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PubMed2000

Molecular cloning of cDNA encoding a drosophila ryanodine receptor and functional studies of the carboxyl-terminal calcium release channel.

Ryanodine is a plant alkaloid that was originally used as an insecticide. To study the function and regulation of the ryanodine receptor (RyR) from insect cells, we have cloned the entire cDNA sequence of RyR from the fruit fly Drosophila melanogaster. The primary sequence of the Drosophila RyR contains 5134 amino acids, which shares approximately 45% identity with RyRs from mammalian cells, with a large cytoplasmic domain at the amino-terminal end and a small transmembrane domain at the carboxyl-terminal end. To characterize the Ca(2+) release channel activity of the cloned Drosophila RyR, we expressed both full-length and a deletion mutant of Drosophila RyR lacking amino acids 277-3650 (Drosophila RyR-C) in Chinese hamster ovary cells. For subcellular localization of the expressed Drosophila RyR and Drosophila RyR-C proteins, green fluorescent protein (GFP)-Drosophila RyR and GFP-Drosophila RyR-C fusion constructs were generated. Confocal microscopic imaging identified GFP-Drosophila RyR and GFP-Drosophila RyR-C on the endoplasmic reticulum membranes of transfected cells. Upon reconstitution into the lipid bilayer membrane, Drosophila RyR-C formed a large conductance cation-selective channel, which was sensitive to modulation by ryanodine. Opening of the Drosophila RyR-C channel required the presence of microM concentration of Ca(2+) in the cytosolic solution, but the channel was insensitive to inhibition by Ca(2+) at concentrations as high as 20 mM. Our data are consistent with our previous observation with the mammalian RyR that the conduction pore of the calcium release channel resides within the carboxyl-terminal end of the protein and further demonstrate that structural and functional features are essentially shared by mammalian and insect RyRs.

Amino Acid Sequence↗
PubMed1977

Transcription and translation of cloned Drosophila DNA fragments in Escherichia coli.

The expression of three unique DNA fragments from Drosophila melanogaster which have been inserted into Escherichia coli (E. coli) via the plasmid, pSC 101, was studied. The hybrid plasmid DNA molecules containing Drosophila DNA were transformed into the minicell producing strain of E. coli, X1411. Drosophila DNA-directed RNA synthesis was studied by hybridizing newly synthesized RNA isolated from the minicells with various DNA fragments which were immobilized on nitrocellulose filters. RNA was synthesized as readily from the inserted Drosophila DNA as from the original bacterial plasmid, pSC 101. In one case, transcription appeared to be initiated preferentially on one of the two strands of a Drosophila DNA fragment regardless of the orientation of that Drosophila DNA fragment with respect to the pSC 101 sequences. Two of the three Drosophila DNA fragments did not induce the synthesis of new polypeptides in minicells as detected by autoradiography of [35S]methionine-labeled polypeptides on polyacrylamide gels. The third Drosophila DNA fragment caused the synthesis of one additional polypeptide of 29 000 daltons. When an 8200 base pair portion of the third inserted Drosophila DNA (63% OF THE TOTAL Drosophila insertion) was removed by digestion with the restriction enzyme, Eco R1, this new polypeptide was no longer synthesized by minicells containing the remaining Drosophila DNA. When the 8200 base pair fragment was placed back into its parent plasmid as an inversion, the new polypeptide did not reappear. In addition, the presence of some, but not all, of the Drosophila DNA insertions affected the relative synthesis of the six polypeptides coded for by the parent plasmid, pSC 101.

Animals↗
PubMed2002

Characterization of a novel Drosophila melanogaster galectin. Expression in developing immune, neural, and muscle tissues.

We have cloned and characterized the first galectin to be identified in Drosophila melanogaster. The amino acid sequence of Drosophila galectin showed striking sequence similarity to invertebrate and vertebrate galectins and contained amino acids that are crucial for binding beta-galactoside sugars. Confirming its identity as a galectin family member, the Drosophila galectin bound beta-galactoside sugars. Structurally, the Drosophila galectin was a tandem repeat galectin containing two carbohydrate recognition domains connected by a unique peptide link. This divalent structure suggests that like mammalian galectins, Drosophila galectin may mediate cell-cell communication or facilitate cross-linking of receptors to trigger signal transduction events. The Drosophila galectin was very abundant in embryonic, larval, and adult Drosophila. During embryogenesis, Drosophila galectin had a unique and specific tissue distribution. Drosophila galectin expression was concentrated in somatic and visceral musculature and in the central nervous system. Similar to other insect lectins, Drosophila galectin may function in both embryogenesis and in host defense. Drosophila galectin was expressed by hemocytes, circulating phagocytic cells, suggesting a role for Drosophila galectin in the innate immune system.

Amino Acid Sequence↗
PubMed2002

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.

Amino Acid Sequence↗
PubMed2002

Characterization and isolation of novel microsatellites from the Drosophila dunni subgroup.

We have isolated and characterized 77 novel microsatellites from two species, Drosophila dunni and Drosophila nigrodunni, which are closely related Caribbean-island endemics from the Drosophila cardini species group. These species are very distantly related to all other Drosophila from which microsatellites have previously been characterized. We find that the average length of microsatellites isolated in these species is quite small, with an overall mean length of 9.8 repeat units for dinucleotide microsatellites in the two study species. The nucleotide composition of dinucleotides differs between the two species: D. nigrodunni has a predominance of (AC/GT)n repeats, whereas D. dunni has equal numbers of (AC/GT)n and (AG/CT)n repeats. Tri- and tetranucleotide repeats are not abundant in either species. We assayed the variability of eight microsatellites in a closely related third species, Drosophila arawakana, using wild-caught individuals from the island of Guadeloupe. We found the microsatellites to be extremely variable in this population, with observed heterozygosities ranging from 0.541 to 0.889. DNA amplification trials suggest that these eight microsatellites are widely conserved across the D. cardini group, with five of the eight producing amplification products in every species tested. However, the loci are very poorly conserved over greater phylogenetic distances. DNA amplification of the microsatellite loci was unreliable in members of the closely related Drosophila quinaria, Drosophila calloptera, Drosophila guarani and Drosophila tripunctata species groups. Furthermore, these microsatellites could not be detected in the genome of Drosophila melanogaster, despite the conservation of microsatellite flanking regions at some loci. These data indicate that Drosophila microsatellite loci are quite short lived over evolutionary timescales relative to many other taxa.

Animals↗
PubMed1997

Temperature-sensitive expression of Drosophila neuronal nicotinic acetylcholine receptors.

Heterologous expression of cloned Drosophila nicotinic acetylcholine receptor (nAChR) subunits indicates that these proteins misfold when expressed in mammalian cell lines at 37 degrees C. This misfolding can, however, be overcome either by growing transfected mammalian cells at lower temperatures or by the expression of Drosophila nAChR subunits in a Drosophila cell line. Whereas the Drosophila nAChR beta subunit (SBD) cDNA, reported previously, lacked part of the SBD coding sequence, here we report the construction and expression of a full-length SBD cDNA. We have examined whether problems in expressing functional Drosophila nAChRs in either Xenopus oocytes or mammalian cell lines can be attributed to an inability of these expression systems to assemble correctly Drosophila nAChRs. Despite expression in what might be considered a more native cellular environment, we have been unable to detect functional nAChRs in a Drosophila cell line unless Drosophila nAChR subunit cDNAs are coexpressed with vertebrate nAChR subunits. Our results indicate that the folding of Drosophila nAChR subunits is temperature-sensitive and strongly suggest that the inability of these Drosophila nAChR subunits to generate functional channels in the absence of vertebrate subunits is due to a requirement for coassembly with as yet unidentified Drosophila nAChR subunits.

Amino Acid Sequence↗
PubMed1994

Simian virus 40 origin- and T-antigen-dependent DNA replication with Drosophila factors in vitro.

DNA replication of double-stranded simian virus 40 (SV40) origin-containing plasmids, which has been previously thought to be a species-specific process that occurs only with factors derived from primate cells, is catalyzed with an extract derived from embryos of the fruit fly Drosophila melanogaster. This reaction is dependent upon both large T antigen, the SV40-encoded replication initiator protein and DNA helicase, and a functional T-antigen binding site at the origin of DNA replication. The efficiency of replication with extracts derived from Drosophila embryos is approximately 10% of that observed with extracts prepared from human 293 cells. This activity is not a unique property of embryonic extracts, as cytoplasmic extracts from Drosophila tissue culture cells also support T-antigen-mediated replication of SV40 DNA. By using highly purified proteins, DNA synthesis is initiated by Drosophila polymerase alpha-primase in a T-antigen-dependent manner in the presence of Drosophila replication protein A (RP-A; also known as single-stranded DNA-binding protein), but neither human RP-A nor Escherichia coli single-stranded DNA-binding protein could substitute for Drosophila RP-A. In reciprocal experiments, however, Drosophila RP-A was able to substitute for human RP-A in reactions carried out with human polymerase alpha-primase. These results collectively indicate that many of the specific functional interactions among T antigen, polymerase alpha-primase, and RP-A are conserved from primates to Drosophila species. Moreover, the observation that SV40 DNA replication can be performed with Drosophila factors provides a useful assay for the study of bidirectional DNA replication in Drosophila species in the context of a complete replication reaction.

Animals↗
PubMed2000

Molecular cloning and genomic organization of an allatostatin preprohormone from Drosophila melanogaster.

The insect allatostatins are neurohormones, acting on the corpora allata (where they block the release of juvenile hormone) and on the insect gut (where they block smooth muscle contraction). We screened the "Drosophila Genome Project" database with electronic sequences corresponding to various insect allatostatins. This resulted in alignment with a DNA sequence coding for some Drosophila allatostatins (drostatins). Using PCR with oligonucleotide primers directed against the presumed exons of this Drosophila allatostatin gene and subsequent 3'- and 5'-RACE, we were able to clone its cDNA. The Drosophila allatostatin preprohormone contains four amino acid sequences that after processing would give rise to four Drosophila allatostatins: Val-Glu-Arg-Tyr-Ala-Phe-Gly-Leu-NH(2) (drostatin-1), Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH(2) (drostatin-2), Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH(2) (drostatin-3), and Thr-Thr-Arg-Pro-Gln-Pro-Phe-Asn-Phe-Gly-Leu-NH(2) (drostatin-4). Drostatin-2 is identical to helicostatin-2 (11-18) and drostatin-3 to helicostatin-3, two neurohormones previously isolated from the moth Helicoverpa armigera. Furthermore, drostatin-3 has previously been isolated from Drosophila itself. Drostatins-1 and -4 are novel members of the insect allatostatin neuropeptide family. The Drosophila allatostatin preprohormone gene contains two introns and three exons. The gene is located on the right arm of the third chromosome, position 96A-B. The existence of at least four different Drosophila allatostatins opens the possibility of a differential action of some of these hormones on the two recently cloned Drosophila allatostatin receptors, DAR-1 and -2. This is the first report on an allatostatin preprohormone from Drosophila.

Amino Acid Sequence↗
PubMed2003

Cloning and characterization of the Drosophila U7 small nuclear RNA.

Base pairing between the 5' end of U7 small nuclear RNA (snRNA) and the histone downstream element (HDE) in replication-dependent histone pre-mRNAs is the key event in 3'-end processing that leads to generation of mature histone mRNAs. We have cloned the Drosophila U7 snRNA and demonstrated that it is required for histone pre-mRNA 3'-end processing in a Drosophila nuclear extract. The 71-nt Drosophila U7 snRNA is encoded by a single gene that is embedded in the direct orientation in an intron of the Eip63E gene. The U7 snRNA gene contains conserved promoter elements typical of other Drosophila snRNA genes, and the coding sequence is followed by a 3' box indicating that the Drosophila U7 snRNA gene is an independent transcription unit. Drosophila U7 snRNA contains a trimethyl-guanosine cap at the 5' end and a putative Sm-binding site similar to the unique Sm-binding site found in other U7 snRNAs. Drosophila U7 snRNA is approximately 10 nt longer than mammalian U7 snRNAs because of an extended 5' sequence and has only a limited potential to form a stem-loop structure near the 3' end. The extended 5' end of Drosophila U7 snRNA can base pair with the HDE in all five Drosophila histone pre-mRNAs. Blocking the 5' end of the U7 snRNA with a complementary oligonucleotide specifically blocks processing of a Drosophila histone pre-mRNA. Changes in the HDE that abolish or decrease processing efficiency result in a reduced ability to recruit U7 snRNA to the pre-mRNA.

Animals↗
PubMed2001

Conversion of the bifunctional 8-oxoguanine/beta-delta apurinic/apyrimidinic DNA repair activities of Drosophila ribosomal protein S3 into the human S3 monofunctional beta-elimination catalyst through a single amino acid change.

The Drosophila S3 ribosomal protein has important roles in both protein translation and DNA repair. In regards to the latter activity, it has been shown that S3 contains vigorous N-glycosylase activity for the removal of 8-oxoguanine residues in DNA that leaves baseless sites in their places. Drosophila S3 also possesses an apurinic/apyrimidinic (AP) lyase activity in which the enzyme catalyzes a beta-elimination reaction that cleaves phosphodiester bonds 3' and adjacent to an AP lesion in DNA. In certain situations, this is followed by a delta-elimination reaction that ultimately leads to the formation of a single nucleotide gap in DNA bordered by 5'- and 3'-phosphate groups. The human S3 protein, although 80% identical to its Drosophila homolog and shorter by only two amino acids, has only marginal N-glycosylase activity. Its lyase activity only cleaves AP DNA by a beta-elimination reaction, thus further distinguishing itself from the Drosophila S3 protein in lacking a delta-elimination activity. Using a hidden Markov model analysis based on the crystal structures of several DNA repair proteins, the enzymatic differences between Drosophila and human S3 were suggested by the absence of a conserved glutamine residue in human S3 that usually resides at the cleft of the deduced active site pocket of DNA glycosylases. Here we show that the replacement of the Drosophila glutamine by an alanine residue leads to the complete loss of glycosylase activity. Unexpectedly, the delta-elimination reaction at AP sites was also abrogated by a change in the Drosophila glutamine residue. Thus, a single amino acid change converted the Drosophila activity into one that is similar to that possessed by the human S3 protein. In support of this were experiments executed in vivo that showed that human S3 and the Drosophila site-directed glutamine-changed S3 performed poorly when compared with Drosophila wild-type S3 and its ability to protect a bacterial mutant from the harmful effects of DNA-damaging agents.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and characterization of the genes encoding the two subunits of Drosophila melanogaster calcineurin.

Genomic clones containing the full coding sequences of the two subunits of the Ca2+/calmodulin-stimulated protein phosphatase, calcineurin, were isolated from a Drosophila melanogaster genomic library using highly conserved human cDNA probes. Three clones encoded a 19.3-kDa protein whose sequence is 88% identical to that of human calcineurin B, the Ca(2+)-binding regulatory subunit of calcineurin. The coding sequences of the Drosophila and human calcineurin B genes are 69% identical. Drosophila calcineurin B is the product of a single intron-less gene located at position 4F on the X chromosome. Drosophila genomic clones encoding a highly conserved region of calcineurin A, the catalytic subunit of calcineurin, were used to locate the calcineurin A gene at position 21 EF on the second chromosome of Drosophila and to isolate calcineurin A cDNA clones from a Drosophila embryonic cDNA library. The structure of the calcineurin A gene was determined by comparison of the genomic and cDNA sequences. Twelve exons, spread over a total of 6.6 kilobases, were found to encode a 64.6-kDa protein 73% identical to either human calcineurin A alpha or beta. At the nucleotide level Drosophila calcineurin A cDNA is 67 and 65% identical to human calcineurin A alpha and beta cDNAs, respectively. Major differences between human and Drosophila calcineurins A are restricted to the amino and carboxyl termini, including two stretches of repetitive sequences in the carboxyl-terminal third of the Drosophila molecule. Motifs characteristic of the putative catalytic centers of protein phosphatase-1 and -2A and calcineurin are almost perfectly conserved. The calmodulin-binding and auto-inhibitory domains, characteristic of all mammalian calcineurins A, are also conserved. A remarkable feature of the calcineurin A gene is the location of the intron/exon junctions at the boundaries of the functional domains and the apparent conservation of the intron/exon junctions from Drosophila to man.

Amino Acid Sequence↗
PubMed2006

Drosophila as a model for the identification of genes causing adult human heart disease.

Drosophila melanogaster genetics provides the advantage of molecularly defined P-element insertions and deletions that span the entire genome. Although Drosophila has been extensively used as a model system to study heart development, it has not been used to dissect the genetics of adult human heart disease because of an inability to phenotype the adult fly heart in vivo. Here we report the development of a strategy to measure cardiac function in awake adult Drosophila that opens the field of Drosophila genetics to the study of human dilated cardiomyopathies. Through the application of optical coherence tomography, we accurately distinguish between normal and abnormal cardiac function based on measurements of internal cardiac chamber dimensions in vivo. Normal Drosophila have a fractional shortening of 87 +/- 4%, whereas cardiomyopathic flies that contain a mutation in troponin I or tropomyosin show severe impairment of systolic function. To determine whether the fly can be used as a model system to recapitulate human dilated cardiomyopathy, we generated transgenic Drosophila with inducible cardiac expression of a mutant of human delta-sarcoglycan (deltasg(S151A)), which has previously been associated with familial dilated cardiomyopathy. Compared to transgenic flies overexpressing wild-type deltasg, or the standard laboratory strain w(1118), Drosophila expressing deltasg(S151A) developed marked impairment of systolic function and significantly enlarged cardiac chambers. These data illustrate the utility of Drosophila as a model system to study dilated cardiomyopathy and the applicability of the vast genetic resources available in Drosophila to systematically study the genetic mechanisms responsible for human cardiac disease.

Animals↗
PubMed2000

Molecular cloning and genomic organization of a second probable allatostatin receptor from Drosophila melanogaster.

We (C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 269, 91-96) and others (N. Birgül et al. (1999) EMBO J. 18, 5892-5900) have recently cloned a Drosophila receptor that was structurally related to the mammalian galanin receptors, but turned out to be a receptor for a Drosophila peptide belonging to the insect allatostatin neuropeptide family. In the present paper, we screened the Berkeley "Drosophila Genome Project" database with "electronic probes" corresponding to the conserved regions of the four rat (delta, kappa, mu, nociceptin/orphanin FQ) opioid receptors. This yielded alignment with a Drosophila genomic database clone that contained a DNA sequence coding for a protein having, again, structural similarities with the rat galanin receptors. Using PCR with primers coding for the presumed exons of this second Drosophila receptor gene, 5'- and 3'-RACE, and Drosophila cDNA as template, we subsequently cloned the cDNA of this receptor. The receptor cDNA codes for a protein that is strongly related to the first Drosophila receptor (60% amino acid sequence identity in the transmembrane region; 47% identity in the overall sequence) and that is, therefore, most likely to be a second Drosophila allatostatin receptor (named DAR-2). The DAR-2 gene has three introns and four exons. Two of these introns coincide with two introns in the first Drosophila receptor (DAR-1) gene, and have the same intron phasing, showing that the two receptor genes are clearly evolutionarily related. The DAR-2 gene is located at the right arm of the third chromosome, position 98 D-E. This is the first report on the existence of two different allatostatin receptors in an animal.

Amino Acid Sequence↗
PubMed2003

Analysis of protein phosphatase function in Drosophila cells using RNA interference.

Double stranded RNA-mediated RNA interference is an effective method to downregulate the levels of protein phosphatases in Drosophila S2 cells. In many cases, nearly complete ablation of the targeted protein can be achieved. RNAi-mediated knockdown of protein phosphatases is akin to pharmacological inhibition with drugs and can be used to determine the roles of specific protein phosphatases in intact cells. RNAi can avoid the problems associated with less than adequate specificity of phosphatase inhibitors. Although information about the signaling pathways present in Drosophila S2 cells is not as well developed as many mammalian cell lines, the Drosophila system is particularly attractive for the study of oligomeric phosphatases like PP2A. Drosophila has far fewer isoforms for the phosphatases we have examined. This is especially true of the genes for PP2A regulatory subunits where over 50 isoforms are present in mammals but only four are present in Drosophila. Once hypotheses regarding phosphatase function have been generated from RNAi experiments in S2 cells, they can potentially be tested utilizing recent advances in the use of siRNAs to conduct RNAi experiments in mammalian cell lines. RNAi in Drosophila S2 cells has proven to be a powerful technique for identifying physiological functions of signaling proteins. The RNAi method is straightforward and works routinely with almost all proteins. RNAi in S2 cells can be used to assess the role of signaling proteins in specific pathways and as a screening tool to identify new roles for signaling molecules. For example, results from RNAi analysis of PP2A show that regulation of MAP kinase signaling involves the R2/B regulatory subunit and that the R5/B56 subunits play a previously unidentified role in apoptosis. While RNAi in Drosophila S2 cells is a powerful tool for analyzing protein function, the method does have limitations. Foremost, cells may exhibit an RNAi response to any nonspecific dsRNA, even in the absence of interferon. Therefore, physiological processes that respond to nonspecific dsRNA will be difficult to study. A second limitation is the need to produce antibodies that react with Drosophila isoforms. We have found that many antibodies to mammalian protein phosphatases do not cross-react with the corresponding Drosophila proteins. Finally, the physiology and signaling pathways of S2 cells have not been extensively studied. This lack of information limits the number of available readouts that can be used when assessing the effects of protein knockdowns.

Animals↗
PubMed1995

The distribution of transposable elements on X chromosomes from a natural population of Drosophila simulans.

The distribution of 13 transposable element families along 15 X chromosomes from an African natural population of Drosophila simulans was determined by in situ hybridization to polytene chromosomes. The transposable elements cloned from Drosophila melanogaster all hybridized with Drosophila simulans chromosomes. The number of copies per family was 3.5 times lower in the latter species and correlated with the copy number per family in Drosophila melanogaster. With the exception of 297, the copy number per chromosome followed a Poisson distribution. Element frequencies per chromosome band were generally low. However, several sites of the distal region and the base of the X chromosome had high frequencies of occupation. Elements had higher abundance at the base of the chromosome compared to distal regions. Overall, the distribution of transposable elements in Drosophila simulans is similar to that found in Drosophila melanogaster. These data provide evidence for the operation of a force (or forces) opposing transpositional increase in copy number, and that this force is weaker at the bases of chromosomes, consistent with the idea that recombination between elements at non-homologous sites contains TE copy number. The reduction in copy number of all TE families in Drosophila simulans compared to Drosophila melanogaster can be explained by stronger selection against transposable element multiplication and/or lower rates of transposition in Drosophila simulans.

Africa↗
PubMed1996

Gliolectin is a novel carbohydrate-binding protein expressed by a subset of glia in the embryonic Drosophila nervous system.

Interactions between embryonic neural cells generate the specific patterns of connectivity observed in nervous systems. Cell surface carbohydrates have been proposed to function in cellular recognition events guiding such interactions. Carbohydrate-binding proteins (lectins) that recognize specific oligosaccharide ligands in embryonic neural tissue provide a molecular mechanism for carbohydrate-mediated cell-cell interactions in neural development. Therefore, we have screened an embryonic Drosophila melanogaster cDNA library, expressed in COS1 cells, for carbohydrate-binding activity. COS1 cells expressing putative Drosophila lectins were identified and recovered based on their adhesion to immobilized preparations of neutral and zwitterionic glycolipids extracted from Drosophila embryos. We have identified an endogenous lectin expressed during Drosophila embryogenesis. The cloned lectin, designated 'gliolectin', possesses a novel protein sequence with a calculated molecular mass of 24,993. When expressed in Drosophila S2 cells, the lectin mediates heterophilic cellular aggregation. In embryos, gliolectin is expressed by a subset of glial cells found at the midline of the developing nervous system. Expression is highest during the formation of the Drosophila embryonic axonal commissures, a process requiring midline glial cell funcion. Immunoprecipitation with a monoclonal antibody against gliolectin yields a protein of Mr=46,600 from Drosophila embryonic membranes, suggesting that post-translational modification of gliolectin is extensive. Epitope- tagged chimericproteins composed of the amino terminal one-half of gliolectin and the Fc region of human IgG bind a small subset of the total glycolipids extracted from Drosophila embryos, demonstrating that the lectin activity of gliolectin can discriminate between oligosaccharide structures. The presence of gliolectin in the developing Drosophila embryonic nervous system further supports a role for cell surface carbohydrates in cell-cell recognition and indicates that the molecular diversity of animal lectins is not yet completely defined.

Amino Acid Sequence↗
PubMed1990

Mitochondrial DNA evolution in the obscura species subgroup of Drosophila.

Mitochondrial DNA (mtDNA) restriction site maps for nine species of the Drosophila obscura subgroup and for Drosophila melanogaster were established. Taking into account all restriction enzymes (12) and strains (45) analyzed, a total of 105 different sites were detected, which corresponds to a sample of 3.49% of the mtDNA genome. Based on nucleotide divergences, two phylogenetic trees were constructed assuming either constant or variable rates of evolution. Both methods led to the same relationships. Five differentiated clusters were found for the obscura subgroup species, one Nearctic, represented by Drosophila pseudoobscura, and four Palearctic, two grouping the related triads of species Drosophila subobscura, Drosophila madeirensis, Drosophila obscura, Drosophila subsilvestris, and two more represented by one species each, Drosophila bifasciata, and Drosophila tristis. The different Palearctic clusters are as distant between themselves as with the Nearctic one. For the related species D. subobscura, D. madeirensis, and D. guanche, the pair D. subobscura-D. madeirensis is the closest one. The relationships found by nucleotide divergence were confirmed by differences in mitochondrial genome size, with related species sharing similar genome lengths and differing from the distant ones. The total mtDNA size range for the obscura subgroup species was from 15.5 kb for D. pseudoobscura to 17.1 for D. tristis.

Animals↗
PubMed2006

Comparative genomic analysis of allatostatin-encoding (Ast) genes in Drosophila species and prediction of regulatory elements by phylogenetic footprinting.

The role of the YXFGLa family of allatostatin (AST) peptides in dipterans is not well-established. The recent completion of sequencing of genomes for multiple Drosophila species provides an opportunity to study the evolutionary variation of the allatostatins and to examine regulatory elements that control gene expression. We performed comparative analyses of Ast genes from seven Drosophila species (Drosophila melanogaster, Drosophila simulans, Drosophila ananassae, Drosophila yakuba, Drosophila pseudoobscura, Drosophila mojavensis, and Drosophila grimshawi) and used phylogenetic footprinting methods to identify conserved noncoding motifs, which are candidates for regulatory regions. The peptides encoded by the Ast precursor are nearly identical across species with the exception of AST-1, in which the leading residue may be either methionine or valine. Phylogenetic footprinting predicts as few as 3, to as many as 17 potential regulatory sites depending on the parameters used during analysis. These include a Hunchback motif approximately 1.2 kb upstream of the open reading frame (ORF), overlapping motifs for two Broad-complex isoforms in the first intron, and a CF2-II motif located in the 3'-UTR. Understanding the regulatory elements involved in Ast expression may provide insight into the function of this neuropeptide family.

Amino Acid Sequence↗