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PubMed2002

3' end processing of Drosophila melanogaster histone pre-mRNAs: requirement for phosphorylated Drosophila stem-loop binding protein and coevolution of the histone pre-mRNA processing system.

Synthetic pre-mRNAs containing the processing signals encoded by Drosophila melanogaster histone genes undergo efficient and faithful endonucleolytic cleavage in nuclear extracts prepared from Drosophila cultured cells and 0- to 13-h-old embryos. Biochemical requirements for the in vitro cleavage are similar to those previously described for the 3' end processing of mammalian histone pre-mRNAs. Drosophila 3' end processing does not require ATP and occurs in the presence of EDTA. However, in contrast to mammalian processing, Drosophila processing generates the final product ending four nucleotides after the stem-loop. Cleavage of the Drosophila substrates is abolished by depleting the extract of the Drosophila stem-loop binding protein (dSLBP), indicating that both dSLBP and the stem-loop structure in histone pre-mRNA are essential components of the processing machinery. Recombinant dSLBP expressed in insect cells by using the baculovirus system efficiently complements the depleted extract. Only the RNA-binding domain plus the 17 amino acids at the C terminus of dSLBP are required for processing. The full-length dSLBP expressed in insect cells is quantitatively phosphorylated on four residues in the C-terminal region. Dephosphorylation of the recombinant dSLBP reduces processing activity. Human and Drosophila SLBPs are not interchangeable and strongly inhibit processing in the heterologous extracts. The RNA-binding domain of the dSLBP does not substitute for the RNA-binding domain of the human SLBP in histone pre-mRNA processing in mammalian extracts. In addition to the stem-loop structure and dSLBP, 3' processing in Drosophila nuclear extracts depends on the presence of a short stretch of purines located ca. 20 nucleotides downstream from the stem, and an Sm-reactive factor, most likely the Drosophila counterpart of vertebrate U7 snRNP.

Amino Acid Sequence↗
PubMed2003

Evolution of the Drosophila broad locus: the Manduca sexta broad Z4 isoform has biological activity in Drosophila.

The Drosophila melanogaster broad locus is essential for normal metamorphic development. Broad encodes three genetically distinct functions (rbp, br, and 2Bc) and a family of four zinc-finger DNA-binding proteins (Z1-Z4). The Z1, Z2, and Z3 protein isoforms are primarily associated with the rbp, br, and 2Bc genetic functions respectively. The Z4 protein isoform also provides some rbp genetic function, however an essential function for the Z4 isoform in metamorphosis has not been identified. To determine the degree of conservation of Z4 function between the tobacco hornworm Manduca sexta and Drosophila we generated transgenic Drosophila expressing the Manduca broad Z4 isoform and used this transgene to rescue rbp mutant lethality during Drosophila metamorphosis. We find that the Manduca Z4 protein has significant biological activity in Drosophila with respect to rescue of rbp-associated lethality. There was also some overlap in effects on cuticle gene expression between the Manduca Z4 and Drosophila Z1 isoforms that was not shared with the Drosophila Z4 isoform. Our findings show that Z4 function has been conserved over the 260-million-year period since the divergence of Diptera and Lepidoptera, and are consistent with the hypothesis that the Drosophila Z4 and Manduca Z4 isoforms have essential roles in metamorphosis.

Animals↗
PubMed1995

The expression of esterase S gene of Drosophila virilis in Drosophila melanogaster.

Drosophila melanogaster was transformed with the esterase S gene from Drosophila virilis. This gene is strongly activated in ejaculatory bulbs of mature males of Drosophila virilis. The closely related gene from Drosophila melanogaster is activated in ejaculatory ducts. The tissue- and stage-specific expression of incomplete genomic copy of the esterase S gene integrated into the Drosophila melanogaster genome is the same as in Drosophila virilis. These data show that tissue and stage specificity is determined by relatively small 5' regulatory region of the esterase S gene. The comparison between deduced amino-acid sequences of the esterase S of Drosophila virilis and esterase 6 of Drosophila melanogaster was performed. These sequences revealed 50% homology.

Amino Acid Sequence↗
PubMed1992

Drosophila C virus cycle during the development of two Drosophila melanogaster strains (Charolles and Champetières) after larval contamination by food.

Drosophila C virus (DCV) cycle during Drosophila melanogaster development was studied after feeding contamination at the first, most sensitive, instar (L1). Two Drosophila strains were examined and compared. Presence of DCVC in apparently healthy animals (L3 larvae bred on a contaminated rearing medium and adults coming from larvae which were grown on medium containing DCVC) was demonstrated by biological tests. Using the immunofluorescence technique, DCV was exhibited in the diseased Charolles larvae, in the lumen of the digestive tract and in the basal part of gut cells which is in contact with the haemolymph. On the contrary, in Charolles larvae which seemed 'healthy', DCV was exhibited only in the lumen of the digestive tract at the apical boundary of the gut cells. But DCV typical protein capsid was not shown in the tissues of Drosophila L3 and adults. However, C virus remained in Drosophila tissues even after host metamorphosis and would seem to interact with Drosophila cells. Hypotheses are proposed concerning the intracellular state of Drosophila C virus in this case.

Animal Feed↗
PubMed1999

The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes.

A fundamental goal of genetics and functional genomics is to identify and mutate every gene in model organisms such as Drosophila melanogaster. The Berkeley Drosophila Genome Project (BDGP) gene disruption project generates single P-element insertion strains that each mutate unique genomic open reading frames. Such strains strongly facilitate further genetic and molecular studies of the disrupted loci, but it has remained unclear if P elements can be used to mutate all Drosophila genes. We now report that the primary collection has grown to contain 1045 strains that disrupt more than 25% of the estimated 3600 Drosophila genes that are essential for adult viability. Of these P insertions, 67% have been verified by genetic tests to cause the associated recessive mutant phenotypes, and the validity of most of the remaining lines is predicted on statistical grounds. Sequences flanking >920 insertions have been determined to exactly position them in the genome and to identify 376 potentially affected transcripts from collections of EST sequences. Strains in the BDGP collection are available from the Bloomington Stock Center and have already assisted the research community in characterizing >250 Drosophila genes. The likely identity of 131 additional genes in the collection is reported here. Our results show that Drosophila genes have a wide range of sensitivity to inactivation by P elements, and provide a rationale for greatly expanding the BDGP primary collection based entirely on insertion site sequencing. We predict that this approach can bring >85% of all Drosophila open reading frames under experimental control.

Alleles↗
PubMed2003

Highly conserved Drosophila ananassae timeless gene functions as a clock component in Drosophila melanogaster.

The behavior and physiology of Drosophila are subject to rhythms that are controlled by the circadian clock genes, period, timeless, clock and cycle, all of which are thought to participate in central pacemaker control. The molecular mechanism of rhythm in Drosophila has been studied in detail. However, rhythm and clock genes have mostly been analyzed in Drosophila melanogaster. To confirm whether the tim gene exists and works as a clock component in other Drosophila species, we cloned a tim homolog from Drosophila ananassae that shared 85.9% similarity with Drosophila melanogaster tim at the amino acid level. In addition, the PER interaction domains and NLS were highly conserved. Introduction of the D. ananassae tim homolog rescued the rhythm of the locomotor activity of about 44% of a population of D. melanogaster tim(01) flies. At the molecular level, hs-tim introduced not only TIM but PER oscillation in transgenic flies. These results indicate that the tim gene in D. ananassae functions as a component of the circadian clock in D. melanogaster.

Amino Acid Sequence↗
PubMed2006

Metabolism of arsenic in Drosophila melanogaster and the genotoxicity of dimethylarsinic acid in the Drosophila wing spot test.

Inorganic arsenic is nongenotoxic in the Drosophila melanogaster wing somatic mutation and recombination test (SMART). Recent evidence in mammalian systems indicates that methylated metabolites of arsenic are more genotoxic than inorganic arsenic. Thus, we hypothesized that inorganic arsenic is nongenotoxic in Drosophila because they are unable to biotransform arsenic to methylated forms. In the present study, we fed trivalent and pentavalent inorganic arsenic to Drosophila larvae and adults and measured the production of methylated derivatives. No biomethylated arsenic species were found in the organisms or in the growth medium, which suggests that Drosophila are unable to biomethylate inorganic arsenic. Exposure of Drosophila to the methylated arsenic derivative dimethylarsinic acid (DMA(V)) resulted in incorporation of this organoarsenic compound without demethylation. In addition, we used the SMART wing spot assay, which measures loss of heterozygosity (LOH) resulting from gene mutation, chromosomal rearrangement, chromosome breakage, and chromosome loss, to evaluate the genotoxicity of DMA. DMA by itself induced significant increases in the frequency of total spots, small spots, and large single spots. These results are consistent with the important role of arsenic biomethylation as a determinant of the genotoxicity of arsenic compounds. The absence of biomethylation in Drosophila could explain the lack of genotoxicity for inorganic arsenic and the genotoxicity of methylated arsenic species in the SMART wing spot assay.

Animals↗
PubMed2001

Identification of four Drosophila allatostatins as the cognate ligands for the Drosophila orphan receptor DAR-2.

The allatostatins are generally inhibitory insect neuropeptides. The Drosophila orphan receptor DAR-2 is a G-protein-coupled receptor, having 47% amino acid residue identity with another Drosophila receptor, DAR-1 (which is also called dros. GPCR, or DGR) that was previously shown to be the receptor for an intrinsic Drosophila A-type (cockroach-type) allatostatin. Here, we have permanently expressed DAR-2 in CHO cells and found that it is the cognate receptor for four Drosophila A-type allatostatins, the drostatins-A1 to -A4. Of all the drostatins, drostatin-A4 (Thr-Thr-Arg-Pro-Gln-Pro-Phe-Asn-Phe-Gly-Leu-NH(2)) is the most effective in causing a second messenger cascade (measured as bioluminescence; threshold, 10(-9) M; EC(50), 10(-8) M), whereas the others are less effective and about equally potent (EC(50), 8 x 10(-8) M). Northern blots showed that the DAR-2 gene is expressed in embryos, larvae, pupae, and adult flies. In adult flies, the receptor is more strongly expressed in the thorax/abdomen than in the head parts, suggesting that DAR-2 is a gut receptor. This is confirmed by Northern blots from 3rd instar larvae, showing that the DAR-2 gene is mainly expressed in the gut and only very weakly in the brain. The Drosophila larval gut also contains about 20-30 endocrine cells, expressing the gene for the drostatins-A1 to -A4. We suggest, therefore, that DAR-2 mediates an allatostatin (drostatin)-induced inhibition of gut motility. This is the first report on the permanent and functional expression of a Drosophila gut neurohormone receptor.

Amino Acid Sequence↗
PubMed1990

Tissue-specific expression phenotypes of Hawaiian Drosophila Adh genes in Drosophila melanogaster transformants.

Interspecific differences in the tissue-specific patterns of expression displayed by the alcohol dehydrogenase (Adh) genes within the Hawaiian picture-winged Drosophila represent a rich source of evolutionary variation in gene regulation. Study of the cis-acting elements responsible for regulatory differences between Adh genes from various species is greatly facilitated by analyzing the behavior of the different Adh genes in a homogeneous background. Accordingly, the Adh gene from Drosophila grimshawi was introduced into the germ line of Drosophila melanogaster by means of P element-mediated transformation, and transformants carrying this gene were compared to transformants carrying the Adh genes from Drosophila affinidisjuncta and Drosophila hawaiiensis. The results indicate that the D. affinidisjuncta and D. grimshawi genes have relatively higher levels of expression and broader tissue distribution of expression than the D. hawaiiensis gene in larvae. All three genes are expressed at similar overall levels in adults, with differences in tissue distribution of enzyme activity corresponding to the pattern in the donor species. However, certain systematic differences between Adh gene expression in transformants and in the Hawaiian Drosophila are noted along with tissue-specific position effects in some cases. The implications of these findings for the understanding of evolved regulatory variation are discussed.

Alcohol Dehydrogenase↗
PubMed2003

Molecular cloning, functional expression, and gene silencing of two Drosophila receptors for the Drosophila neuropeptide pyrokinin-2.

The database of the Drosophila Genome Project contains the sequences of two genes, CG8784 and CG8795, predicted to code for two structurally related G protein-coupled receptors. We have cloned these genes and expressed their coding parts in Chinese hamster ovary cells. We found that both receptors can be activated by low concentrations of the Drosophila neuropeptide pyrokinin-2 (CG8784, EC(50) for pyrokinin-2, 1x10(-9)M; CG8795, EC(50) for pyrokinin-2, 5 x 10(-10)M). The precise role of Drosophila pyrokinin-2 (SVPFKPRLamide) in Drosophila is unknown, but in other insects, pyrokinins have diverse myotropic actions and are also initiating sex pheromone biosynthesis and embryonic diapause. Gene silencing, using the RNA-mediated interference technique, showed that CG8784 gene silencing caused lethality in embryos, whereas CG8795 gene silencing resulted in strongly reduced viability for both embryos and first instar larvae. In addition to the two Drosophila receptors, we also identified two probable pyrokinin receptors in the genomic database from the malaria mosquito Anopheles gambiae. The two Drosophila pyrokinin receptors are, to our knowledge, the first invertebrate pyrokinin receptors to be identified.

Amino Acid Sequence↗
PubMed1986

Isolation of a Drosophila genomic sequence homologous to the kinase domain of the human insulin receptor and detection of the phosphorylated Drosophila receptor with an anti-peptide antibody.

A Drosophila genomic fragment has been isolated with a deduced amino acid sequence that is strikingly homologous to that of the kinase domain of the human insulin receptor. The Drosophila DNA hybridizes with an 11-kilobase mRNA that is most prominent in 8- to 12-hr embryos. An anti-peptide antibody prepared to a sequence in the human insulin receptor kinase domain that is conserved in the Drosophila sequence immunoprecipitates a single 95-kDa Drosophila protein whose phosphorylation on tyrosine residues is dependent on insulin. We conclude that the DNA sequence is that of the kinase domain of the Drosophila insulin receptor and that the 95-kDa phosphoprotein is the autophosphorylated beta subunit of that receptor. The results are compatible with our previous reports demonstrating a specific insulin-binding Drosophila glycoprotein and an insulin-dependent tyrosine protein kinase whose activity is greatest during embryogenesis. The observations suggest a role for insulin-dependent protein tyrosine phosphorylation during embryogenesis.

Amino Acid Sequence↗
PubMed1999

The Drosophila poly(A)-binding protein II is ubiquitous throughout Drosophila development and has the same function in mRNA polyadenylation as its bovine homolog in vitro.

The poly(A)-binding protein II (PABP2) is one of the polyadenylation factors required for proper 3'-end formation of mammalian mRNAs. We have cloned Pabp2, the gene encoding the Drosophila homolog of mammalian PABP2, by using a molecular screen to identify new Drosophila proteins with RNP-type RNA-binding domains. Sequence comparison of PABP2 from Drosophila and mammals indicates that the most conserved domains are the RNA-binding domain and a coiled-coil like domain which could be involved in protein-protein interactions. Pabp2 produces four mRNAs which result from utilization of alternative poly(A) sites and encode the same protein. Using an antibody raised against Drosophila PABP2, we show that the protein accumulates in nuclei of all transcriptionally active cells throughout Drosophila development. This is consistent with a general role of PABP2 in mRNA polyadenylation. Analysis of Drosophila PABP2 function in a reconstituted mammalian polyadenylation system shows that the protein has the same functions as its bovine homolog in vitro : it stimulates poly(A) polymerase and is able to control poly(A) tail length.

Amino Acid Sequence↗
PubMed1994

The organization of the gene for Drosophila lamin C: limited homology with vertebrate lamin genes and lack of homology versus the Drosophila lamin Dmo gene.

Drosophila melanogaster has, in addition to the well characterized lamin Dmo, a second nuclear lamin called lamin C, which was recently defined by cDNA cloning. Here we characterize the gene encoding Drosophila lamin C. It is composed of 4 exons and 3 introns, which all interrupt the coding sequence. No intron position of the Drosophila lamin C gene has a counterpart in the Drosophila lamin Dmo gene, which has a structure unrelated to the highly conserved structures of vertebrate lamin genes. Surprisingly, however, two of the three intron positions of the Drosophila lamin C gene are also found in all currently known vertebrate lamin genes, which have an additional 8 to 9 introns. Thus, lamin genes from vertebrates and invertebrates can be more closely related in organization than earlier thought on the basis of only two invertebrate genes. The striking difference in structure of the two Drosophila lamin genes indicates that their ancestral genes separated earlier in metazoan evolution and that more than one lamin gene may also be expected in other invertebrates.

Amino Acid Sequence↗
PubMed1997

FlyView, a Drosophila image database, and other Drosophila databases

FlyView is an image database for Drosophila development and genetics, particularly for gene-expression patterns. Thousands of enhancer-trap lines have now been isolated by different methods, particularly by using a variety of transposons with the resulting flies being kept in a great many laboratories. We are collecting pictures of expression patterns of all lines that are available for use by Drosophila researchers and now offer about 1800 images of about 350 enhancer-trap lines in FlyView. This article also summarizes information on the other main Drosophila resources: FlyBase-the main Drosophila database, Flybrain, an online atlas and database of the Drosophila nervous system, and Interactive Fly, a cyberspace guide to Drosophila genes and their roles in development.Copyright 1997 Academic Press Limited Copyright 1997Academic Press Limited

Journal Article↗
PubMed1988

Differing amounts of genetic polymorphism in testes and male accessory glands of Drosophila melanogaster and Drosophila simulans.

We surveyed genetic polymorphism by two-dimensional gel electrophoresis of male reproductive tract proteins in 20 isofemale lines each of Drosophila melanogaster and Drosophila simulans. After classifying 244 such proteins of Drosophila melanogaster and 271 of Drosophila simulans by their distribution between testes and accessory glands within the reproductive tract, significant correlations were found between genetic polymorphism and tissue distribution. In both species, gland-specific proteins were significantly more polymorphic than testis-specific proteins, as well as those found in both testes and glands. Simultaneously, in Drosophila simulans, proteins found in roughly equivalent relative abundance in both testes and glands were significantly less variable than gland-specific and testis-specific proteins, as well as those with a quantitative difference in relative abundance between testes and glands. These correlations may reflect general differences in variability between extracellular and intracellular proteins and between proteins with broad as opposed to tissue-specific distributions.

Animals↗