences do not efficiently inhibit telomerase activity or significantly alter cell growth and viability. The in vivo efficacy of this PS-ODN was evaluated in a xenograft human-nude mouse model. Once tumors were established these animals were administered the telomere mimic, 5'-d(TTAGGG)-3', a control scrambled sequence 5'-d(TGTGAG)-3', or saline for 14 days via a subcutaneous osmotic pumps in a blinded study monitoring tumor size with dose and time. A significant decrease in tumor size was observed in animals given 50 micrograms/mouse/day 5'-d(TTAGGG)-3', but not following 5'-d(TGTGAG)-3', relative to the saline-treated animals. The antitumor activity of the 6-mer telomere mimic demonstrated a dose dependency including a reduction in metastatic nodules in the spleen. No activity was observed with the scrambled controls. In addition to antitumor activity we observed an increase in the mouse hematopoietic progenitor cell populations, BFU-e and CFU-GM. These results demonstrated the effects of a short hexameric oligonucleotide telomere mimic in vitro and in vivo and the potential utility of short oligonucleotides as telomerase inhibitors.     

A genetic analysis of hermaphrodite, a pleiotropic sex determination gene in Drosophila melanogaster

Sex determination in Drosophila is controlled by a cascade of regulatory genes. Here we describe hermaphrodite (her), a new component of this regulatory cascade with pleiotropic zygotic and maternal functions. Zygotically, her+ function is required for female sexual differentiation: when zygotic her+ function is lacking, females are transformed to intersexes. Zygotic her+ function may also play a role in male sexual differentiation. Maternally, her+ function is needed to ensure the viability of female progeny: a partial loss of her+ function preferentially kills daughters. In addition, her has both zygotic and maternal functions required for viability in both sexes. Temperature sensitivity prevails for all known her alleles and for all of the her phenotypes described above, suggesting that her may participate in an intrinsically temperature-sensitive process. This analysis of four her alleles also indicates that the zygotic and maternal components of of her function are differentially mutable. We have localized her cytologically to 36A3-36A11.     

Regulation of a dpp target gene in the Drosophila embryo

EnvZ is an inner membrane protein present in Escherichia coli that is important for osmosensing and required for porin gene regulation. EnvZ is phosphorylated by intracellular ATP, and EnvZ-P phosphorylates OmpR, which then binds to the porin promoters to regulate their expression. An overexpressed, truncated form of the enzyme, EnvZ115, was used to characterize the kinase reaction in vitro. Using a filter binding assay, we report the first direct measurements of the kinase activity, including the apparent affinity for ATP of 200 microM. The phosphorylation reaction is dependent on MgCl2, and the phosphoenzyme has the expected stability of a phosphohistidine; i.e., it is stable in base and less stable in acid at room temperature. The addition of OmpR and ATP to solutions containing EnvZ resulted in an OmpR-stimulated, EnvZ-dependent ATPase activity that was not vanadate-sensitive. The in vivo kinase activity of EnvZ and two mutants that were deficient in porin expression were studied using an immune complex kinase reaction. Interestingly, a mutation located in the periplasmic domain of EnvZ exhibited kinase activity that was identical to that of the wild-type enzyme, while a mutation located close to the phosphorylation site showed a significant decrease in both kinase and phosphotransferase activities. These data provide support for models of EnvZ consisting of separate sensing and kinase domains.     

DSP1 gene of Drosophila melanogaster encodes an HMG-domain protein that plays multiple roles in development

Historically, juvenile justice policy has oscillated between rehabilitative and punitive approaches to managing young offenders. Policy and practice in the 1970s and 1980s emphasized individual treatment for young offenders in nonsecure, community-based programs. An increase in violent youth crime during the past decade has renewed interest in punishing delinquent youths. Cyclic fluctuations in juvenile justice policy and their relationship to policy, practice, and youth crime are examined. Our analysis suggests that overall crime rates have remained relatively stable over the past three decades and are independent of prevailing juvenile justice policies. The findings support the need for targeted prevention efforts addressing the root causes of juvenile crime. Needed policy reforms, public education efforts, and practice approaches are outlined.     

Amyrel, a paralogous gene of the amylase gene family in Drosophila melanogaster and the Sophophora subgenus

We describe a gene from Drosophila melanogaster related to the alpha-amylase gene Amy. This gene, which exists as a single copy, was named Amyrel. It is strikingly divergent from Amy because the amino acid divergence is 40%. The coding sequence is interrupted by a short intron at position 655, which is unusual in amylase genes. Amyrel has also been cloned in Drosophila ananassae, Drosophila pseudoobscura, and Drosophila subobscura and is likely to be present throughout the Sophophora subgenus, but, to our knowledge, it has not been detected outside. Unexpectedly, there is a strong conservation of 5' and 3' flanking regions between Amyrel genes from different species, which is not the case for Amy and which suggests that selection acts on these regions. In contrast to the Amy genes, Amyrel is transcribed in larvae of D. melanogaster but not in adults. However, the protein has not been detected yet. Amyrel evolves about twice as fast as Amy in the several species studied. We suggest that this gene could result from a duplication of Amy followed by accelerated and selected divergence toward a new adaptation.     

Genetic characterization of the Drosophila melanogaster Suppressor of deltex gene: A regulator of notch signaling

The Notch receptor signaling pathway regulates cell differentiation during the development of multicellular organisms. A number of genes are known to be components of the pathway or regulators of the Notch signal. One candidate for a modifier of Notch function is the Drosophila Suppressor of deltex gene [Su(dx)]. We have isolated four new alleles of Su(dx) and mapped the gene between 22B4 and 22C2. Loss-of-function Su(dx) mutations were found to suppress phenotypes resulting from loss-of-function of Notch signaling and to enhance gain-of-function Notch mutations. Hairless, a mutation in a known negative regulator of the Notch pathway, was also enhanced by Su(dx). Phenotypes were identified for Su(dx) in wing vein development, and a role was demonstrated for the gene between 20 and 30 hr after puparium formation. This corresponds to the period when the Notch protein is involved in refining the vein competent territories. Taken together, our data indicate a role for Su(dx) as a negative regulator of Notch function.     

Genetic and developmental characterization of Dmca1D, a calcium channel alpha1 subunit gene in Drosophila melanogaster

To begin unraveling the functional significance of calcium channel diversity, we identified mutations in Dmca1D, a Drosophila calcium channel alpha1 subunit cDNA that we recently cloned. These mutations constitute the l(2)35Fa lethal locus, which we rename Dmca1D. A severe allele, Dmca1D(X10), truncates the channel after the IV-S4 transmembrane domain. These mutants die as late embryos because they lack vigorous hatching movements. In the weaker allele, Dmca1D(AR66), a cysteine in transmembrane domain I-S1 is changed to tyrosine. Dmca1D(AR66) embryos hatch but pharate adults have difficulty eclosing. Those that do eclose have difficulty in fluid-filling of the wings. These studies show that this member of the calcium channel alpha1 subunit gene family plays a nonredundant, vital role in larvae and adults.     

Maternal Nanos regulates zygotic gene expression in germline progenitors of Drosophila melanogaster

Maternal Nanos (Nos) protein is required for germline development in Drosophila embryos. Here we show that Nos regulates zygotic gene expression in the germline progenitors, or pole cells. In order to probe the gene expression in pole cells, we screened ten enhancer-trap lines which showed beta-gal expression in pole cells. All of these enhancer-trap markers were fully activated in pole cells after their migration to the embryonic gonads. In the pole cells lacking Nos, the expression of nine out of ten enhancer-trap markers was affected. Among nine markers, five (Type-A) were prematurely expressed in the pole cells during the course of their migration. The expression of other four markers (Type-B) initiated correctly after pole-cell migration, but their expression was significantly reduced. Thus, we conclude that the maternal Nos plays a dual role in zygotic gene regulation in pole cells: to define the stages of expression for Type-A markers, and to enhance expression for Type-B markers. Contrary to our results, "Heller and Steinmann-Zwicky (1998)" have recently reported that no premature expression of Type-A markers occurs in the pole cells of embryos derived from nos mutant females. This discrepancy is due to the difference in the nos mutant alleles used for these analyses. We used the much stronger allele, nosBN.     

Divergent and conserved features in the spatial expression of the Drosophila pseudoobscura esterase-5B gene and the esterase-6 gene of Drosophila melanogaster

The regulatory regions of homologous genes encoding esterase 6 (Est-6) of Drosophila melanogaster and esterase 5B (Est-5B) of Drosophila pseudoobscura show very little similarity. We have undertaken a comparative study of the pattern of expression directed by the Est-5B and Est-6 5'-flanking DNA to attempt to reveal conserved elements regulating tissue-specific expression in adults. Esterase regulatory sequences were linked to a lacZ reporter gene and transformed into D. melanogaster embryos. Est-5B, 5' upstream elements, give rise to a beta-galactosidase expression pattern that coincides with the wild-type expression of Est-5B in D. pseudoobscura. The expression patterns of the Est-5B/lacZ construct are different from those of a fusion gene containing the upstream region of Est-6. Common sites of expression for both kinds of constructs are the third segment of antenna, the maxillary palps, and salivary glands. In vitro deletion mutagenesis has shown that the two genes have a different organization of regulatory elements controlling expression in both the third segment of antenna and maxillary palps. The results suggest that the conservation of the expression pattern in genes that evolved from a common ancestor may not be accompanied by preservation of the corresponding cis-regulatory elements.     

Extended reproductive roles of the fruitless gene in Drosophila melanogaster revealed by behavioral analysis of new fru mutants

The fruitless mutants fru3 and fru4 were assessed for sex-specific reproductive-behavioral phenotypes and compared to the previously reported fru mutants. Among the several behavioral anomalies exhibited by males expressing these relatively new mutations, some are unique. fru3 and fru4 males are less stimulated to court females than fru1 and fru2. No courtship pulse song is generated by either fru3 or fru4 males, even though they perform brief wing extensions. fru3 and fru4 males display significantly less chaining behavior than do fru1 males. The hierarchy of courtship responses by fru males directed toward females vs. males, when presented with both sexes simultaneously, is that fru1 males perform vigorous and indiscriminant courtship directed at either sex; fru4 males are similarly indiscriminant, but courtship levels were lower than fru1; fru2 males prefer females; fru3 males show a courtship bias toward males. fru3 and fru4 males essentially lack the Muscle of Lawrence (MOL). On several reproductive criteria, there was no difference between fru-variant females and fru+. The increases in phenotypic severity measured for the new mutants are discussed in the context of the emerging molecular genetics of fru and with regard to the gene's position within the sex-determination pathway.