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PubMed2001

[The sbr gene product in Drosophila melanogaster and its orthologs in yeast (Mex67p) and human (TAP)].

A DNA sequence from the 9F region of Drosophila melanogaster polytene chromosomes was cloned. Sequencing the cloned region and its comparison with the known sequences of the D. melanogaster genome showed that the cloned DNA part contains gene sbr and adjacent sequences. The literature data on the structure and functions of genes TAP in humans and Mex67 in yeast are discussed. These genes are orthologous to the sbr gene of Drosophila and control mRNA export from the nucleus to the cytoplasm. The literature evidence is consistent with the recessive expression of mutation l(1)ts403 (sbr10) upon heat treatment that is manifested as impaired HSP synthesis at the posttranscriptional level. However, it fails to explain the semidominant effect of the mutation manifested in high frequency of meiotic sex-chromosome nondisjunction in heat-treated females. A comparison of amino-acid sequences corresponding to the products of the three orthologous genes, TAP, Mex67, and sbr, showed that the sbr gene product of Drosophila is more similar to the human TAP factor than to the Mex67 factor in yeast.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

A functional genomic screen for cardiogenic genes using RNA interference in developing Drosophila embryos.

Identifying genetic components is an essential step toward understanding complex developmental processes. The primitive heart of the fruit fly, the dorsal vessel, which is a hemolymph-pumping organ, has provided a unique model system to identify cardiogenic genes and to further our understanding of the molecular mechanisms of cardiogenesis. Using RNA interference in developing Drosophila embryos, we performed a genomewide search for cardiogenic genes. Through analyses of the >5800 genes that cover approximately 40% of all predicted Drosophila genes, we identified a variety of genes encoding transcription factors and cell signaling proteins required for different steps during heart development. Analysis of mutant heart phenotypes and identified genes suggests that the Drosophila heart tube is segmentally patterned, like axial patterning, but assembled with regional modules. One of the identified genes, simjang, was further characterized. In the simjang mutant embryo, we found that within each segment a subset of cardial cells is missing. Interestingly, the simjang gene encodes a protein that is a component of the chromatin remodeling complex recruited by methyl-CpG-DNA binding proteins, suggesting that epigenetic information is crucial for specifying cardiac precursors. Together, these studies not only identify key regulators but also reveal mechanisms underlying heart development.

Animals↗

PubMed1997

A framework physical map of Drosophila virilis based on P1 clones: applications in genome evolution.

The analysis of patterns of genome evolution may help to evaluate the evolutionary forces that shape the composition and organization of the genome. Comparisons between the physical maps of divergent species can be used to identify conserved blocks of closely linked genes whose synteny is possibly under selective constraint. We have used in situ hybridization to determine the genomic position of 732 randomly selected clones from a bacteriophage P1 library of Drosophila virilis. The resulting map includes at least one clone in each of 69% of the subdivisions into which the D. virilis polytene chromosomes are divided. A subset of these clones was used to carry out a comparative physical analysis of chromosome 2 from D. virilis and from Drosophila montana. A number of discrepancies with the classical scenario of chromosome evolution were noted. The D. virilis P1 clones were also used to determine the physical relations between ten genes that are located in the X chromosome of Drosophila melanogaster between the markers crn (2F1) and omb (4C5-6). In this region, which is approximately 2 Mb in length, there have been at least six breakpoints since the divergence of the species, and six of the genes are found at widely scattered locations in the D. virilis X chromosome. However, a block of four functionally unrelated genes, including white, roughest, Notch, and dunce, seems to be conserved between the two species.

Animals↗

PubMed1998

Oncoviral DNAs induce transposition of endogenous mobile elements in the genome of Drosophila melanogaster.

Previously, we have shown that particles of Rous sarcoma virus or cloned fragments of RSV cDNA as well as DNA of oncogenic simian adenovirus Sa7, injected into the polar plasm of early Drosophila melanogaster embryos, were able to induce, with high frequency, unstable visible mutations in different groups of genetic loci. The genetic instability of the recovered mutations, i.e., their ability to revert to normal state or to generate new mutant alleles at the affected locus, was manifest in mutant lines through several generations. The molecular analysis undertaken in this study of the yellow-scute loci region which is highly sensitive to the microinjected Sa7 DNA, and of the white locus, that frequently mutates under the influence of RSV cDNA, clearly shows that the induced mutations and reversions are accompanied by insertion/excision of endogenous mobile elements. This conclusion is confirmed by in situ hybridization experiments which demonstrate that the adenovirus DNA is able to change, though with different efficiency, the chromosomal localization of certain Drosophila retrotransposons. These results partially elucidate the molecular mechanism of the genetic instability in D. melanogaster induced by microinjection of oncoviruses into early embryos, implying that is results from mobilization of endogenous transposons which play the role of insertional elements directly causing unstable mutations.

Adenoviruses, Simian↗

PubMed1998

Chromatin complexes as aperiodic microcrystalline arrays that regulate genome organisation and expression.

The current understanding of chromatin-mediated repression in Metazoa stems largely from work on two systems in Drosophila: heterochromatin-induced position-effect variegation and repression of the homeotic genes by the Polycomb-group of genes. A common feature of these two systems is the cooperative assembly of multimeric complexes which can epigenetically silence gene activity. Moreover, both older and more recent work has suggested that these complexes can themselves associate to give rise to larger complexes: The specificity of the association is likely to be determined by complementarity of the structural components of the complexes. Here, we aim to accommodate these, and other, features of chromatin-mediated repression in a single hypothesis, namely the crystallisation hypothesis. This hypothesis views the nucleus as being an environment that favours the formation of chromatin complexes which behave as aperiodic microcrystalline arrays constructed through the cooperative assembly of different types of lattice unit. The lattice units possess regions of structural complementarity that allow interactions between complexes. Aperiodicity confers specificity on the complexes and is a key feature of the model which, we suggest, provides a gene with a "chromosomal address." The chromosomal address allows the side-by-side alignment of homologous chromosomal regions, a properly that may be important in a variety of biologically relevant situations. Aperiodicity is also a feature of the hypothesis that is directly testable.

Animals↗

PubMed2001

Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes.

BACKGROUND: The availability of complete genome sequences enables all the members of a gene family to be identified without limitations imposed by temporal, spatial or quantitative aspects of mRNA expression. Using the nearly completed human genome sequence, we combined in silico and experimental approaches to define the complete human nuclear receptor (NR) set. This information was used to carry out a comparative genomic study of the NR superfamily. RESULTS: Our analysis of the human genome identified two novel NR sequences. Both these contained stop codons within the coding regions, indicating that both are pseudogenes. One (HNF4 gamma-related) contained no introns and expressed no detectable mRNA, whereas the other (FXR-related) produced mRNA at relatively high levels in testis. If translated, the latter is predicted to encode a short, non-functional protein. Our analysis indicates that there are fewer than 50 functional human NRs, dramatically fewer than in Caenorhabditis elegans and about twice as many as in Drosophila. Using the complete human NR set we made comparisons with the NR sets of C. elegans and Drosophila. Searches for the >200 NRs unique to C. elegans revealed no human homologs. The comparative analysis also revealed a Drosophila member of NR subfamily NR3, confirming an ancient metazoan origin for this subfamily. CONCLUSIONS: This work provides the basis for new insights into the evolution and functional relationships of NR superfamily members.

Amino Acid Sequence↗

PubMed1983

The cloning and expression of the gene encoding organ-specific esterase S from the genome of Drosophila virilis.

We have cloned the gene for the esterase S isozymes complex from the genome of Drosophila virilis in pBR322. Esterase S is an enzyme which is specifically synthesized in the ejaculatory bulbs of D. virilis adult males. The gene for the esterase S isozyme complex (estS) has been localized in band 2G5e of chromosome II. Poly(A)+ RNA prepared from ejaculatory bulbs actively hybridizes with this band. A cloned 15-kb fragment of D. virilis DNA (pVE9) also hybridizes with band 2G5e. The area encoding the poly(A)+ RNA is located in the middle part of the cloned fragment whose ends are not transcribed in vivo. Only one poly(A)+ RNA which is 1.9 kb long and complementary to pVE9 DNA can be revealed in the cytoplasm. The mRNA preselected by hybridization to pVE9 DNA was microinjected into the cytoplasm of Xenopus laevis oocytes. In other experiments, the pVE9 DNA itself was microinjected into oocyte nuclei. In both cases, esterase S is synthesized in the oocytes, and the major part of the protein is transported from the oocytes and accumulated in the incubation medium.

Animals↗

PubMed2001

Analysis of conserved noncoding DNA in Drosophila reveals similar constraints in intergenic and intronic sequences.

Comparative genomic approaches to gene and cis-regulatory prediction are based on the principle that differential DNA sequence conservation reflects variation in functional constraint. Using this principle, we analyze noncoding sequence conservation in Drosophila for 40 loci with known or suspected cis-regulatory function encompassing >100 kb of DNA. We estimate the fraction of noncoding DNA conserved in both intergenic and intronic regions and describe the length distribution of ungapped conserved noncoding blocks. On average, 22%-26% of noncoding sequences surveyed are conserved in Drosophila, with median block length approximately 19 bp. We show that point substitution in conserved noncoding blocks exhibits transition bias as well as lineage effects in base composition, and occurs more than an order of magnitude more frequently than insertion/deletion (indel) substitution. Overall, patterns of noncoding DNA structure and evolution differ remarkably little between intergenic and intronic conserved blocks, suggesting that the effects of transcription per se contribute minimally to the constraints operating on these sequences. The results of this study have implications for the development of alignment and prediction algorithms specific to noncoding DNA, as well as for models of cis-regulatory DNA sequence evolution.

Animals↗

PubMed2002

Evolutionary conservation of otd/Otx2 transcription factor action: a genome-wide microarray analysis in Drosophila.

BACKGROUND: Homeobox genes of the orthodenticle (otd)/Otx family have conserved roles in the embryogenesis of head and brain. Gene replacement experiments show that the Drosophila otd gene and orthologous mammalian Otx genes are functionally equivalent, in that overexpression of either gene in null mutants of Drosophila or mouse can restore defects in cephalic and brain development. This suggests that otd and Otx genes control a comparable subset of downstream target genes in either organism. Here we use quantitative transcript imaging to analyze this equivalence of otd and Otx gene action at a genomic level. RESULTS: Oligonucleotide arrays representing 13,400 annotated Drosophila genes were used to study differential gene expression in flies in which either the Drosophila otd gene or the human Otx2 gene was overexpressed. Two hundred and eighty-seven identified transcripts showed highly significant changes in expression levels in response to otd overexpression, and 682 identified transcripts showed highly significant changes in expression levels in response to Otx2 overexpression. Among these, 93 showed differential expression changes following overexpression of either otd or Otx2, and for 90 of these, comparable changes were observed under both experimental conditions. We postulate that these transcripts are common downstream targets of the fly otd gene and the human Otx2 gene in Drosophila. CONCLUSION: Our experiments indicate that approximately one third of the otd-regulated transcripts also respond to overexpression of the human Otx2 gene in Drosophila. These common otd/Otx2 downstream genes are likely to represent the molecular basis of the functional equivalence of otd and Otx2 gene action in Drosophila.

Animals↗

PubMed1994

PCR-based homology probing reveals a family of GABA receptor-like genes in Drosophila melanogaster.

A polymerase chain reaction (PCR)-based homology probing strategy was employed to screen Drosophila melanogaster genomic DNA for sequences encoding a conserved amino acid 'signature motif' known to be present in vertebrate GABA receptor and glycine receptor subunit genes. This approach yielded three discrete amplified sequence elements (designated LCCH1, LCCH2, and LCCH3) that contained open reading frames and > 40% amino acid sequence identity to the corresponding regions of vertebrate ligand-gated chloride channel genes. Genomic DNA clones corresponding to each element were isolated and sequenced, and predicted amino acid sequences corresponding to the second (M2) and third (M3) transmembrane domains of vertebrate genes were analyzed for identity or similarity to known sequences. LCCH1 was identical to the Rdl gene, a known GABA receptor subunit gene from D. melanogaster, whereas LCCH2 and LCCH3 were novel D. melanogaster sequences that exhibited structural similarity to other members of the ligand-gated chloride channel gene family. LCCH2 was equally divergent in M2 and M3 (46-49% amino acid identity) from all other known members of this family and may therefore represent a new subunit or receptor class within this family. LCCH2 was localized by in situ hybridization to cytogenetic region 75A on the left arm of chromosome 3. LCCH3 was closely related to mammalian (79% amino acid identity) and snail (96% amino acid identity) GABA receptor beta subunits and may therefore be the homologue in D. melanogaster of this subunit class. LCCH3 was localized by in situ hybridization to cytogenetic region 13F on the X chromosome.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2002

The role of lineage-specific gene family expansion in the evolution of eukaryotes.

A computational procedure was developed for systematic detection of lineage-specific expansions (LSEs) of protein families in sequenced genomes and applied to obtain a census of LSEs in five eukaryotic species, the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the green plant Arabidopsis thaliana. A significant fraction of the proteins encoded in each of these genomes, up to 80% in A. thaliana, belong to LSEs. Many paralogous gene families in each of the analyzed species are almost entirely comprised of LSEs, indicating that their diversification occurred after the divergence of the major lineages of the eukaryotic crown group. The LSEs show readily discernible patterns of protein functions. The functional categories most prone to LSE are structural proteins, enzymes involved in an organism's response to pathogens and environmental stress, and various components of signaling pathways responsible for specificity, including ubiquitin ligase E3 subunits and transcription factors. The functions of several previously uncharacterized, vastly expanded protein families were predicted through in-depth protein sequence analysis, for example, small-molecule kinases and methylases that are expanded independently in the fly and in the nematode. The functions of several other major LSEs remain mysterious; these protein families are attractive targets for experimental discovery of novel, lineage-specific functions in eukaryotes. LSEs seem to be one of the principal means of adaptation and one of the most important sources of organizational and regulatory diversity in crown-group eukaryotes.

Animals↗

PubMed2003

A functional genomic analysis of cell morphology using RNA interference.

BACKGROUND: The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies. RESULTS: We adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA. CONCLUSIONS: Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology.

Animals↗

PubMed1995

Structure and tissue-specific expression of the Drosophila melanogaster organellar-type Ca(2+)-ATPase gene.

A 14 kb genomic clone covering the organellar-type Ca(2+)-ATPase gene of Drosophila melanogaster has been isolated and characterized. The sequence of a 7132 bp region extending from 1.1 kb 5' upstream of the initiation ATG codon over the polyadenylation signal at the 3' end has been determined. The gene consists of nine exons including one with an exceptional size of 2172 bp representing 72% of the protein coding region. Introns are relatively small (< 100 bp) except for the 3' intron which has a size of 2239 bp, an exceptionally large size among Drosophila introns. Five of the introns are in the same positions in Drosophila, Artemia and rabbit SERCA1 Ca(2+)-ATPase genes. There is only one organellar-type Ca(2+)-ATPase gene in the Drosophila genome, as was shown by Southern-blot analysis [Váradi, Gilmore-Hebert and Benz (1989) FEBS Lett. 258, 203-207] and by chromosomal localization [Magyar and Váradi (1990) Biochem. Biophys. Res. Commun. 173, 872-877]. Primer extension and S1-nuclease assays revealed a potential transcription initiation site 876 bp upstream of the translation initiation ATG with a TATA-box 23 bp upstream of this site. Analysis of the 5' region of the Drosophila organellar-type Ca(2+)-ATPase gene suggests the presence of potential recognition sequences of various muscle-specific transcription factors and shows a region with remarkable similarity to that in the rabbit SERCA2 gene. The tissue distribution of expression of the organellar-type Ca(2+)-ATPase gene has been studied by in situ RNA-RNA hybridization on microscopic sections. A low mRNA abundance can be detected in each tissue of adult flies, suggesting a housekeeping function for the gene. On the other hand a pronounced tissue specificity of expression has also been found as the organellar-type Ca(2+)-ATPase is expressed at a very high level in cell bodies of the central nervous system and in various muscles.

Amino Acid Sequence↗

PubMed2002

B-ZIP proteins encoded by the Drosophila genome: evaluation of potential dimerization partners.

The basic region-leucine zipper (B-ZIP) (bZIP) protein motif dimerizes to bind specific DNA sequences. We have identified 27 B-ZIP proteins in the recently sequenced Drosophila melanogaster genome. The dimerization specificity of these 27 B-ZIP proteins was evaluated using two structural criteria: (1) the presence of attractive or repulsive interhelical g<-->e' electrostatic interactions and (2) the presence of polar or charged amino acids in the 'a' and 'd' positions of the hydrophobic interface. None of the B-ZIP proteins contain only aliphatic amino acids in the'a' and 'd' position. Only six of the Drosophila B-ZIP proteins contain a "canonical" hydrophobic interface like the yeast GCN4, and the mammalian JUN, ATF2, CREB, C/EBP, and PAR leucine zippers, characterized by asparagine in the second 'a' position. Twelve leucine zippers contain polar amino acids in the first, third, and fourth 'a' positions. Circular dichroism spectroscopy, used to monitor thermal denaturations of a heterodimerizing leucine zipper system containing either valine (V) or asparagine (N) in the 'a' position, indicates that the V-N interaction is 2.3 kcal/mole less stable than an N-N interaction and 5.3 kcal/mole less stable than a V-V interaction. Thus, we propose that the presence of polar amino acids in novel positions of the 'a' position of Drosophila B-ZIP proteins has led to leucine zippers that homodimerize rather than heterodimerize.

Amino Acid Sequence↗

PubMed2003

Extrachromosomal circular DNA of tandemly repeated genomic sequences in Drosophila.

One characteristic of genomic plasticity is the presence of extrachromosomal circular DNA (eccDNA). This DNA is found in various eukaryotes from yeast to humans, and its levels are elevated by exposure to carcinogens. eccDNA is heterogeneous in size and composed of chromosomal sequences. In this study we used two-dimensional gel electrophoresis to detect and characterize eccDNA in Drosophila. We found eccDNA throughout the fly's life cycle. These molecules comprise up to 10% of the total repetitive DNA content, and their size ranges from <1 kb to >20 kb. The eccDNA population contains circular multimers of tandemly repeated genes such as histones, rDNA, Stellate, and the Suppressor of Stellate. Multimers of centromeric heterochromatin sequences are included in eccDNA as well. Our findings are consistent with the hypothesis that intramolecular homologous recombination between direct tandem repeats is a favorite mechanism for eccDNA formation. The level of eccDNA increased following MMS treatment of wild-type larvae, consistent with phenomena observed in cultured mammalian cells. This shows mutagen-induced eccDNA formation in the context of the whole organism for the first time. Mutations in the genes okra, mus309, and mei41 did not affect eccDNA under normal conditions or following mutagen treatment, implying that eccDNA formation is different from known pathways of DNA repair.

Animals↗

PubMed2004

Population genetics of autocidal control and strain replacement.

The concept that an insect species' genome could be altered in a manner that would result in the control of that species (i.e., autocidal control) or in the replacement of a pestiferous strain of the species with a more benign genotype was first proposed in the mid-twentieth century. A major research effort in population genetics and ecology followed and led to the development of a set of classical genetic control approaches that included use of sterile males, conditional lethal genes, translocations, compound chromosomes, and microbe-mediated infertility. Although there have been a number of major successes in application of classical genetic control, research in this area has declined in the past 20 years for technical and societal reasons. Recent advances in molecular biology and transgenesis research have renewed interest in genetically based control methods because these advances may remove some major technical problems that have constrained effective genetic manipulation of pest species. Population genetic analyses suggest that transgenic manipulations may enable development of strains that would be 10 to over 100 times more efficient than strains developed by classical methods. Some of the proposed molecular approaches to genetic control involve modifications of classical approaches such as conditional lethality, whereas others are novel. Experience from the classical era of genetic control research indicates that the population structure and population dynamics of the target population will determine which, if any, genetic control approaches would be appropriate for addressing a specific problem. As such, there continues to be a need for ongoing communication between scientists who are developing strains and those who study the native pest populations.

Animals↗

PubMed2006

Division of labour and colony efficiency in social insects: effects of interactions between genetic architecture, colony kin structure and rate of perturbations.

The efficiency of social insect colonies critically depends on their ability to efficiently allocate workers to the various tasks which need to be performed. While numerous models have investigated the mechanisms allowing an efficient colony response to external changes in the environment and internal perturbations, little attention has been devoted to the genetic architecture underlying task specialization. We used artificial evolution to compare the performances of three simple genetic architectures underlying within-colony variation in response thresholds of workers to five tasks. In the 'deterministic mapping' system, the thresholds of individuals for each of the five tasks is strictly genetically determined. In the second genetic architecture ('probabilistic mapping'), the genes only influence the probability of engaging in one of the tasks. Finally, in the 'dynamic mapping' system, the propensity of workers to engage in one of the five tasks depends not only on their own genotype, but also on the behavioural phenotypes of other colony members. We found that the deterministic mapping system performed well only when colonies consisted of unrelated individuals and were not subjected to perturbations in task allocation. The probabilistic mapping system performed well for colonies of related and unrelated individuals when there were no perturbations. Finally, the dynamic mapping system performed well under all conditions and was much more efficient than the two other mapping systems when there were perturbations. Overall, our simulations reveal that the type of mapping between genotype and individual behaviour greatly influences the dynamics of task specialization and colony productivity. Our simulations also reveal complex interactions between the mode of mapping, level of within-colony relatedness and risk of colony perturbations.

Animals↗

PubMed1998

Molecular cloning, genomic organization and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to gonadotropin-releasing hormone receptors for vertebrates.

After screening the data base of the Berkeley Drosophila Genome Project with a sequence coding for the transmembrane region of a G protein-coupled receptor, we found that Drosophila might contain a gene coding for a receptor that is structurally related to the Gonadotropin-Releasing Hormone (GnRH) receptors from vertebrates. Using the polymerase chain reaction, with Drosophila cDNA as a template, and oligonucleotide probes coding for the presumed exons of this gene, we were able to clone the cDNA coding for this receptor. The transmembrane region of the receptor shows 36% amino acid residue identity with the transmembrane region of the catfish and 31% amino acid residue identity with that of the rat GnRH receptor. The Drosophila receptor gene contains six introns, whereas the rat gene contains two: one intron in the Drosophila gene occurs at exactly the same position and has the same intron phasing as one intron in the rat gene, suggesting that the Drosophila and mammalian GnRH receptor genes are evolutionarily related. Northern blot analyses show that the Drosophila receptor gene is progressively expressed during larval development with a prominent maximum at the 3rd instar larval stage. Pupae contain low amounts of receptor mRNA, while adult flies contain higher levels, with males having about five times more receptor mRNA than females flies. Southern blot analyses show that Drosophila contains only one copy of the receptor gene, which is located at position 27A2-B1 of chromosome 2. This paper is the first report on the molecular cloning of a member of the GnRH receptor family from invertebrates.

Amino Acid Sequence↗