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PubMed1994

Characterization of class II and class III ADP-ribosylation factor genes and proteins in Drosophila melanogaster.

ADP-ribosylation factors (ARFs) are ubiquitous approximately 20-kDa guanine nucleotide-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin and are involved in intracellular vesicular transport. Based on size, phylogenetic analysis, amino acid identity, and gene structure, mammalian ARFs fall into three classes (class I, ARF1, -2, and -3; class II, ARF4 and -5; class III, ARF6). A class I ARF had been identified in Drosophila melanogaster. To search for ARFs of other classes in Drosophila, polymerase chain reaction-based techniques were used, resulting in cloning of Drosophila ARF (dARF) II and dARF III with deduced amino acid sequences similar to those of class II and class III mammalian ARFs, respectively. The three Drosophila ARF genes map to different chromosomes and the coding regions have different splicing sites. dARF II mRNA, like ARF I mRNA, is fairly uniformly distributed throughout adult flies, whereas dARF III mRNA is significantly more abundant in heads than in legs or bodies. Recombinant dARF II and dARF III have biochemical and immunological properties similar to those of human ARF5 (hARF5) and hARF6, respectively. These observations are consistent with the conclusion that the three classes of ARFs are present in non-mammalian as well as mammalian species.

ADP-Ribosylation Factor 1↗
PubMed1995

wingless and DWnt4, 2 Drosophila Wnt genes, have related expression, regulation and function during the embryonic development.

The multigenic Wnt family encode secreted signalling molecules with important regulatory functions in various developmental processes. This paper reports an analysis of the relationships, in terms of structure, expression and function, that exist between the Drosophila genes wingless, the orthologue of the mammalian Wnt1 proto-oncogene, and DWnt4, a new member of the Wnt family. The 2 genes are physically clustered, are transcribed in overlapping embryonic territories under the control of the same regulatory molecules. Co-expression and co-regulation suggest first, that the close physical linkage results from the sharing of cis-control elements and second, that the 2 Wnt signals cooperate in developmental patterning events. Antisense RNA experiments revealed that signalling by DWnt4 is essential for cells from the anterior compartment of each parasegment to adopt a denticled fate. We propose that wingless and DWnt4 achieve opposite, but complementary functions in intrasegmental cell patterning of the embryonic ectoderm.

Animals↗
PubMed1996

[Effect of genetic background on mutation frequency of insertional alleles of the lozenge in Drosophila melanogaster].

We studied the effect of genetic background on mutation frequency of an unstable lz75V allele of the lozenge gene (lz; 1-27.7) isolated from natural populations of Drosophila melanogaster and its mutant derivatives lzB abd lzsl. Genetic composition of the X chromosome containing unstable alleles (X75V chromosome) was shown to affect their mutability. The region of the chromosome proximal to lozenge contains factors required for high mutability of lz75V and lzB. Substitution of a distal part of the X chromosome from a laboratory strain for a homologous part of the X75V chromosome also resulted in stabilizing lz75V, but caused an increase in mutation frequency of lzB. Association between instability of lz75V and the presence of P element with the locus was revealed by in situ hybridization. Studying effects of regulatory elements from a pi 2 P strain showed that the P cytotype is associated with a twofold to threefold decrease in mutation frequency of lzB and lzsl, but P-M hybrid dysgenesis is associated with its slight increase. Regulation of instability of the lozenge gene within the X75V chromosome was assumed to involve three levels: (1) character and topography of a mobile element inserted into the locus, (2) regulatory factors of other X-chromosomal regions, and (3) cytoplasmic factors. The results obtained are discussed in terms of regulation of transposition of mobile genetic elements.

Alleles↗
PubMed1997

Mutations of the PATCHED gene in several types of sporadic extracutaneous tumors.

Patients with basal cell nevus syndrome have a high incidence of multiple basal cell carcinomas, medulloblastomas, and meningiomas. Because somatic PATCHED (PTCH) mutations have been found in sporadic basal cell carcinomas, we have screened for PTCH mutations in several types of sporadic extracutaneous tumors. We found that 2 of 14 sporadic medulloblastomas bear somatic nonsense mutations in one copy of the gene and also deletion of the other copy. In addition, we identified missense mutations in PTCH in two of seven breast carcinomas, one of nine meningiomas, and one colon cancer cell line. No PTCH gene mutations were detected in 10 primary colon carcinomas and eighteen bladder carcinomas.

Amino Acid Sequence↗
PubMed1996

Transcription of gypsy elements in a Y-chromosome male fertility gene of Drosophila hydei.

We have found that defective gypsy retrotransposons are a major constituent of the lampbrush loop pair Nooses in the short arm of the Y chromosome of Drosophila hydei. The loop pair is formed by male fertility gene Q during the primary spermatocyte stage of spermatogenesis, each loop being a single transcription unit with an estimated length of 260 kb. Using fluorescent in situ hybridization, we show that throughout the loop transcripts gypsy elements are interspersed with blocks of a tandemly repetitive Y-specific DNA sequence, ay1. Nooses transcripts containing both sequence types show a wide size range on Northern blots, do not migrate to the cytoplasm, and are degraded just before the first meiotic division. Only one strand of ay1 and only the coding strand of gypsy can be detected in the loop transcripts. However, as cloned genomic DNA fragments also display opposite orientations of ay1 and gypsy, such DNA sections cannot be part of the Nooses. Hence, they are most likely derived from the flanking heterochromatin. The direction of transcription of ay1 and gypsy thus appears to be of a functional significance.

Animals↗
PubMed1995

The Drosophila E74 gene is required for metamorphosis and plays a role in the polytene chromosome puffing response to ecdysone.

The steroid hormone ecdysone initiates Drosophila metamorphosis by reprogramming gene expression during late larval and prepupal development. The ecdysone-inducible gene E74, a member of the ets proto-oncogene family, has been proposed to play a key role in this process. E74 is encoded within the 74EF early puff and consists of two overlapping transcription units, E74A and E74B. To assess the function(s) of E74 during metamorphosis, we have isolated and characterized recessive loss-of-function mutations specific to each transcription unit. We find that mutations in E74A and E74B are predominantly lethal during prepupal and pupal development, consistent with a critical role for their gene products in metamorphosis. Phenotypic analysis reveals that E74 function is required for both pupariation and pupation, and for the metamorphosis of both larval and imaginal tissues. E74B mutants are defective in puparium formation and head eversion and die as prepupae or cryptocephalic pupae, while E74A mutants pupariate normally and die either as prepupae or pharate adults. We have also investigated the effects of the E74 mutations on gene expression by examining the puffing pattern of the salivary gland polytene chromosomes in newly formed mutant prepupae. Most puffs are only modestly affected by the E74B mutation, whereas a subset of late puffs are sub-maximally induced in E74A mutant prepupae. These observations are consistent with Ashburner's proposal that early puff proteins induce the formation of late puffs, and define E74A as a regulator of late puff activity. They also demonstrate that E74 plays a wide role in reshaping the insect during metamorphosis, affecting tissues other than the salivary gland in which it was originally identified.

Animals↗
PubMed1991

Molecular cloning and transformation of cyclodiene resistance in Drosophila: an invertebrate gamma-aminobutyric acid subtype A receptor locus.

Cyclodiene resistance represents 60% of the reported cases of insecticide resistance and is also present in vertebrates. Resistance is due to insensitivity of the cyclodiene/picrotoxinin binding site on the gamma-aminobutyric acid subtype A (GABAA) receptor-chloride ionophore complex. Following isolation of cyclodiene-resistant Drosophila mutants, we report the cloning of the locus conferring resistance via a "chromosomal walk" and rescue of the susceptible phenotype by P-element-mediated germ-line transformation. Amino acid sequence analysis of a cDNA from the locus reveals homology with vertebrate GABAA subunits. To our knowledge, this represents the first cloning of an invertebrate GABA receptor and also allows us to manipulate the resistance status of an insect via germ-line transformation. This gene may be useful as a selectable marker in other insect systems.

Amino Acid Sequence↗
PubMed1998

Squash procedure for protein immunolocalization in meiotic cells.

Several techniques have been developed for protein immunolocalization in meiotic cells. However, most of them include treatments that lead to cell disruption and are only suitable for prophase-I cells. We describe a novel squash procedure of cell preparation for protein immunolabelling of different meiotic stages. This procedure is an alternative to both cryosectioning and whole spreading procedures. We present results obtained in mouse spermatocytes with three different antibodies: the MPM-2 mAb against mitotic phosphoepitopes, an anticentromere serum and a polyclonal serum against the SCP3 protein of the axial elements and lateral elements of the synaptonemal complex. The procedure was tested for single and double immunolabelling. With this technique a large number of cells at different meiotic stages can be analysed. Cell stages are easily identified and cell and chromosome structures are preserved. Thus, it allows the study of chromosome behaviour and the relationships between the different structural elements of the cell throughout meiotic divisions. Our procedure is also suitable for three-dimensional (3D) analyses and proved to be reliable in a wide range of systems including insects and mammals. In addition, the procedure may be interesting to obtain a rapid immunological diagnosis.

Animals↗
PubMed1980

Behavioral mutants of Drosophila melanogaster. III. Isolation and mapping of mutations by direct visual observations of behavioral phenotypes.

Sex-linked behavioral mutants were induced in Drosophila melanogaster with ethyl methanesulfonate (EMS) and isolated by direct visual observation of abnormal phenotypes. The four behavioral phenotypes used were flight-reduction, hyperactivity, hypoactivity and stress-sensitivity, and are easily discernable in either single or small populations of mutant flies. In one screen, forty-two behavioral mutants were recovered from strains derived from 800 mutagen-treated X chromosomes In a second screen, 139 behavioral mutants were obtained from 2369 X chromosomes. The high rate at which behavioral mutants were recovered in the second screen, when compared to new visibles (28) and new temperature-sensitive lethals (124), suggests that the isolation of behavioral mutations on the autosomes of Drosophila and in the genomes of larger insects should be practical.

Animals↗
PubMed2005

The evolutionary origin of insect telomeric repeats, (TTAGG)n.

The (TTAGG)n sequence is supposed to be an ancestral DNA motif of telomeres in insects. Here we examined the occurrence of TTAGG telomeric repeats in other arthropods and their close relatives by Southern hybridization of genomic DNAs and fluorescence in-situ hybridization (FISH) of chromosomes with (TTAGG)n probes or, alternatively, with the 'vertebrate' telomeric probe, (TTAGGG)n. Our results show that the (TTAGG)n motif is conserved in entognathous hexapods (Diplura and Collembola), crustaceans (Malacostraca, Branchiura, Pentastomida, and Branchiopoda), myriapods (Diplopoda and Chilopoda), pycnogonids, and most chelicerates (Palpigradi, Amblypygi, Acari, Opiliones, Scorpiones, Pseudoscorpiones, and Solifugae) but not in spiders (Araneae). The presence of TTAGG repeats in these groups suggests that the sequence is an ancestral motif of telomeres not only in insects but in Arthropoda. We failed, however, to detect the TTAGG repeats in close relatives of the arthropods, Tardigrada and Onychophora. But while Onychophora had the 'vertebrate' (TTAGGG)n motif instead, the Tardigrada did not. The (TTAGG)n motif probably evolved from the (TTAGGG)n motif. Based on our and compiled data, we presume that the 'vertebrate' motif (TTAGGG)n is an ancestral motif of telomeres in bilaterian animals and possibly also in the superclade including animals, fungi and amoebozoans.

Acari↗
PubMed2006

Intron length evolution in Drosophila.

I present data on the evolution of intron lengths among 3 closely related Drosophila species, D. melanogaster, Drosophila simulans, and Drosophila yakuba. Using D. yakuba as an outgroup, I mapped insertion and deletion mutations in 148 introns (spanning approximately 30 kb) to the D. melanogaster and D. simulans lineages. Intron length evolution in the 2 sister species has been different: in D. melanogaster, X-linked introns have increased slightly in size, whereas autosomal ones have decreased slightly in size; in D. simulans, both X-linked and autosomal introns have decreased in size. To understand the possible evolutionary causes of these lineage- and chromosome-specific patterns of intron evolution, I studied insertion-deletion (indel) polymorphism and divergence in D. melanogaster. Small insertion mutations segregate at elevated frequencies and enjoy elevated probabilities of fixation, particularly on the X chromosome. In contrast, there is no detectable X chromosome effect on fixations in D. simulans. These findings suggest X chromosome-specific selection or biased gene conversion-gap repair favoring insertions in D. melanogaster but not in D. simulans. These chromosome- and lineage-specific patterns of indel substitution are not easily explained by existing general population genetic models of intron length evolution. Genomic data from D. melanogaster further suggest that the forces described here affect introns and intergenic regions similarly.

Animals↗
PubMed2006

Misregulation of sex-lethal and disruption of male-specific lethal complex localization in Drosophila species hybrids.

A major model system for the study of evolutionary divergence between closely related species has been the unisexual lethality resulting from reciprocal crosses of Drosophila melanogaster and D. simulans. Sex-lethal (Sxl), a critical gene for sex determination, is misregulated in these hybrids. In hybrid males from D. melanogaster mothers, there is an abnormal expression of Sxl and a failure of localization of the male-specific lethal (MSL) complex to the X chromosome, which causes changes in gene expression. Introduction of a Sxl mutation into this hybrid genotype will allow expression of the MSL complex but there is no sequestration to the X chromosome. Lethal hybrid rescue (Lhr), which allows hybrid males from this cross to survive, corrects the SXL and MSL defects. The reciprocal cross of D. simulans mothers by D. melanogaster males exhibits underexpression of Sxl in embryos.

Animals↗
PubMed1997

Evidence for an inducible repair-recombination system in the female germ line of Drosophila melanogaster. III. Correlation between reactivity levels, crossover frequency and repair efficiency.

We previously reported evidence that the so-called reactivity level, a peculiar cellular state of oocytes that regulates the frequency of transposition of I factor, a LINE element-like retrotransposon, might be one manifestation of a DNA repair system. In this article, we report data showing that the reactivity level is correlated with the frequency of crossing over, at least on the X chromosome and on the pericentromeric region of the third chromosome. Moreover, a check for X-chromosome losses and recessive lethals produced after gamma irradiation in flies with different reactivity levels, but common genetic backgrounds, brings more precise evidence for the relationship between reactivity levels and DNA repair. Those results support the existence of a repair-recombination system whose efficiency is modulated by endogenous and environmental factors. The implications of this biological system in connecting genomic variability and environment may shed new lights on adaptative mechanisms. We propose to call it VAMOS for variability modulation system.

Aging↗
PubMed1993

Localization of Shaw-related K+ channel genes on mouse and human chromosomes.

Four related genes, Shaker, Shab, Shaw, and Shal, encode voltage-gated K+ channels in Drosophila. Multigene subfamilies corresponding to each of these Drosophila genes have been identified in rodents and primates; this suggests that the four genes are older than the common ancestor of present-day insects and mammals and that the expansion of each into a family occurred before the divergence of rodents and primates. In order to define these evolutionary relationships more precisely and to facilitate the search for mammalian candidate K+ channel gene mutations, we have mapped members of the Shaw-homologous gene family in humans and mice. Fluorescence in situ hybridization analysis of human metaphase chromosomes mapped KCNC2 (KShIIIA, KV3.2) and KCNC3 (KShIIID, KV3.3) to Chromosome (Chr) 19q13.3-q13.4. Inheritance patterns of DNA restriction fragment length variants in recombinant inbred strains of mice placed the homologous mouse genes on distal Chr 10 near Ms15-8 and Mdm-1. The mouse Kcnc1 (KShIIIB, NGK2-KV4, KV3.1) gene mapped to Chr7 near Tam-1. These results are consistent with the hypothesis that the generation of the mammalian KCNC gene family included both duplication events to generate family members in tandem arrays (KCNC2, KCNC3) and dispersion of family members to unlinked chromosomal sites (KCNC1). The KNCN2 and KCNC3 genes define a new synteny group between humans and mice.

Animals↗
PubMed1999

Identification of nuclear genes encoding mitochondrial proteins: isolation of a collection of D. melanogaster cDNAs homologous to sequences in the Human Gene Index database.

As a first step towards using cross-species comparison to complete the inventory of the nuclear genes that encode mitochondrial polypeptides, and ultimately to understand their function through systematic molecular and genetic analysis in a model organism of choice, we report here the characterization of 41 Drosophila melanogaster cDNAs. These cDNAs were isolated by screening an ovarian expression library with antibodies against mitochondrial proteins and identify 17 novel Drosophila genes. The deduced amino acid sequences encoded by the majority of these cDNAs turned out to show significant homology to mitochondrial proteins previously identified in other species. Among others, ORFs putatively encoding six different subunits of ATP synthase and three NADH:ubiquinone reductase subunits were detected. By in situ hybridization, all cDNAs were mapped to single bands on polytene chromosomes, thus identifying candidate Drosophila genes required for mitochondrial biogenesis and maintenance. A search of the Human Gene Index database made it possible in most cases to align the entire Drosophila coding sequence with a human consensus sequence, suggesting that the cDNAs originate from insect counterparts of expressed mammalian genes. Our experimental strategy represents an efficient approach to the identification and interspecies comparison of genes encoding products targeted to the mitochondrion.

Animals↗
PubMed2001

Electrophoretic karyotypes of some species of the genus Leptomonas (kinetoplastida, trypanosomatidae), homoxenous trypanosomatids parasites of insects.

Electrophoretic karyotypes of homoxenous trypanosomatids Leptomonas peterhoffi, L. mycophilus, L. nabiculae and Leptomonas sp. have been studied by transverse alternating-field electrophoresis under varying electrophoretic conditions. From 12 to 17 chromosomal DNA bands, ranging from 370 to more than 1500 kb were detected in the karyograms of the species compared. In each pattern, some intensely stained bands could represent more than one chromosome. Taking into account the number of intensely stained bands, the karyotype of L. peterhoffi was estimated to contain at least 18 chromosomes, the karyotypes of L. mycophilus and L. nabiculae, at least 21 chromosome each, and the karyotype of Leptomonas sp. up to 20 chromosomes. Interclonal variations of electrophoretic karyotypes of 10 clones of Leptomonas sp. (cfmI-cfmX) were studied. Seven of ten clones had identical electrophoretic patterns. In the karyograms of three clones (cfmI, cfmVI, cfmVII), additional chromosomal DNA bands were observed. The obtained results suggest, that electrophoretic karyotypes cannot be used as reliable markers of species of homoxenous trypanosomatids, since intraspecies variability does occur in these parasites.

Animals↗
PubMed2005

Pest control by genetic manipulation of sex ratio.

We model the release of insects carrying an allele at multiple loci that shifts sex ratios in favor of males. We model two approaches to sex ratio alteration. In the first (denoted SD), meiotic segregation (or sperm fertility) is distorted in favor of gametes carrying the male-determining genetic element (e.g., Y-chromosome). It is assumed that any male carrying at least one copy of the SD allele produces only genotypically male offspring. In the second approach (denoted PM), the inserted allele alters sex ratio by causing genetically female individuals to become phenotypically male. It is assumed that any insect carrying at least one copy of the PM allele is phenotypically male. Both approaches reduce future population growth by reducing the number of phenotypic females. The models allow variation in the number of loci used in the release, the size of the release, and the negative fitness effect caused by insertion of each sex ratio altering allele. We show that such releases may be at least 2 orders of magnitude more effective than sterile male releases (SIT) in terms of numbers of surviving insects. For example, a single SD release with two released insects for every wild insect and a 5% fitness cost per inserted allele could reduce the target population to 1/1000th of the no-release population size, whereas a similar-sized SIT release would only reduce the population to one-fifth of its original size. We also compare these two sex ratio alteration approaches to a female-killing (FK) system and the sterile male technique when there are repeated releases over a number of generations. In these comparisons, the SD approach is the most efficient with equivalent pest suppression achieved by release of approximately 1 SD, 1.5-20 PM, 2-70 FK, and 16-3,000 SIT insects, depending on conditions. We also calculate the optimal number of SD and PM allele insertions to be used under various conditions, assuming that there is an additional genetic load incurred for each allelic insertion.

Alleles↗