Expression of xanthine dehydrogenase activity during embryonic development of Drosophila melanogaster

The contributions of oogenesis and zygotic genome expression to xanthine dehydrogenase activity during embryogenesis were investigated utilizing the mal and ry² mutants. In vitro complementation experiments demonstrated the presence of the mal⁺ complementation factor in the oocyte, suggesting an explanation for the mal maternal effect. The ry⁺ complementation factor synthesized from paternal template was detected at gastrulation. This is the earliest detection of a paternal enzyme during nonmammalian embryonic development.     

Anatomy of the stomatogastric nervous system associated with the foregut in Drosophila melanogaster and Calliphora vicina third instar larvae

The stomatogastric nervous system (SNS) associated with the foregut was studied in 3rd instar larvae of Drosophila melanogaster and Calliphora vicina (blowfly). In both species, the foregut comprises pharynx, esophagus, and proventriculus. Only in Calliphora does the esophagus form a crop. The position of nerves and neurons was investigated with neuronal tracers in both species and GFP expression in Drosophila. The SNS is nearly identical in both species. Neurons are located in the proventricular and the hypocerebral ganglion (HCG), which are connected to each other by the proventricular nerve. Motor neurons for pharyngeal muscles are located in the brain not, as in other insect groups, in the frontal ganglion. The position of the frontal ganglion is taken by a nerve junction devoid of neurons. The junction is composed of four nerves: the frontal connectives that fuse with the antennal nerves (ANs), the frontal nerve innervating the cibarial dilator muscles and the recurrent nerve that innervates the esophagus and projects to the HCG. Differences in the SNS are restricted to a crop nerve only present in Calliphora and an esophageal ganglion that only exists in Drosophila. The ganglia of the dorsal organs give rise to the ANs, which project to the brain. The extensive conformity of the SNS of both species suggests functional parallels. Future electrophysiological studies of the motor circuits in the SNS of Drosophila will profit from parallel studies of the homologous but more accessible structures in Calliphora.     

Evolution of vitellins in the ovary and during development in three species of Diptera, Drosophila melanogaster, Calliphora vicina and Prasarcophaga argyrostoma

The SDS electrophoresis reveals the asynchronous appearance of the three vitellins V1, V2, V3 during the ovarian cycle. It is always the V3 which appears first with the beginning of previtellogenesis. The vitellin degradation occurs late at the end of embryogenesis. The presence of a protein migrating at the level of V3 during the whole development is discussed.     

Functional characterization of a Drosophila melanogaster succinic semialdehyde dehydrogenase and a non-specific aldehyde dehydrogenase

The putative Drosophila (D.) melanogaster gene ortholog of mammalian succinic semialdehyde dehydrogenase (SSADH, EC1.2.1.24; NM_143151) that is involved in the degradation of the neurotransmitter GABA, and the putative D. melanogaster aldehyde dehydrogenase gene Aldh (NM_135441) were cloned and expressed as enzymatically active maltose binding protein (MalE) fusion products in Escherichia coli. The identities of the NM_143151 gene product as NAD+-dependent SSADH and of the Aldh gene product as NAD+-dependent non-specific aldehyde dehydrogenase (ALDH, EC1.2.1.3) were established by substrate specificity studies using 30 different aldehydes. In the case of D. melanogaster MalE-SSADH, the Michaelis constants (K(M)s) for the specific substrates succinic semialdehyde and NAD+ was 4.7 and 90.9 microM, respectively. For D. melanogaster MalE-ALDH the K(M) of the putative in vivo substrate acetaldehyde was 0.9 microM while for NAD+, a K(M) of 62.7 microM was determined. Site-directed mutagenesis studies on D. melanogaster MalE-SSADH suggest that cysteine 311 and glutamic acid 277 of this enzyme are likely candidates for the active site residues directly involved in catalysis.     

Purification and partial characterization of alcohol dehydrogenase,fructose-1,6-bisphosphate aldolase and the cytoplasmic form of malate dehydrogenase from Drosophila melanogaster

A purification scheme for the cytoplasmic form of malate dehydrogenase (s-MDH) of Drosophila melanogaster is presented which is superior to any previously reported method. In addition, this scheme can also be used to obtain alcohol dehydrogenase (ADH) and FDP aldolase. Gel filtration experiments reveal an oligomeric molecular weight of 69,000 for s-MDH, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate indicates subunit molcular weights of 32,100 for s-MDH, 24,600 for ADH and 34,000 for FDP aldolase. The amino acid composition of Drosophila melanogaster s-MDH and FDP aldolase are reported.     

Effects of adenine on the stability and expression of xanthine dehydrogenase from Drosophila melanogaster

The in vivo effect of adenine on xanthine dehydrogenase from Drosophila melanogaster has been studied by feeding adult flies for 48 hr on regular undefined media buffered with phosphate and containing 15 mM adenine. Extracts of adenine-fed flies show 25–30% reduction in xanthine dehydrogenase activity, and 35–40% reduction in uric acid production. The adenine exposed enzyme, however, is now more stable during overnight dialysis with the loss of activity never more than 30%, while controls show losses of over 50%.Thermal stability studies reveal that the enzyme from adenine-fed flies, in contrast to that from controls, is resistant to high temperature (50°C). During the 48-hr period of feeding, enzyme activity decreases in the control flies whereas this ageing effect is less pronounced in the adenine-fed flies. Column chromatography and gel electrophoresis, using [8-¹⁴C]adenine show that adenine appears to bind to xanthine dehydrogenase, with the radioactivity peaks always corresponding with enzyme activity peaks.     

Biosynthesis of dihydroxanthommatin in Drosophila melanogaster: Possible involvement of xanthine dehydrogenase

The aim of this research was to clarify the lower dihydroxanthommatin content in the mal mutant of Drosophila melanogaster as compared to its wild type (w.t.). By means of a new technique, the dihydroxanthommatin content was measured in w.t., mal, bw, mal bw and again in HPP-treated bw and mal bw. The results show that xanthine dehydrogenase (XDH) is indirectly involved in the biosynthesis of dihydroxanthommatin. This may be explained by the fact that the cofactor NADH, required by kynurenine-3-hydroxylase, is at least partially dependent on the XDH activity. This fully agrees with the chemotype of the mutants studied. Moreover, the remarkable decrease in dihydroxanthommatin content observed in both bw and mal bw as a consequence of the HPP treatment indicates that also in Drosophila melanogaster HPP may be an inhibitor in vivo of tryptophan pyrrolase.     

Molybdenum hydroxylases in Drosophila. III. Further characterization of the low xanthine dehydrogenase gene

The biochemical effects of several newly induced low xanthine dehydrogenase (lxd) mutations in Drosophila melanogaster were investigated. When homozygous, all lxd alleles simultaneously interrupt each of the molybdoenzyme activities to approximately the same levels: xanthine dehydrogenase, 25%; aldehyde oxidase, 12%; pyridoxal oxidase, 0%; and sulfite oxidase, 2% as compared to the wild type. In order to evaluate potentially small complementation or dosage effects, mutant stains were made coisogenic for 3R. These enzymes require a molybdenum cofactor, and lxd cofactor levels are also reduced to less than 10% of the wild type. These low levels of molybdoenzyme activities and cofactor activity are maintained throughout development from late larval to adult stages. The lxd alleles exhibit a dosage-dependent effect on molybdoenzyme activities, indicating that these mutants are leaky for wild-type function. In addition, cofactor activity is dependent upon the number of lxd ⁺ genes present. The lxd mutation results in the production of more thermolabile XDH and AO enzyme activities, but this thermolability is not transferred with the cofactor to a reconstituted Neurospora molybdoenzyme. The lxd gene is localized to salivary region 68 A4-9, 0.1 map unit distal to the superoxide dismutase (Sod) gene.     

Cloning and dosage compensation of the 6-phosphogluconate dehydrogenase gene (Pgd+) of Drosophila melanogaster

Using a heterologous rat cDNA probe, we have identified a 14.7 kbp Drosophila melanogaster genomic clone containing the X-linked gene Pgd+, which encodes the enzyme 6-phosphogluconate dehydrogenase (6PGD). We used in situ hybridization to larval polytene chromosomes, a somatic transient expression assay for enzyme activity, and the rescue of the lethal Pgd- phenotype by germline transformation to verify the identity of the gene. A 7.4 kbp fragment including the gene and approximately 1.2 kbp of upstream and 1.8 kbp of downstream sequences was relocated to autosomal ectopic sites by germline transformation; this transduced gene exhibits levels of enhanced activity in males comparable to those of the indigenous gene at its normal X chromosome locus. We conclude that the sequences responsible for dosage compensation of Pgd+ are included in this fragment.     

Cloning and characterization of the homologue of the Saccharomyces cerevisiae gene in Drosophila melanogaster]

RAD23 is an evolutionary conserved protein, which is essential for DNA excision repair. It is believed that this protein is present in all eukaryotic organisms from yeast to mammals. In this work, molecular cloning of the Drosophila melanogaster RAD23 gene and an analysis of the encoded protein are reported.     

Cloning,characterization, and embryonic expression analysis of the Drosophila melanogaster gene encoding insulin/relaxin-like peptide

Insulin is one of the key peptide hormones that regulates growth and metabolism in vertebrates. Evolutionary conservation of many elements of the insulin/IGF signaling network makes it possible to study the basic genetic function of this pathway in lower metazoan models such as Drosophila. Here we report the cloning and characterization of the gene for Drosophila insulin/relaxin-like peptide (DIRLP). The predicted protein structure of DIRLP greatly resembles typical insulin structure and contains features that differentiate it from the Drosophila juvenile hormone, another member of the insulin family. The Dirlp gene is represented as a single copy in the Drosophila melanogaster genome (compared to multiple copies for Drosophila juvenile hormone) and shows evolutionary conservation of genetic structure. The gene was mapped to the Drosophila chromosome 3, region 67D2. In situ hybridization of whole-mount Drosophila embryos with Dirlp antisense RNA probe reveals early embryonic mesodermal/ventral furrow expression pattern, consistent with earlier observation of the insulin protein immunoreactivity in Drosophila embryos. The in situ hybridization pattern was found to be identical to that obtained during immunohistochemistry analysis of the Drosophila embryos using various insulin monoclonal and polyclonal antibodies that do not recognize Drosophila juvenile hormone, supporting the idea that Dirlp is a possible Drosophila insulin ortholog. Identification of the gene for DIRLP provides a new approach for study of the regulatory pathway of the insulin family of peptides.