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PubMed2002

Decoding cis-regulatory DNAs in the Drosophila genome.

Cis-regulatory DNAs control the timing and sites of gene expression during metazoan development. Changes in gene expression are responsible for the morphological diversification of metazoan body plans. However, traditional methods for the identification and characterization of cis-regulatory DNAs are tedious. During the past year, computational methods have been used to identify novel cis-DNAs within the entire Drosophila genome. These methods change the way that cis-DNAs will be analyzed in future studies and offer the promise of unraveling complex gene networks.

Animals↗

PubMed2002

MRD: a microsatellite repeats database for prokaryotic and eukaryotic genomes.

MRD is a database system to access the microsatellite repeats information of genomes such as archea, eubacteria, and other eukaryotic genomes whose sequence information is available in public domains. MRD stores information about simple tandemly repeated k-mer sequences where k= 1 to 6, i.e. monomer to hexamer. The web interface allows the users to search for the repeat of their interest and to know about the association of the repeat with genes and genomic regions in the specific organism. The data contains the abundance and distribution of microsatellites in the coding and non-coding regions of the genome. The exact location of repeats with respect to genomic regions of interest (such as UTR, exon, intron or intergenic regions) whichever is applicable to organism is highlighted. MRD is available on the World Wide Web at http://www.ccmb.res.in/mrd and/or http://www.ingenovis.com/mrd. The database is designed as an open-ended system to accommodate the microsatellite repeats information of other genomes whose complete sequences will be available in future through public domain.

Animals↗

PubMed2006

Loss of genomic imprinting in Drosophila clones.

Genomic imprinting is a process that genetically distinguishes maternal and paternal genomes, and can result in parent-of-origin-dependent monoallelic expression of a gene that is dependent on the parent of origin. As such, an otherwise functional maternally inherited allele may be silenced so that the gene is expressed exclusively from the paternal allele, or vice versa. Once thought to be restricted to mammals, genomic imprinting has been documented in angiosperm plants (J.L. Kermicle. 1970. Genetics, 66: 69-85), zebrafish (C.C. Martin and R. McGowan. 1995. Genet. Res. 65: 21-28), insects, and C. elegans (C.J. Bean, C.E. Schaner, and W.G. Kelly. 2004. Nat. Genet. 36: 100-105.). In each case, it appears to rely on differential chromatin structure. Aberrant imprinting has been implicated in various human cancers and has been detected in a number of cloned mammals, potentially limiting the usefulness of somatic nuclear transfer. Here we show that genomic imprinting associated with a mini-X chromosome is lost in Drosophila melanogaster clones.

Adaptor Protein Complex 3↗

PubMedDate not supplied

Comparisons of genetic variability and genome structure among mosquito strains selected for refractoriness to a malaria parasite.

Restriction fragment length polymorphism (RFLP) markers were used to evaluate Aedes aegypti genome structure and genetic variability within and between substrains selected for different levels of refractoriness to the malaria parasite, Plasmodium gallinaceum. The MOYO-R substrain was previously selected for complete refractoriness and the MOYO-IS substrain for intermediate susceptibility from the Moyo-In-Dry (MOYO) strain by selective inbreeding (F = 0.5). Eighteen mapped RFLP markers were used to provide coverage of the mosquito genome. The two substrains showed reduced genetic diversity compared with the MOYO strain, including significant reductions in mean heterozygosity, number of alleles per locus, and proportion of polymorphic loci. Genetic differentiation between the two substrains was statistically significant, as reflected by differences in allele frequencies. Significant pairwise linkage disequillbrium among the RFLP loci was detected in all three strains, most evidently in the MOYO strain. This is surprising because the RFLP loci examined are separated by large map distances, and therefore linkage disequilibrium should decay to zero after many generations of laboratory culture. Our hypothesis to explain this phenomena is that lack of recombination, or low recombination rates in some regions of the A. aegypti genome, is a result of chromosome inversions. Finally, we used graphical genotyping, wherein whole genome genotypic information for individual mosquitoes is represented in a simple graphic format, to illustrate genome structure and allelic variation within and among the mosquito strains. Our analysis revealed an apparent chromosomal deletion on chromosome 3 for some individuals in the MOYO strain and MOYO-IS substrain.

Aedes↗

PubMed2002

Molecular anatomy of Chilo iridescent virus genome and the evolution of viral genes.

Chilo iridescent virus (CIV) or Insect iridescent virus 6 (IIV-6) is the type species of the genus iridovirus, a member of the Iridoviridae family. CIV is highly pathogenic for a variety of insect larvae and this implicates a possible use as a biological insecticide. CIV progeny and assembly occur in the cytoplasm of the infected cell and accumulate in the fatbody of the infected insects. Since the discovery of CIV in 1966, many attempts were made to elucidate the viral genome structure and the amino acid sequences of different viral gene products. The elucidation of the coding capacity and strategy of CIV was the first step towards understanding the underlying mechanisms of viral infection, replication and virus-host interaction. The virions contain a single linear ds DNA molecule that is circularly permuted and terminally redundant. The coding capacity of the CIV genome was determined by the analysis of the complete DNA nucleotide sequence consisting of 212,482 bp that represent 468 open reading frames encoding for polypeptides ranging from 40 to 2432 amino acid residues. The analysis of the coding capacity of the CIV genome revealed that 50% (234 ORFs) of all identified ORFs (468 ORFs) were non-overlapping. The identification of several putative viral gene products including a DNA ligase and a viral antibiotic peptide is a powerful tool for the investigation of the phylogenetic relatedness of this evolutionary and ecologically relevant eukaryotic virus.

Amino Acid Sequence↗

PubMed1999

Circular configuration of the genome of ascoviruses.

A circular configuration of genomic DNA was observed in ascoviruses isolated from two species of insects of the family Noctuidae [fall armyworm (Spodoptera frugiperda) and cotton bollworm (Helicoverpa zea)] using restriction endonuclease (REN) digestion, conventional gel electrophoresis, pulsed-field gel electrophoresis and Southern hybridization analysis. This circular configuration of ascovirus genomic DNA was established based on the difference between linear and circular DNA in the numbers of fragments resolved on agarose gel electrophoresis after single and double REN digestion. Genomic DNA of ascoviruses was found to be sheared after purification.

Animals↗

PubMed1996

Genomic imprinting of chromatin in Drosophila melanogaster.

During gametogenesis, chromosomes may become imprinted with information which facilitates proper expression of the DNA in offspring. We have used a position effect variegation mutant as a reporter system to investigate the possibility of imprinting in Drosophila melanogaster. Genetic crosses were performed in which the variegating gene and a strong modifier of variegation were present either within the same parental genome or in opposite parental genomes in all possible combinations. Our results indicate that the presence of the variegating chromosome and a modifier chromosome in the same parental genome can alter the amount of variegation formed in progeny. The genomic imprinting we observed is not determined by the parental origin of the variegating chromosome but is instead determined by the genetic background the variegating chromosome is subjected to during gametogenesis.

Animals↗

PubMed1995

Molecular and genetic dissection of a reproductive isolation gene, zygotic hybrid rescue, of Drosophila melanogaster.

Hybrids from the cross between males of Drosophila melanogaster and females of its sibling species (D. simulans, D. mauritiana, or D. sechellia) are embryonic lethal when they carry the wild type allele of zygotic hybrid rescue (zhr) from D. melanogaster. The zhr gene has been mapped in the proximal region of the X heterochromatin slightly distal to the proximal breakpoint of In(1)sc8, the region rich in 1.688 g/cm3 satellite DNA. Since this satellite DNA does not exist in the sibling species, the satellite DNA was considered to be involved in the hybrid lethality. We examined the hypothesis molecular cytogenetically. The results are (1) three Df(1)zhr chromosomes carried this satellite DNA, and (2) hybrids were viable even if the amount of the satellite DNA in hybrids was increased by adding minichromosomes Dp(1;f)1205 and Dp(1;f)1187 into the genome. These results do not support the above hypothesis.

Alleles↗

PubMed1996

[A new method of cloning Drosophila melanogaster genes marked with a highly copies mobile genetic element].

A new method for cloning Drosophila melanogaster genes genetically localized and marked with the insert of a highly copied mobile element is described. A clue step of the method involves a genetic scheme that allows two series of D. melanogaster strains differing only in genomic location of the studied gene to be obtained. This method allowed the e(y)1 gene marked with an insert of the Stalker highly copied transposon to be cloned [1].

Animals↗

PubMed2002

Chromosomal elements evolve at different rates in the Drosophila genome.

Recent results indicate that the rate of chromosomal rearrangement in the genus Drosophila is the highest found so far in any eukaryote. This conclusion is based chiefly on the comparative mapping analysis of a single chromosomal element (Muller's element E) in two species, D. melanogaster and D. repleta, representing the two farthest lineages within the genus (the Sophophora and Drosophila subgenera, respectively). We have extended the analysis to two other chromosomal elements (Muller's elements A and D) and tested for differences in rate of evolution among chromosomes. With this purpose, detailed physical maps of chromosomes X and 4 of D. repleta were constructed by in situ hybridization of 145 DNA probes (gene clones, cosmids, and P1 phages) and their gene arrangements compared with those of the homologous chromosomes X and 3L of D. melanogaster. Both chromosomal elements have been extensively reshuffled over their entire length. The number of paracentric inversions fixed has been estimated as 118 +/- 17 for element A and 56 +/- 8 for element D. Comparison with previous data for elements E and B shows that there are fourfold differences in evolution rate among chromosomal elements, with chromosome X exhibiting the highest rate of rearrangement. Combining all results, we estimated that 393 paracentric inversions have been fixed in the whole genome since the divergence between D. repleta and D. melanogaster. This amounts to an average rate of 0.053 disruptions/Mb/myr, corroborating the high rate of rearrangement in the genus Drosophila.

Animals↗

PubMed2005

[Genomic DNA sequences and functional expression, purification of BmalphaTX14 neurotoxin from scorpion Buthus martensii Karsch].

Based on the full-length cDNA of BmalphaTX14 from Chinese scorpion Buthus martensii Karsch (BmK), gene of the mature peptide of BmalphaTX14 was cloned into the yeast expression vector pPIC9K. After transforming, screening and inducing, tricine-SDS-PAGE and Western blot proved that rBmalphaTX14 protein was expressed in the medium for up to 84 hours, getting nearly 120 mg/L. Recombinant BmalphaTX14 was purified rapidly and efficiently through Ni-NTA-agarose, polyethylene glycol precipitation and gel filtration chromatography. The purified rBmalphaTX14 proved to have the anti-insect activity by toxicity assay. Meanwhile, genomic gene of BmalphaTX14 was cloned and sequenced by PCR method, sequence analysis of this gene showed that BmalphaTX14 had an intron of 408 base pairs located at the signal peptide encoding region, which was similar with the characteristic of other alpha-type sodium ion-channel toxin. Considering both the genomic organization and the peptide function, BmaTX14 proved to be a membership belonging to alpha-type sodium ion-channel toxin.

Amino Acid Sequence↗

PubMed1991

Retrotransposable elements R1 and R2 interrupt the rRNA genes of most insects.

A large number of insect species have been screened for the presence of the retrotransposable elements R1 and R2. These elements integrate independently at specific sites in the 28S rRNA genes. Genomic blots indicated that 43 of 47 insect species from nine orders contained insertions, ranging in frequency from a few percent to greater than 50% of the 28S genes. Sequence analysis of these insertions from 8 species revealed 22 elements, 21 of which corresponded to R1 or R2 elements. Surprisingly, many species appeared to contain highly divergent copies of R1 and R2 elements. For example, a parasitic wasp contained at least four families of R1 elements; the Japanese beetle contained at least five families of R2 elements. The presence of these retrotransposable elements throughout Insecta and the observation that single species can harbor divergent families within its rRNA-encoding DNA loci present interesting questions concerning the age of these elements and the possibility of cross-species transfer.

Amino Acid Sequence↗

PubMed2001

GST-PRIME: a genome-wide primer design software for the generation of gene sequence tags.

The availability of sequenced genomes has generated a need for experimental approaches that allow the simultaneous analysis of large, or even complete, sets of genes. To facilitate such analyses, we have developed GST-PRIME, a software package for retrieving and assembling gene sequences, even from complex genomes, using the NCBI public database, and then designing sets of primer pairs for use in gene amplification. Primers were designed by the program for the direct amplification of gene sequence tags (GSTs) from either genomic DNA or cDNA. Test runs of GST-PRIME on 2000 randomly selected Arabidopsis and Drosophila genes demonstrate that 93 and 88% of resulting GSTs, respectively, fulfilled imposed length criteria. GST-PRIME primer pairs were tested on a set of 1900 Arabidopsis genes coding for chloroplast-targeted proteins: 95% of the primer pairs used in PCRs with genomic DNA generated the correct amplicons. GST-PRIME can thus be reliably used for large-scale or specific amplification of intron-containing genes of multicellular eukaryotes.

Arabidopsis Proteins↗

PubMed2006

The evolution of hexapod engrailed-family genes: evidence for conservation and concerted evolution.

Phylogenetic analyses imply that multiple engrailed-family gene duplications occurred during hexapod evolution, a view supported by previous reports of only a single engrailed-family gene in members of the grasshopper genus Schistocerca and in the beetle Tribolium castaneum. Here, we report the cloning of a second engrailed-family gene from Schistocerca gregaria and present evidence for two engrailed-family genes from four additional hexapod species. We also report the existence of a second engrailed-family gene in the Tribolium genome. We suggest that the engrailed and invected genes of Drosophila melanogaster have existed as a conserved gene cassette throughout holometabolous insect evolution. In total 11 phylogenetically diverse hexapod orders are now known to contain species that possess two engrailed-family paralogues, with in each case only one paralogue encoding the RS-motif, a characteristic feature of holometabolous insect invected proteins. We propose that the homeoboxes of hexapod engrailed-family paralogues are evolving in a concerted fashion, resulting in gene trees that overestimate the frequency of gene duplication. We present new phylogenetic analyses using non-homeodomain amino acid sequence that support this view. The S. gregaria engrailed-family paralogues provide strong evidence that concerted evolution might in part be explained by recurrent gene conversion. Finally, we hypothesize that the RS-motif is part of a serine-rich domain targeted for phosphorylation.

Amino Acid Motifs↗

PubMed2005

[Retrotransposon gtwin: structural analysis and distribution in Drosophila melanogaster strains].

A search for noncanonical variants of the gypsy retrotransposon (MDG4) in the genome of the Drosophila melanogaster strain G32 led to the cloning of four copies of the poorly studied 7411-bp gtwin element. Sequence analysis showed that gtwin belongs to a family of endogeneous retroviruses, which are widespread in the Drosophila genome and have recently been termed insect erantiviruses. The gtwin retrotransposon is evolutionarily closest to MDG4, as evident from a good alignment of their nucleotide sequences including ORF1 (the pol gene) and ORF3 (the env gene), as well as the amino acid sequences of their protein products. These regions showed more than 75% homology. The distribution of gtwin was studied in several strains of the genus Drosophila. While strain G32 contained more than 20 copies of the element, ten other D. melanogaster strains carried gtwin in two to six copies per genome. The gtwin element was not detected in D. hydei or D. virilis. Comparison of the cloned gtwin sequences with the gtwin sequence available from the D. melanogaster genome database showed that the two variants of the mobile element differ by the presence or absence of a stop codon in the central region of ORF3. Its absence from the gtwin copies cloned from the strain G32 may indicate an association between the functional state of ORF3 and amplification of the element.

Animals↗

PubMed2000

Estimation of spontaneous genome-wide mutation rate parameters: whither beneficial mutations?

Empirical estimates of genome-wide mutation rates and of the distribution of mutational effects are needed to illuminate various topics ranging from evolutionary biology to conservation. Methods for inferring genome-wide mutation parameters are presented, and results stemming from these studies are reviewed. It is argued that, although most if not all mutations detected in mutation accumulation experiments are deleterious, the question of the rate of favourable mutations (and their effects) is still a matter for debate.

Animals↗

PubMed1993

ELISA for antibody to hepatitis E virus (HEV) based on complete open-reading frame-2 protein expressed in insect cells: identification of HEV infection in primates.

A recombinant baculovirus containing the complete open-reading frame (ORF)-2 region of the hepatitis E virus (HEV) genome was constructed. The major protein synthesized in insect cells infected with recombinant virus was about the size expected for the complete ORF-2 product. This protein reacted in a Western blot assay with plasma from an HEV-infected chimpanzee. Lysates of the recombinant virus-infected insect cells were used in ELISA to monitor seroconversion of eight primate species (chimpanzees, four species of Old World monkeys, and three species of New World monkeys) inoculated with HEV. Homologous detector anti-immunoglobulin was more sensitive than heterologous anti-immunoglobulin for detecting anti-HEV by ELISA. All primate species except tamarins seroconverted after inoculation with HEV, although anti-HEV titers of Old World monkey species were generally higher than those of New World monkey species. The ELISA with complete ORF-2 antigen appeared to be a sensitive and practical method for detecting anti-HEV.

Animals↗

PubMed2002

Drosophila melanogaster as a model system for drug discovery and pathway screening.

Although the freakish nightmare world of Mr Samsa may seem completely divorced from our view of the real world, the comparisons between the Drosophila and human genomes indicate that we are a lot closer to insects than we like to think. The use of sophisticated genetic approaches combined with emerging genomics technologies suggest that the fly has much to offer as a tool for understanding basic cellular processes and provides an attractive and complex model system for exploring the molecular basis of human diseases such as cancer, Alzheimer's disease and Huntington's disease.

Animals↗