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PubMed2002

The evolution of sex determination systems in dipteran insects other than Drosophila.

The multitude of sex determination mechanisms displayed in dipteran insects has usually been described in terms of variations on a single principle in which the primary signal of the primitive pathway consists of a single allelic difference at one locus. Evolution of sex determination mechanisms is thought to have occurred by the addition of genes below the top gene of the pathway. The elucidation of the complex sex determination pathway of Drosophila melanogaster, as well as recent evidence that the basal genes of the pathway seem to be conserved across metazoan genera both in structure and, to a lesser degree, in function, points towards the possibility that sex determination pathways may have evolved from the bottom-up. Further to this is the question of whether the dominant male-determining factor, M, which is found in a number of insect species, represents part of the ancient sex determination pathway or is a later addition to the pathway. This, together with the possibility that the M factors found in numerous dipteran insect species may have a common origin, is discussed. The similarities of the sex determination pathways under the control of M and the implications in relation to the construction of genetic sexing strains for biological control are also discussed.

Animals↗
PubMed1996

Origins of immunity: Relish, a compound Rel-like gene in the antibacterial defense of Drosophila.

NF-kappa B/Rel transcription factors are central regulators of mammalian immunity and are also implicated in the induction of cecropins and other antibacterial peptides in insects. We identified the gene for Relish, a compound Drosophila protein that, like mammalian p105 and p100, contains both a Rel homology domain and an I kappa B-like domain. Relish is strongly induced in infected flies, and it can activate transcription from the Cecropin A1 promoter. A Relish transcript is also detected in early embryos, suggesting that it acts in both immunity and embryogenesis. The presence of a compound Rel protein in Drosophila indicates that similar proteins were likely present in primordial immune systems and may serve unique signaling functions.

Amino Acid Sequence↗
PubMed2002

What's buzzing? Mosquito genomics and transgenic mosquitoes.

Genome projects and associated technologies are now being established for mosquito species that are vectors of human disease. The recent announcement of an award by the National Institute of Allergy and Infectious Diseases (NIAID) to Celera Genomics to sequence the Anopheles gambiae genome will further accelerate the completion of the sequencing of this genome. Completion of the An. gambiae sequence will mean that the genomes of all three organisms involved in the transmission of falciparum malaria--the mosquito, the parasite, and the human--will have been sequenced. This will greatly facilitate the identification of genes and pathways involved in the transmission of malaria. The recent genetic transformation of An. gambiae with the piggyBac transposable element and the transformation of another important malarial vector, Anopheles stephensi using the Minos element, now provide researchers with powerful tools with which to genetically manipulate these medically important vector species. Here we review the recent progress made in the extension of contemporary tools of modern genetics and genomics into these medically important insects.

Aedes↗
PubMed1995

Analysis and chromosomal localization of retrotransposons in sugar beet (Beta vulgaris L.): LINEs and Ty1-copia-like elements as major components of the genome.

DNA sequences of the reverse transcriptase gene of long terminal repeat (LTR) and non-LTR (non-viral) retrotransposons have been isolated and cloned from the genome of sugar beet (Beta vulgaris). Both retrotransposon types are highly amplified in sugar beet and may account for 2-5% of the genome. The BNR1 family, representing the first non-viral retrotransposon reported from a dicotyledonous species, shows homology to the mammalian L1 family of long interspersed repeated sequences (LINEs) and to retrotransposable elements from maize and lily. Sequences of the Tbv family are homologous to the Ty1-copia class of LTR retrotransposons. The BNR1 and Tbv retrotransposon families are characterized by sequence heterogeneity and are probably defective. The deduced peptide sequences were used to investigate the relation to other retroelements from plants, insects and mammals. Fluorescence in situ hybridization was used to investigate the physical distribution and revealed that both retrotransposon families are present on all sugar beet chromosomes and largely excluded from chromosomal regions harbouring the 18S-5.8S-25S rRNA genes. The BNR1 family is organized in discrete clusters, while the Tbv family of Ty1-copia-like retrotransposons shows a more uniform distribution along chromosome arms and is absent from some chromosomal regions. These contrasting distributions emphasize the differences in evolutionary amplification and dispersion mechanisms between the two types of retrotransposons. The in situ results of both elements reflect significant features of a higher order structure of the genome, as it is known for both short interspersed repeated sequences (SINEs) and LINEs in human.

Amino Acid Sequence↗
PubMed1976

Naturally occuring mutagens.

Naturally occurring mutagens have usually been discovered as a result of outbreaks of disease in agricultural livestock, or as a result of epidemiological studies of cancer of the liver in man. Subsequent work has then shown that the toxic agents responsible often have mutagenic properties. Examples are the pyrrolizidine alkaloids, cyasin, a range of mycotoxins produced by various fungi, and at least two unidentified toxic agents in bracken. Commonly the toxic agent itself does not show high biological activity, but after ingestion it is converted by metabolic processes into the active mutagen or carcinogen. Some of these toxic substances have been responsible for considerable losses of agricultural livestock and therefore are of economic significance. From the view-point of genetic hazards to man, the most significant compounds are probably the mycotoxins, e.g. aflatoxin, because of the common risk of fungal contamination of food, especially in tropical regions. No information is yet available on the effects of these mutagens on natural populations of animals. Plants containing the pyrrolizidine alkaloids are found in areas of Africa and the Middle East where plagues of the migratory locust occur. Although it is known that some of the alkaloids can induce chromosomal damage in grasshoppers, whether such damage ever becomes a significant factor under ecological conditions is not known. In some cases, insects have not only evolved resistance towards mutagenic alkaloids but have become dependent on them for certain purposes. The males of certain Danaid butterflies feed on plants containing pyrrolizidine alkaloids. After ingestion, the alkaloids are metabolised to dihydropyrrolizine derivatives, which are then secreted on special organs (hair pencils) and, following dissemination into the atmosphere, act as pheromones for the stimulation of mating behaviour.

Aflatoxins↗
PubMed2002

Genomic expansion and clustering of ZAD-containing C2H2 zinc-finger genes in Drosophila.

C2H2 zinc-finger proteins (ZFPs) constitute the largest family of nucleic acid binding factors in higher eukaryotes. In silico analysis identified a total of 326 putative ZFP genes in the Drosophila genome, corresponding to approximately 2.3% of the annotated genes. Approximately 29% of the Drosophila ZFPs are evolutionary conserved in humans and/or Caenorhabditis elegans. In addition, approximately 28% of the ZFPs contain an N-terminal zinc-finger-associated C4DM domain (ZAD) consisting of approximately 75 amino acid residues. The ZAD is restricted to ZFPs of dipteran and closely related insects. The evolutionary restriction, an expansion of ZAD-containing ZFP genes in the Drosophila genome and their clustering at few chromosomal sites are features reminiscent of vertebrate KRAB-ZFPs. ZADs are likely to represent protein-protein interaction domains. We propose that ZAD-containing ZFP genes participate in transcriptional regulation either directly or through site-specific modification and/or regulation of chromatin.

Amino Acid Sequence↗
PubMed1992

Selection of dieldrin-resistant strains of Lucilia cuprina (Diptera: Calliphoridae) after ethyl methanesulfonate mutagenesis of a susceptible strain.

After ethyl methanesulfonate (EMS) mutagenesis of a susceptible strain (SWT), selective screening of Lucilia cuprina (Wiedemann) resulted in four strains that were resistant to the insecticide dieldrin. Concentrations used for selection were greater than LC99 of susceptible phenotypes. No resistant variants were screened from the standard laboratory strain (SWT) not treated with EMS. The resistance phenotypes of the four resistant strains were similar to each other and to that of a field-selected resistant strain. The genetic basis of resistance is monogenic in all strains and the data are consistent with the same locus, Rdl, determining resistance status in each strain. The Rdl locus maps to chromosome V, approximately 3.5 map units distal to the Sut locus. Dieldrin resistance may be caused by less effective blocking of insect neuronal GABA receptors by the chemical in resistant strains. The data indicate that the evolution of resistance to an insecticide in the field may be constrained by a limited number of genetical and biochemical options if a monogenic response is selected for and that the spontaneous mutation rate to the Rdl allele is less than 1 in 10(6) in the laboratory.

Animals↗
PubMed2003

"Candidatus Phytoplasma oryzae", a novel phytoplasma taxon associated with rice yellow dwarf disease.

In addition to rice yellow dwarf (RYD) phytoplasma, several phytoplasmas infect gramineous plants, including rice orange leaf, bermuda grass white leaf, brachiaria grass white leaf and sugarcane white leaf phytoplasmas. To investigate whether the RYD phytoplasma is a discrete, species-level taxon, several isolates of the aforementioned phytoplasmas were analysed using PCR-amplified 16S rDNA sequences. Two RYD isolates, RYD-J(T) and RYD-Th, were almost identical (99.2 %), but were distinct (similarities of 96.3-97.9 %) from other phytoplasma isolates of the RYD 16S-group. The notion that the RYD phytoplasma constitutes a unique taxon is also supported by its unique insect vector (Nephotettix sp.), its unique host plant in nature (rice) and its limited geographical distribution (Asia). In Southern blot analysis, chromosomal and extrachromosomal DNA probes of the RYD phytoplasma reportedly did not hybridize with those of closely related phytoplasmas. These properties of the RYD phytoplasma clearly indicate that it represents a novel taxon, 'Candidatus Phytoplasma oryzae'.

Base Sequence↗
PubMed1987

Observations on the Anopheles gambiae complex in the Senegal River Basin, West Africa.

1. Three sibling species of mosquitoes of the Anopheles gambiae complex are found in the Senegal River Basin: An. melas Theobald, An. gambiae Giles and An. arabiensis Patton. 2. An. melas is restricted to the river delta and environs where saltwater breeding places are present. 3. An. gambiae and An. arabiensis are sympatric in the study area; An. arabiensis predominates in coastal zones where it breeds also during the dry season; An. gambiae predominates in inland areas where breeding is mostly restricted to the rainy season (July-September). 4. An. arabiensis is chromosomally polymorphic all over the study area, with much variation of inversion frequencies, particularly for the 2Ra arrangement. 5. An. gambiae is characterized by a very high frequency of the 2Rb-2La inversion arrangement which is typical of the Savanna chromosomal form.

Animals↗
PubMed2005

The Drosophila meiotic mutant mei-352 is an allele of klp3A and reveals a role for a kinesin-like protein in crossover distribution.

The semisterile meiotic mutant mei-352 alters the distribution of meiotic exchanges without greatly affecting their total frequency. We show that the mei-352 mutation is an allele of the klp3A gene, which encodes a kinesin-like protein of the Kinesin-4 family. The semisterility observed in mei-352 females results from a known defect of klp3A oocytes in mediating pronuclear fusion. Interestingly, other klp3A alleles also exhibit defects in meiotic recombination similar to those of mei-352. Finally, we show that the Klp3A protein localizes within the oocyte nucleus during meiotic prophase, the time at which exchange distribution is established, and extensively colocalizes with DNA. The parallel of the klp3A phenotype with a meiotic defect observed for kar3 mutants in yeast suggests a role for kinesins in early meiosis and might reflect a previously suggested role for this class of kinesins in chromosome condensation.

Alleles↗
PubMed2003

[Cloning and sequencing of chitinase gene from Bacillus thuringiensis subsp israelensis].

Chitin, a linear homopolymer of N-acetyl-D-glucosamine, is a common constituent of fungal cell walls, exoskeletons of insects, and shells of invertebrates. Thus, chitinase, a chitin hydrolytic enzyme, has become of interest for potential use as biopesticides for controlling insect pests. Using a pair of specific primers, chitinase gene (ichi) was amplified by touchdown PCR from Bacillus thuringiensis subsp israelensis chromosomal DNA, and then subcloned into pGEM-T easy vector. ichi sequence (GenBank Accession Number: AF526379) with a length of 2570 bp included an open reading frame (ORF) of 2067 bp, which contained 688 amino acids with Mr = 75.79 kDa and pI = 5.90. The amino acid sequence of ichi gene shows 97.24%, 97.18%, 97.63% and 63.07% identity to that of Bacillus cereus strain 28-9 chitinase CW, Bacillus cereus CH chitinase B, Bt subsp Mexican chitinase, and Bt subsp pakistani chitinase A71, respectively. It was demonstrated that Ichi contains a signal peptide (46 amino acid residues) and three functional domains: an N-terminal catalytic domain (105 amino acid residues), a fibronectin type III like domain (74 amino acid residues), and a C-terminal chitin-binding domain (40 amino acid residues).

Amino Acid Sequence↗
PubMed2001

Isolation of a deet-insensitive mutant of Drosophila melanogaster (Diptera: Drosophilidae).

Despite the widespread use of N,N,-diethyl-3-methylbenzamide (deet) in insect repellent products, nothing is known about the molecular basis for the repellency of deet, we initiated a molecular genetics program to elucidate the molecular mechanism of deet repellency in Drosophila melanogaster (Meigen). Deet repellency was apparently due to airborne vapors, as wild type flies were repelled by a deet-treated surface in the absence of physical contact and in the dark. A mutant was isolated using chemical mutagenesis and at choice assay. In a choice assay, mutant flies entered 82 +/- 1% of deet-containing tubes, whereas wild type flies entered only 6 +/- 2% of deet-containing tubes. The mutant was repelled by other repellents, benzaldehyde and citronellal. The mutation was recessive and located on the X chromosome.

Animals↗
PubMed1993

Localization of the lampbrush loop pair Nooses on the Y chromosome of Drosophila hydei by fluorescence in situ hybridization.

We have used fluorescence in situ hybridization to map the positions of the different repetitive DNA sequences from the region forming the lampbrush loop pair Nooses on the Y chromosome of Drosophila hydei. This region harbours a megabase cluster of tandemly organized repeats of the Y-specific ay1 family and a megabase cluster of tandem repeats of the related Y-specific YsI family. In addition, ay1 repeats also occur in short blocks that are interspersed by other repetitive DNA sequences that we call Y-associated, since they have additional copies on other chromosomes. Using specific probes for ay1, YsI and Y-associated DNA sequences, we show that there is one large proximal cluster of YsI repeats and one, more distally located, large cluster of ay1 repeats. The Y-chromosomal copies of the Y-associated sequences are located in the most distal part of the ay1 cluster. This is consistent with the juxtaposition of ay1 and Y-associated sequences in more than 300 kb of cloned genomic DNA. Since both ay1 and Y-associated sequences have been shown to be transcribed in the Nooses, the lampbrush loop is formed in a distal region of the short arm of the Y chromosome, adjacent to the terminally located nucleolus organizer region. The clusters of homogeneous ay1 and YsI repeats are of no functional significance for the formation of the lampbrush loop.

Animals↗
PubMed2003

Spiralin is not essential for helicity, motility, or pathogenicity but is required for efficient transmission of Spiroplasma citri by its leafhopper vector Circulifer haematoceps.

Spiralin is the most abundant protein at the surface of the plant pathogenic mollicute Spiroplasma citri and hence might play a role in the interactions of the spiroplasma with its host plant and/or its insect vector. To study spiralin function, mutants were produced by inactivating the spiralin gene through homologous recombination. A spiralin-green fluorescent protein (GFP) translational fusion was engineered and introduced into S. citri by using an oriC-based targeting vector. According to the strategy used, integration of the plasmid by a single-crossover recombination at the spiralin gene resulted in the expression of the spiralin-GFP fusion protein. Two distinct mutants were isolated. Western and colony immunoblot analyses showed that one mutant (GII3-9a5) did produce the spiralin-GFP fusion protein, which was found not to fluoresce, whereas the other (GII3-9a2) produced neither the fusion protein nor the wild-type spiralin. Both mutants displayed helical morphology and motility, similarly to the wild-type strain GII-3. Genomic DNA analyses revealed that GII3-9a5 was unstable and that GII3-9a2 was probably derived from GII3-9a5 by a double-crossover recombination between plasmid sequences integrated into the GII3-9a5 chromosome and free plasmid. When injected into the leafhopper vector Circulifer haematoceps, the spiralinless mutant GII3-9a2 multiplied to high titers in the insects (1.1 x 10(6) to 2.8 x 10(6) CFU/insect) but was transmitted to the host plant 100 times less efficiently than the wild-type strain. As a result, not all plants were infected, and symptom production in these plants was delayed for 2 to 4 weeks compared to that in the wild-type strain. In the infected plants however, the mutant multiplied to high titers (1.2 x 10(6) to 1.4 x 10(7) CFU/g of midribs) and produced the typical symptoms of the disease. These results indicate that spiralin is not essential for pathogenicity but is required for efficient transmission of S. citri by its insect vector.

Animals↗
PubMed2006

Chromosome-wide gene-specific targeting of the Drosophila dosage compensation complex.

The dosage compensation complex (DCC) of Drosophila melanogaster is capable of distinguishing the single male X from the other chromosomes in the nucleus. It selectively interacts in a discontinuous pattern with much of the X chromosome. How the DCC identifies and binds the X, including binding to the many genes that require dosage compensation, is currently unknown. To identify bound genes and attempt to isolate the targeting cues, we visualized male-specific lethal 1 (MSL1) protein binding along the X chromosome by combining chromatin immunoprecipitation with high-resolution microarrays. More than 700 binding regions for the DCC were observed, encompassing more than half the genes found on the X chromosome. In addition, several rare autosomal binding sites were identified. Essential genes are preferred targets, and genes binding high levels of DCC appear to experience the most compensation (i.e., greatest increase in expression). DCC binding clearly favors genes over intergenic regions, and binds most strongly to the 3' end of transcription units. Within the targeted genes, the DCC exhibits a strong preference for exons and coding sequences. Our results demonstrate gene-specific binding of the DCC, and identify several sequence elements that may partly direct its targeting.

Animals↗
PubMed1983

Utilization of Chinese hamster cells in vitro and in vivo in genetic toxicology: a multiphasic approach.

An approach often used to study genetic risk is comparative mutagenesis in different organisms. We have begun the development and validation of a multiphasic genetic toxicity system using the Chinese hamster and its somatic cells to measure mutational events in the same (or similar) gene, the same chromosome derived from the same animal (Fig. 1). This system will eliminate much of the uncertainty generated when different mutational events in such evolutionally divergent organisms as bacteria, insects, and mammals are compared. Using CHO cells we have defined an assay, CHO/HGPRT, to determine mutation at the hgprt locus. Coupled with S9 metabolic activation system, the CHO/HGPRT assay can quantify gene mutation and cytotoxicity induced by various classes of chemicals, physical agents, and the combination thereof. The quantitative nature of this assay permits elucidation of the structure-activity relationship for a given class of direct-acting agents. By incorporating the cytogenetics of CHO cells into this assay we can simultaneously measure induced chromosome aberrations and SCE. Using the stable CHO/human hybrid cell line AL-J1 measurement of chromosome deletion and/loss can be also performed. In order to further expand the usefulness of this genetic toxicity system to the molecular and whole animal levels we have begun development and validation of two additional systems. To study the molecular events which may result in mutation we are developing a CHOpSVgpt system. A Chinese hamster system with treatment in vivo is being developed to study mutation at the hgprt locus, chromosome aberration, and SCE in spleen cells in vitro. The use of this multiphasic genetic toxicity system at the cellular, molecular, and animal levels may soon provide reliable and rapid identification of suspected environmental mutagens.

Alkylating Agents↗
PubMed2000

Solh, the mouse homologue of the Drosophila melanogaster small optic lobes gene: organization, chromosomal mapping, and localization of gene product to the olfactory bulb.

The Drosophila melanogaster small optic lobes gene (sol) is required for normal development of the neuropiles of the medulla and lobula complexes of the adult optic lobes. The predicted protein products of sol and its human homologue SOLH contain zinc-finger-like repeats, a calpain-like protease domain, and a C-terminal domain of unknown function. Long-distance PCR was used to amplify genomic DNA for Solh, the mouse homologue of sol, following the identification of mouse Solh expressed sequence tags. The nucleotide sequence of the Solh coding region (6.0 kb) was determined. The predicted Solh protein of 1095 amino acid residues shows 89% identity (93% similarity) to the human homologue. Solh was localized by in situ hybridization to band A3.3 on mouse Chromosome 17, in a region of maintained homology with human 16p13.3. Antipeptide antibodies were prepared and verified by demonstration of specific reactivity with recombinant human SOLH protein prepared by in vitro transcription/translation and expression in insect cells using the baculovirus system. The antibodies were used to show that the Solh protein localizes to the olfactory bulb in mouse and rat brain, suggesting that it could have an analogous role in development of sensory system neurons in Drosophila and in mammals.

Amino Acid Sequence↗
PubMed1993

Mutations in the Drosophila gene encoding ribosomal protein S6 cause tissue overgrowth.

We have characterized two P-element-induced, lethal mutations in Drosophila melanogaster which affect the larval hemocytes, mediators of the insect immune response. Each mutant displays larval melanotic tumors characteristic of mutations affecting the insect cellular immune system, and the moribund animals develop grossly hypertrophied hematopoietic organs because of increased cell proliferation and extra rounds of endoreduplication in some hematopoietic cells. Surprisingly, these mutations are due to P element insertions in the 5' regulatory region of the Drosophila gene encoding ribosomal protein S6 and cause a reduction of S6 transcript abundance in mutant larvae.

Animals↗