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PubMed2000

Expression and translocation of Drosophila nuclear encoded cytochrome b(5) proteins to mitochondria.

DNA sequence studies of cytochrome b(5) (Cyt-b) genes from Drosophila melanogaster and Drosophila virilis predict that the Drosophila Cyt-b proteins are extremely hydrophobic and have at least eight potential transmembrane spanning domains. Primary protein sequence analysis also predicts that the Cyt-b proteins have mitochondrial targeting sequences and they contain sites for potential post-translational modification similar to other cytochrome proteins. We report the characterization of the cytochrome b(5) proteins from Drosophila melanogaster and Drosophila virilis. We have used a Drosophila cytochrome b(5) specific antibody to demonstrate that cytochrome b(5) proteins are expressed in muscle-containing tissues in the fly. We also provide evidence that the nuclear encoded cytochrome b(5) protein that contains a mitochondrial targeting sequence is translocated to mitochondria.

Animals↗
PubMed1998

Molecular characterization of myosin V from Drosophila melanogaster.

Recent studies have revealed unconventional myosin V to be an important actin-based molecular motor involved in vesicular movement. In this paper we report the molecular characterization of the Drosophila myosin V, identified by reverse genetics. The gene encodes a 1792-residue, 207 kDa heavy chain polypeptide which possesses a typical head or motor domain of 771 residues, a region of six IQ motifs (139 residues) which serve as potential calmodulin/light chain binding sites at the head/tail junction, and a tail domain of 882 residues containing sequences of putative alpha-helical coiled-coils required for dimerization of the molecule and sequences of non-helical structure at the C-terminal end. Based on Southern blot analyses and chromosomal localization, evidence is presented for a single Drosophila myosin V gene. RNA analyses revealed a doublet of transcripts of about 6 kb, expressed throughout the lifetime of a fly but particularly abundant in the early stages of embryonic development (maternally contributed), in the ectodermic tissue of the hindgut starting at stage 16, and in the adult head. These results suggest that myosin V may be involved in processes required in a variety of cell types in Drosophila. We have also mapped the Drosophila myosin V locus to chromosome 2 at the position 43C-D, and we are currently searching for known mutations in this region. Finally, phylogenetic analysis of the head domain reveals that Drosophila myosin V is more closely related to mammalian myosin Va and Vb than to other invertebrate class-V myosins; nevertheless, it is not significantly more related to myosin Va than to myosin Vb. While vertebrates would need two different myosin V isoforms to accomplish specific functions, we speculate that Drosophila myosin V might provide the equivalent functions by itself.

Amino Acid Sequence↗
PubMed2000

Parental imprinting in Drosophila.

Genetic imprinting is a form of epigenetic silencing. But with a twist. The twist is that while imprinting results in the silencing of genes, chromosome regions or entire chromosome sets, this silencing occurs only after transmission of the imprinted region by one sex of parent. Thus genetic imprinting reflects intertwined levels of epigenetic and developmental modulation of gene expression. Imprinting has been well documented and studied in Drosophila, however, these studies have remained largely unknown due to nothing more significant than differences in terminology. Imprinting in Drosophila is invariably associated with heterochromatin or regions with unusual chromatin structure. The imprint appears to spread from imprinted centers that reside within heterochromatin and these are, seemingly, the only regions that are normally imprinted in Drosophila. This is significant as it implies that while imprinting occurs in Drosophila, it is generally without phenotypic consequence. Hence the evolution of imprinting, at least in Drosophila, is unlikely to be driven by the function of specific imprinted genes. Thus, the study of imprinting in Drosophila has the potential to illuminate the mechanism and biological function of imprinting, and challenge models based solely on imprinting of mammalian genes.

Animals↗
PubMed1996

Expression of human dopamine beta-hydroxylase in Drosophila Schneider 2 cells.

Human dopamine beta-hydroxylase (DBH) has been expressed in transformed Drosophila Schneider 2 (S2) cells with yields of > 16 mg/l. Most of the activity was found in the culture fluid. Similarly, human neuroblastoma cells also secrete native DBH into the medium, but at a much lower level than recombinant Drosophila cells. We have purified native and recombinant human DBH by a modified purification procedure using SP-Sepharose, lentil lectin-Sepharose and gel-filtration chromatography and carried out studies to compare the two enzymes. Two variants of human DBH that differ by a single amino acid (either serine or alanine) at position 304 were expressed in Drosophila cells, purified, and found to have no significant difference in enzyme activity. The molecular mass of human DBH monomer has been determined from SDS/PAGE to be 73 kDa, but the recombinant DBH from Drosophila is smaller at 66 kDa. The difference may be due to glycosylation as deglycosylated enzymes from both sources are identical in size (61 kDa). The Km of tyramine for native and recombinant human enzymes are virtually the same but higher than bovine DBH by about 3-fold. Likewise, the inhibition of native and recombinant human DBH by fusaric acid and SKF102698 is not significantly different but IC50 values are 2-3-fold higher than that for the bovine enzyme. These results strongly support the conclusion that recombinant human DBH from Drosophila S2 cells can be used in place of human neuroblastoma-derived DBH for drug screening, characterization of the enzyme's physicochemical properties, and determination of structure-function relationships. The Drosophila expression system has thus provided a convenient source for large quantities of human DBH enzyme.

Amino Acid Sequence↗
PubMed2003

Myogenic differentiation of Drosophila Schneider cells by DNA double-strand break-inducing drugs.

Drosophila melanogaster has been widely used as a model organism to study various aspects of development. Apart from the whole Drosophila embryo, there are a number of cultured cell lines derived from Drosophila embryo that have also been used for elucidating various aspects of development. Drosophila Schneider line 2 cells were derived from the late stages of the embryo (Schneider, 1972). We found that the Schneider cells undergo myogenic differentiation upon treatment with neocarzinostatin (NCS), DNA double-strand break (DSB)-inducing drug, as indicated by elongated morphology, myosin heavy chain protein expression, multinucleation and exit from the cell cycle. No induction of differentiation was observed when cell proliferation was inhibited with drugs that do not cause DNA DSBs. Pre-treatment of Schneider cells with inhibitors of PKC, PP 1/2A, p38 MAPK, JNK and proteasomes resulted in the inhibition of morphological differentiation induced by NCS. These results indicate that DNA DSBs can turn on the myogenic program in Drosophila Schneider cells and the process is dependent on PK C-, PP 1/2A-, p38 MAPK-, and JNK- mediated signaling and proteasomal activity. The molting hormone, 20-hydroxyecdysone (20-HE), also showed an anti-myogenic effect on the process. This is the first report of insect cells undergoing differentiation by DNA DSB-inducing drugs as far as we know, and it provides a very useful and convenient in vitro system to study various aspects of Drosophila myogenesis.

Acetylcysteine↗
PubMed1996

Novel neonicotinoid-agarose affinity column for Drosophila and Musca nicotinic acetylcholine receptors.

Neonicotinoids such as the insecticide imidacloprid (IMI) act as agonists at the insect nicotinic acetylcholine receptor (nAChR). Head membranes of Drosophila melanogaster and Musca domestica have a single high-affinity binding site for [3H]IMI with KD values of 1-2 nM and Bmax values of 560-850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an alpha-bungarotoxin (alpha-BGT)-agarose affinity column are known to be alpha-subunit homooligomers. This study uses 1-[N-(6-chloro-3-pyridylmethyl)-N-ethyl]amino-1-amino-2-nitroethene++ + (which inhibits [3H]IMI binding to Drosophila and Musca head membranes at 2-3 nM) to develop a neonicotinoid-agarose affinity column. The procedure-introduction of Triton-solubilized Drosophila or Musca head membranes into this neonicotinoid-based column, elution with IMI, and analysis by lithium dodecyl sulfate-polyacrylamicle gel electrophoresis-gives only three proteins (69, 66, and 61 kDa) tentatively assigned as putative subunits of the nAChR; the same three proteins are obtained with Musca using the alpha-BGT-agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with 125I-alpha-BGT-4-azidosalicylic acid gives a labeled derivative of 66-69 kDa. The yield is 2-5 micrograms of receptor protein from 1 g of Drosophila or Musca heads. Neonicotinoid affinity chromatography to isolate native Drosophila and Musca receptors will facilitate studies on the structure and function of insect nAChRs.

Animals↗
PubMed1981

Integration, transcription, and control of a Drosophila heat shock gene in mouse cells.

Mouse L cells were transformed with a cloned 3.6-kilobase (kb) segment of Drosophila melanogaster DNA carrying the 2.25-kb transcribed sequence for the Drosophila 70,000-dalton heat shock protein (hsp70) and 1.1 kb and 0.2 kb of 5' and 3' flanking DNA, respectively. Heat shock of one of three such transformed cell lines containing multiple copies of the intact Drosophila segment induced the abundant accumulation of transcripts of the Drosophila gene, with correct or nearly correct 5' and 3' termini. This provides evidence, in accord with earlier indications, that diverse eukaryotes, including vertebrates, have heat shock systems similar to that studied extensively in Drosophila. Our results suggest that the signals for heat shock transcription and the chromosomal sites with which they interact have been highly conserved in evolution and that the regulatory sequences controlling transcription of the gene for hsp70 lie within the 3.6-kb Drosophila segment.

Animals↗
PubMed1982

Small nuclear ribonucleoprotein complexes of Drosophila melanogaster.

We report here on the isolation and characterization of small nuclear ribonucleoproteins (snRNPs) and corresponding small nuclear RNA (snRNA) species from nuclei of Drosophila melanogaster. Velocity sedimentation in sucrose gradients was used to partially fractionate the RNPs; analysis of fractions so obtained suggests that, in general, one snRNP contains one snRNA. At least 11 species of snRNA are present in Drosophila nuclei; among them we identify a potential mammalian U1 homolog based on sequence homology. Autoimmune antiserum designated anti-Sm from patients with systemic lupus erythematosus recognizes nuclear antigens in Drosophila and precipitates seven species of snRNPs. The antigens in HeLa and Drosophila nuclei recognized by anti-Sm antibodies have been identified and compared. Anti-Sm antibodies at least bind to a 26,000-dalton polypeptide in HeLa extracts and to two polypeptides, one of 18,000 daltons and one of 26,000 daltons, in Drosophila extracts. This suggests that the 26,000-dalton polypeptide is an evolutionarily conserved antigenic component of Drosophila and HeLa snRNPs.

Animals↗
PubMed1983

Identification of a cytoplasmic tropomyosin gene linked to two muscle tropomyosin genes in Drosophila.

A Drosophila cytoplasmic tropomyosin gene has been identified and is located on the same genomic DNA clone as two Drosophila muscle tropomyosin genes previously identified. A positive hybrid-selection translation assay using the subcloned gene and RNA from non-muscle cell sources yielded a protein with a size (Mr, 31,000) and isoelectric point (5.0) similar to vertebrate cytoplasmic tropomyosin. A modified protocol for the purification of vertebrate cytoplasmic tropomyosin was used to partially purify cytoplasmic tropomyosin from the Drosophila Kc cell line. The Kc cell protein was identified as a cytoplasmic form of tropomyosin on the basis of its size, isoelectric point, and crossreactivity with a polyclonal vertebrate antitropomyosin antibody in a two-step binding assay. The Kc cell cytoplasmic tropomyosin comigrates in two dimensions with the hybrid-selected in vitro translation product of the region 3 gene, and both proteins show a mobility shift in NaDodSO4/urea/polyacrylamide gels that is characteristic of vertebrate tropomyosins. The cytoplasmic tropomyosin gene hybridizes to both Drosophila muscle tropomyosin genes under decreased stringency conditions. This cross-hybridization spans several internal restriction endonuclease sites in each muscle tropomyosin gene and indicates an overall partial homology among the three Drosophila tropomyosin genes. These results show that Drosophila tropomyosins are encoded by a closely linked family of differentially regulated genes.

Animals↗
PubMed1984

Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous.

The Escherichia coli dnaK gene is homologous to the major heat shock-induced gene in Drosophila (Hsp70). The primary DNA sequence of the entire protein-coding region of the dnaK gene was determined and compared with that of the Hsp70 gene of Drosophila. The two sequences are homologous; the dnaK gene could encode a 69,121-Da polypeptide, 48% identical to the hsp70 protein of Drosophila. The homology between the Hsp70 gene of Drosophila and the E. coli dnaK gene illustrates the remarkable conservation of the heat shock genes in evolution. In contrast to Drosophila and Saccharomyces cerevisiae, both of which contain multigene families related to the Hsp70 gene, hybridization analyses indicate that E. coli contains only a single Hsp70-related gene, dnaK. Hybridization between the DNA of an archaebacterium Methanosarcina barkeri and the Hsp70 genes of Drosophila, Saccharomyces, and E. coli has been detected, suggesting the existence of Hsp70-related genes in the three "primary kingdoms": eukaryotes, eubacteria, and archaebacteria.

Amino Acid Sequence↗
PubMed1985

A human acetylcholinesterase gene identified by homology to the Ace region of Drosophila.

The Ace locus of the Drosophila genome controls biosynthesis of the neurotransmitter-hydrolyzing enzyme acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). We injected the mRNA species hybridizing with DNA fragments from this region into Xenopus oocytes, in which acetylcholinesterase mRNA is translated into active acetylcholinesterase. A 2.0-kilobase (kb) fragment of DNA from this region selectively hybridizes with Drosophila mRNA capable of inducing the biosynthesis of acetylcholinesterase in oocytes. This Drosophila DNA fragment cross-hybridized with human brain poly(A)+ RNA. We therefore used this DNA fragment as a probe for homologous sequence(s) in a human genomic DNA library and thus selected a 13.5-kb human DNA segment. DNA blot-hybridization revealed that a 2.6-kb fragment of this human DNA segment hybridizes with the Drosophila 2.0-kb DNA fragment. Both Drosophila and human fragments hybridized with a human brain mRNA species of about 7.0-kb that was barely detectable in the acetylcholinesterase-deficient HEp carcinoma. A fraction containing mRNA of similar size, extracted from human brain, induced acetylcholinesterase biosynthesis in oocytes. The human DNA fragment also was used in hybridization-selection experiments. In oocytes, hybrid-selected human brain mRNA induced acetylcholinesterase activity that was completely inhibited by 1,5-bis[4-allyldimethylammonium)phenyl]pentan-3-one dibromide but not by tetraisopropyl pyrophosphamide, a differential response to these inhibitors characteristic of "true" human brain acetylcholinesterase. These findings strongly suggest that both the Drosophila and the human DNA fragments are directly involved in controlling acetylcholinesterase biosynthesis.

Acetylcholinesterase↗
PubMed1986

Discordance of nuclear and mitochondrial DNA phylogenies in Hawaiian Drosophila.

Drosophila differens, endemic to Molokai, Drosophila planitibia of Maui, and Drosophila silvestris and Drosophila heteroneura from the island of Hawaii are chromosomally homosequential species that presumably have colonized the newer islands of the Hawaiian archipelago by sequential founder events. We have examined the phylogenetic relationships of these four species by using mitochondrial DNA restriction site data for 23 enzymes. Both distance and character-state analyses indicate that a sequential or monotonic branching relationship exists for mtDNA restriction site data from the four species. The mtDNA data suggest that the maternal lineage that gave rise to D. differens is ancestral to the D. planitibia maternal lineage, which in turn shares the most recent common ancestor with the D. silvestris and D. heteroneura maternal lineages [with Drosophila hemipeza (Oahu) and Drosophila neopicta (Molokai and Maui) as outside references]. We also discuss the phylogenetic implications of the mtDNA data in comparison with other sources of phylogenetic data. We conclude that hybridization of the species in this group has been an important factor in the evolution of the nuclear genomes. Because of small population sizes and mating asymmetries, it is possible that the nuclear genetic distance of species that are physically capable of hybridizing (e.g., on the same island or island complex) is depressed. Consequently the mtDNA genetic distance appears to be more sensitive in establishing the sequence of evolutionary events responsible for the present distribution and population structure of these species.

Animals↗
PubMed1988

Functional expression of a Drosophila gene in yeast: genetic complementation of DNA topoisomerase II.

Since DNA topoisomerase II (EC 5.99.1.3) is an essential enzyme in yeast, heterologous topoisomerase II gene expression in yeast cells can provide a system for analyzing the structure and function of topoisomerase II genes from other species. A series of yeast expression plasmids was constructed in which segments of the cDNA sequences encoding Drosophila DNA topoisomerase II were inserted under the transcriptional control of yeast GAL1 promoter. Expression of the functional form of Drosophila topoisomerase II cDNA can complement conditionally lethal, temperature-sensitive mutations in the yeast topoisomerase II gene (TOP2), as well as mutations in which the TOP2 locus was disrupted. The survival of these yeast cells depends upon the continuous expression of Drosophila topoisomerase II. Repression of Drosophila gene expression by glucose causes these yeast cells to cease dividing after a few generations. In addition to these genetic complementation data, the expression of the Drosophila topoisomerase II gene in yeast cells with a disruption in TOP2 can also be detected by immunochemical methods with an antibody specific for Drosophila topoisomerase II.

Alleles↗
PubMed1988

Isolation and characterization of the gene encoding the heavy chain of Drosophila kinesin.

An antiserum that recognizes the heavy chain of Drosophila kinesin was used to isolate Drosophila cDNA clones. Immunoblot analysis of the proteolytic fragments of the protein produced by one of the cDNA clones has demonstrated that the cDNA clones encode the heavy chain of Drosophila kinesin. The in vitro-synthesized product of the largest cDNA comigrates with Drosophila kinesin heavy chain on NaDodSO4/polyacrylamide gels and binds to taxol-stabilized microtubules in the presence of the nonhydrolyzable analogue of ATP, 5'-adenylyl imidodiphosphate, but not in the presence of ATP or 0.1 M KCl. Analysis of the cDNA clones suggests that there is a single gene encoding kinesin heavy chain in Drosophila located at polytene chromosome position 53A. However, Southern hybridization analyses suggest the presence of related sequences in the Drosophila genome.

Animals↗
PubMed1989

The homeotic gene spalt (sal) evolved during Drosophila speciation.

The region-specific homeotic gene spalt (sal) acts in two separate domains in the head and tail region of the Drosophila melanogaster embryo. Based on comparative morphology, sal is likely to be involved in the establishment of the head during the evolution of invertebrates and thus, it should be conserved. We have analyzed the conservation of the segmentation genes Krüppel (Kr) and even-skipped (eve) in parallel with sal coding sequences in several Drosophila species that are evolutionarily separated by up to 60 million years. To our surprise, sal sequences appear to be conserved in the Sophophora subgenus of the Drosophila genus but not in the Drosophila subgenus. On the other hand, the segmentation and other homeotic genes are conserved in the Drosophila subgroup as well. Our data suggest that sal encodes an accessory function that evolved relatively late during Drosophila speciation rather than playing a fundamental evolutionary role similar to that of other homeotic genes.

Amino Acid Sequence↗
PubMed2000

A novel activation mechanism of caspase-activated DNase from Drosophila melanogaster.

Caspase-activated DNase (CAD) is an enzyme that cleaves chromosomal DNA in apoptotic cells. Here, we identified a DNase in Drosophila Schneider cells that can be activated by caspase 3, and purified it as a complex of two subunits (p32 and p20). Using primers based on the amino acid sequence of the purified proteins, a cDNA coding for Drosophila CAD (dCAD) was cloned. The polypeptide encoded by the cDNA contained 450 amino acids with a calculated M(r) of 52,057, and showed significant homology with human and mouse CAD (22% identity). Mammalian CADs carry a nuclear localization signal at the C terminus. In contrast, dCAD lacked the corresponding sequence, and the purified dCAD did not cause DNA fragmentation in nuclei in a cell-free system. When dCAD was co-expressed in COS cells with Drosophila inhibitor of CAD (dICAD), a 52-kDa dCAD was produced as a heterotetrameric complex with dICAD. When the complex was treated with human caspase 3 or Drosophila caspase (drICE), the dICAD was cleaved, and released from dCAD. In addition, dCAD was also cleaved by these caspases, and behaved as a (p32)(2)(p20)(2) complex in gel filtration. When a Drosophila neuronal cell line was induced to apoptosis by treatment with a kinase inhibitor, both dCAD and dICAD were cleaved. These results indicated that unlike mammalian CAD, Drosophila CAD must be cleaved by caspases to be activated.

Amino Acid Sequence↗
PubMed2000

Identification and characterization of a SUMO-1 conjugation system that modifies neuronal calcium/calmodulin-dependent protein kinase II in Drosophila melanogaster.

Drosophila Uba2 and Ubc9 SUMO-1 conjugation enzyme homologs (DmUba2 and DmUbc9) were isolated as calcium/calmodulin-dependent kinase II (CaMKII) interacting proteins by yeast two-hybrid screening of an adult head cDNA library. We found that at least one isoform of Drosophila neuronal CaMKII is conjugated to DmSUMO-1 in vivo. The interactions observed in the two-hybrid screen may therefore reflect catalytic events. To understand the role of SUMO conjugation in the brain, we undertook a characterization of the system. The other required components of the system, Drosophila Aos1 and SUMO-1 (DmAos1 and DmSUMO-1), were identified in expressed sequence tag data base searches. Purified recombinant DmUba2/DmAos1 dimer can activate DmSUMO-1 in vitro and transfer DmSUMO-1 to recombinant DmUbc9. DmSUMO-1 conjugation occurs in all developmental stages of Drosophila and in the adult central nervous system. Overexpression of a putative dominant negative DmUba2(C175S) mutant protein in the Drosophila central nervous system resulted in an increase in overall DmSUMO-1 conjugates and a base-sensitive p120 species, which is likely to be DmUba2(C175S) linked to endogenous DmSUMO-1 through an oxygen ester bond. Overexpression of DmUba2(wt) protein in vivo also led to increased levels of DmSUMO-1 conjugates. High level overexpression of either DmUba2(wt) or DmUba2(C175S) in the Drosophila central nervous system caused pupal and earlier stage lethality. Expression in the developing eye led to a rough eye phenotype with retinal degeneration. These results suggest that normal SUMO conjugation is essential in the differentiated nervous system and reveal a potential novel mechanism that regulates neuronal calcium/calmodulin-dependent protein kinase II function.

Amino Acid Sequence↗
PubMed2001

Identification and characterization of a Drosophila nuclear proteasome regulator. A homolog of human 11 S REGgamma (PA28gamma ).

We report the cloning and characterization of a Drosophila proteasome 11 S REGgamma (PA28) homolog. The 28-kDa protein shows 47% identity to the human REGgamma and strongly enhances the trypsin-like activities of both Drosophila and mammalian 20 S proteasomes. Surprisingly, the Drosophila REG was found to inhibit the proteasome's chymotrypsin-like activity against the fluorogenic peptide succinyl-LLVY-7-amino-4-methylcoumarin. Immunocytological analysis reveals that the Drosophila REG is localized to the nucleus but is distributed throughout the cell when nuclear envelope breakdown occurs during mitosis. Through site-directed mutagenesis studies, we have identified a functional nuclear localization signal present in the homolog-specific insert region. The Drosophila PA28 NLS is similar to the oncogene c-Myc nuclear localization motif. Comparison between uninduced and innate immune induced Drosophila cells suggests that the REGgamma proteasome activator has a role independent of the invertebrate immune system. Our results support the idea that gamma class proteasome activators have an ancient conserved function within metazoans and were present prior to the emergence of the alpha and beta REG classes.

Amino Acid Sequence↗