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PubMed2001

Gene expression and the evolution of insect polyphenisms.

Polyphenic differences between individuals arise not through differences at the genome level but as a result of specific cues received during development. Polyphenisms often involve entire suites of characters, as shown dramatically by the polyphenic castes found in many social insect colonies. An understanding of the genetic architecture behind polyphenisms provides a novel means of studying the interplay between genomes, gene expression and phenotypes. Here we discuss polyphenisms and molecular genetic tools now available to unravel their developmental bases in insects. We focus on several recent studies that have tracked gene-expression patterns during social insect caste determination. BioEssays 23:62-68, 2001. Published 2001 John Wiley & Sons, Inc.

Animals↗

PubMed2000

Complete sequence and organization of Periplaneta fuliginosa densovirus genome.

The replicative form (RF) of the Periplaneta fuliginosa densovirus (PfDNV) genome was cloned and its complete nucleotide sequence was determined. The PfDNV genome is 5454 nucleotides (nt) in length with distal 201-nt long inverted terminal repeats (ITRs). The first 122 nt at the 5'-end and the terminal 122 nt at the 3'-end of both strands are palindromes with identical sequences, of which each can fold into a typical U-shaped hairpin structure. The coding regions of PfDNV genome are evenly distributed in the 5'-halves of both strands. PfDNV genome contains seven major open reading frames (ORFs), four of which on the plus DNA strand may encode non-structural (NS) proteins, while the others on the minus DNA strand may encode structural proteins. Two potential functional promoters (P3 and P97) were found within the ITRs on both strands. The ORF2 polypeptide contains a highly conserved NTP-binding domain of NS proteins of parvoviruses. The ORF5 polypeptide has significant homology to the conserved PGY region of coat proteins of parvoviruses. The ORF6 polypeptide has distinct homology to the structural polypeptides of insect parvoviruses. The organization of PfDNV genome was compared to those of other parvoviruses.

Amino Acid Sequence↗

PubMed2000

Histone H1 genes and histone gene clusters in the genus Drosophila.

Whereas the genomes of many organisms contain several nonallelic types of linker histone genes, one single histone H1 type is known in Drosophila melanogaster that occurs in about 100 copies per genome. Amplification of H1 gene sequences from genomic DNA of wild type strains of D. melanogaster from Oregon, Australia, and central Africa yielded numerous clones that all exhibited restriction patterns identical to each other and to those of the known H1 gene sequence. Nucleotide sequences encoding the evolutionarily variable domains of H1 were determined in two gene copies of strain Niamey from central Africa and were found to be identical to the known H1 sequence. Most likely therefore, the translated sequences of D. melanogaster H1 genes do not exhibit intragenomic or intergenomic variations. In contrast, three different histone H1 genes were isolated from D. virilis and found to encode proteins that differ remarkably from each other and from the H1 of D. melanogaster and D. hydei. About 40 copies of H1 genes are organized in the D. virilis genome with copies of core histone genes in gene quintets that were found to be located in band 25F of chromosome 2. Another type of histone gene cluster is present in about 15 copies per genome and contains a variable intergenic sequence instead of an H1 gene. The H1 heterogeneity in D. virilis may have arisen from higher recombination rates than occur near the H1 locus in D. melanogaster and might provide a basis for formation of different chromatin subtypes.

Amino Acid Sequence↗

PubMed2005

Pteropsin: a vertebrate-like non-visual opsin expressed in the honey bee brain.

Insects have excellent color vision based on the expression of different opsins in specific sets of photoreceptive cells. Opsins are members of the rhodopsin superfamily of G-protein coupled receptors, and are transmembrane proteins found coupled to light-sensitive chromophores in animal photoreceptors. Diversification of opsins during animal evolution provided the basis for the development of wavelength-specific behavior and color vision, but with the exception of the recently discovered non-visual melanopsins, vertebrate and invertebrate opsins have generally been viewed as representing distinct lineages. We report a novel lineage of insect opsins, designated pteropsins. On the basis of sequence analysis and intron location, pteropsins are more closely related to vertebrate visual opsins than to invertebrate opsins. Of note is that the pteropsins are missing entirely from the genome of drosophilid flies. In situ hybridization studies of the honey bee, Apis mellifera, revealed that pteropsin is expressed in the brain of this species and not in either the simple or compound eyes. It was also possible, on the basis of in situ hybridization studies, to assign different long wavelength opsins to the compound eyes (AmLop1) and ocelli (AmLop2). Insect pteropsin might be orthologous to a ciliary opsin recently described from the annelid Platynereis, and therefore represents the presence of this vertebrate-like light-detecting system in insects.

Animals↗

PubMed1993

Expression of a Bombyx cytoplasmic actin gene in cultured Drosophila cells: influence of 20-hydroxyecdysone and interference with expression of endogenous cytoplasmic actin genes.

The expression of the Bombyx cytoplasmic actin A3 gene and its response to 20-hydroxyecdysone are studied after transfection in hormone responsive Drosophila cells and are compared to the expression of homologous resident genes. The host cells accumulate correct transcripts of the Bombyx gene in a gene dosage dependent way. The relative amount of endogenous cytoplasmic actin mRNAs is decreased in transfected cells, whether the transgene is integrated into the genome or not. When 20-hydroxyecdysone is added to the culture medium, the accumulation of the foreign mRNA is decreased whereas those of endogenous cytoplasmic actin transcripts are increased. These results are discussed in terms of competition for transcription and regulatory factors.

Actins↗

PubMed2006

The capsid protein of beak and feather disease virus binds to the viral DNA and is responsible for transporting the replication-associated protein into the nucleus.

Circoviruses lack an autonomous DNA polymerase and are dependent on the replication machinery of the host cell for de novo DNA synthesis. Accordingly, the viral DNA needs to cross both the plasma membrane and the nuclear envelope before replication can occur. Here we report on the subcellular distribution of the beak and feather disease virus (BFDV) capsid protein (CP) and replication-associated protein (Rep) expressed via recombinant baculoviruses in an insect cell system and test the hypothesis that the CP is responsible for transporting the viral genome, as well as Rep, across the nuclear envelope. The intracellular localization of the BFDV CP was found to be directed by three partially overlapping bipartite nuclear localization signals (NLSs) situated between residues 16 and 56 at the N terminus of the protein. Moreover, a DNA binding region was also mapped to the N terminus of the protein and falls within the region containing the three putative NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome. Interestingly, whereas Rep expressed on its own in insect cells is restricted to the cytoplasm, coexpression with CP alters the subcellular localization of Rep to the nucleus, strongly suggesting that an interaction with CP facilitates movement of Rep into the nucleus.

Active Transport, Cell Nucleus↗

PubMed2002

Genomics in pure and applied entomology.

Genomics is the study of the structure and function of the genome: the set of genetic information encoded in the DNA of the nucleus and organelles of an organism. It is a dynamic field that combines traditional paths of inquiry with new approaches that would have been impossible without recent technological developments. Much of the recent focus has been on obtaining the sequence of entire genomes, determining the order and organization of the genes, and developing libraries that provide immediate physical access to any desired DNA fragment. This has enabled functional studies on a genome-wide level, including analysis of the genetic basis of complex traits, quantification of global patterns of gene expression, and systematic gene disruption projects. The successful contribution of genomics to problems in applied entomology requires the cooperation of the private and public sectors to build upon the knowledge derived from the Drosophila genome and effectively develop models for other insect Orders.

Animals↗

PubMed2003

Exogenous prostaglandin F2alpha as a mediator in the regulation of silkworm growth and silk gland genome.

Prostaglandins are locally acting hormones that have remarkable variety of physiological functions. They are rapidly synthesized in several types of vertebrate cells as oxygenated metabolites of arachidonic acid in response to various stimuli. In many insect species they are biosynthesized in fat body and hemocytes mainly in response to bacterial infections. In the present study, we administered synthetic analog of prostaglandin F2alpha, the most prominent of the prostaglandins to the 48 h old fifth instar silkworm, Bombyx mori L. at a single dose of 4 microg per larva to study its effects on the larval growth pattern and silk synthesis. The possible role of PGF2alpha at altering the quantum of silk synthesis by controlling the silk gene expression was also studied. The genomic DNA was isolated from the posterior silk gland on Days 5 and 7 of the fifth instar from the prostaglandin treated and the control larvae and were random amplified with arbitrary primers. The result presented notable variation in the amplified product suggesting the participation of PGF2alpha in the silk biosynthesis controlling the silk gene expression. The feeding period of treated larvae was unaffected while the cocoon characters exhibited considerable improvement. The filament traits also were improved notably in the treated larvae. The participation of PGF2alpha analog in the silk biosynthetic process with its physiological and molecular implications are discussed.

Animals↗

PubMed2002

Assessing the impact of comparative genomic sequence data on the functional annotation of the Drosophila genome.

BACKGROUND: It is widely accepted that comparative sequence data can aid the functional annotation of genome sequences; however, the most informative species and features of genome evolution for comparison remain to be determined. RESULTS: We analyzed conservation in eight genomic regions (apterous, even-skipped, fushi tarazu, twist, and Rhodopsins 1, 2, 3 and 4) from four Drosophila species (D. erecta, D. pseudoobscura, D. willistoni, and D. littoralis) covering more than 500 kb of the D. melanogaster genome. All D. melanogaster genes (and 78-82% of coding exons) identified in divergent species such as D. pseudoobscura show evidence of functional constraint. Addition of a third species can reveal functional constraint in otherwise non-significant pairwise exon comparisons. Microsynteny is largely conserved, with rearrangement breakpoints, novel transposable element insertions, and gene transpositions occurring in similar numbers. Rates of amino-acid substitution are higher in uncharacterized genes relative to genes that have previously been studied. Conserved non-coding sequences (CNCSs) tend to be spatially clustered with conserved spacing between CNCSs, and clusters of CNCSs can be used to predict enhancer sequences. CONCLUSIONS: Our results provide the basis for choosing species whose genome sequences would be most useful in aiding the functional annotation of coding and cis-regulatory sequences in Drosophila. Furthermore, this work shows how decoding the spatial organization of conserved sequences, such as the clustering of CNCSs, can complement efforts to annotate eukaryotic genomes on the basis of sequence conservation alone.

Animals↗

PubMed2002

Genomic effects of nucleotide substitutions in Drosophila simulans.

Selective fixation of beneficial mutations reduces levels of linked, neutral variation. The magnitude of this "hitchhiking effect" is determined by the strength of selection and the recombination rate between selected and neutral sites. Thus, depending on the values of these parameters and the frequency with which directional selection occurs, the genomic scale over which directional selection reduces levels of linked variation may vary widely. Here we present a permutation-based analysis of nucleotide polymorphisms and fixations in Drosophila simulans. We show evidence of pervasive small-scale hitchhiking effects in this lineage. Furthermore, our results reveal that different types of fixations are associated with different levels of linked variation.

Animals↗

PubMed2000

Phylogenetic position of the Diadromus pulchellus ascovirus DNA polymerase among viruses with large double-stranded DNA genomes.

The ASCOVIRIDAE: is a family of large double-stranded (ds) DNA insect viruses that contains four species, the Spodoptera frugiperda (SfAV1), Trichoplusia ni (TnAV2), Heliothis virescens (HvAV3) and Diadromus pulchellus (DpAV4) ascoviruses. These are unique among insect viruses in that the primary means of transmission among their lepidopteran hosts is generally by being vectored mechanically by hymenopteran parasitoids. Ascoviruses are similar in virion structure, but their relationships with their parasitoid vectors vary from being opportunistic to obligate. Little is known, however, about the relatedness of these viruses to one another or to other large dsDNA viruses. We therefore cloned and sequenced the delta DNA polymerase gene of DpAV4, characterized it and compared it to 59 eukaryotic and viral delta and epsilon DNA polymerases. Phylogenetic analyses based on these genes revealed that the ascoviruses DpAV4 and SfAV1 formed a group of virus species distinct from, but closely related to, species of the family IRIDOVIRIDAE: Detailed analyses of the relatedness of ascovirus species based on conserved delta DNA polymerase motifs showed two groups within the family ASCOVIRIDAE:, one containing DpAV4 and the other containing SfAV1, TnAV2 and HvAV3, which was consistent with their host-vector relationships. Despite significant differences in capsid symmetry between ascoviruses and iridoviruses, these results suggest that these viruses may have originated from a common ancestral virus.

Amino Acid Sequence↗

PubMed1997

Use of ordered deletions in genome sequencing.

Previous attempts to use the non-random approach for sequencing long DNA fragments have met with little success. As a result, nearly all genomic sequencing is done by the random (shotgun) approach, and the economy promised by the non-random approach has so far not materialized. Here we describe a simple system based on the use of ordered deletions that can be incorporated in the common strategies for genome sequencing. Long genomic fragments are cloned in the pAL-F cosmid and fragmented by digestion with specific restriction endonucleases. The digests are religated to subclone individual restriction fragments. The subclones are then subdivided by overlapping deletions and used for sequencing. We present the nucleotide sequences of two cosmid inserts from chromosome IV of Drosophila (containing the ci gene and the 5' end of the zfh-2 gene) that were determined by this method. This is the first report of successful sequencing of long genomic fragments by the use of overlapping deletions. Our calculations show that, with the present approach, sequence data can be acquired at a rate comparable to the shotgun approach but with significantly reduced numbers (approximately 30%) of sequencing runs. Hence, the use of ordered deletions should allow significant savings in both the amount and cost of sequencing work.

Animals↗

PubMed2003

Paucity of genes on the Drosophila X chromosome showing male-biased expression.

Sex chromosomes are primary determinants of sexual dimorphism in many organisms. These chromosomes are thought to arise via the divergence of an ancestral autosome pair and are almost certainly influenced by differing selection in males and females. Exploring how sex chromosomes differ from autosomes is highly amenable to genomic analysis. We examined global gene expression in Drosophila melanogaster and report a dramatic underrepresentation of X-chromosome genes showing high relative expression in males. Using comparative genomics, we find that these same X-chromosome genes are exceptionally poorly conserved in the mosquito Anopheles gambiae. These data indicate that the X chromosome is a disfavored location for genes selectively expressed in males.

Animals↗

PubMed2006

Identification and characterization of piggyBac-like elements in the genome of domesticated silkworm, Bombyx mori.

piggyBac is a short inverted terminal repeat (ITR) transposable element originally discovered in Trichoplusia ni. It is currently the preferred vector of choice for enhancer trapping, gene discovery and identifying gene function in insects and mammals. Many piggyBac-like sequences have been found in the genomes of phylogenetically species from fungi to mammals. We have identified 98 piggyBac-like sequences (BmPBLE1-98) from the genome data of domesticated silkworm (Bombyx mori) and 17 fragments from expressed sequence tags (ESTs). Most of the BmPBLE1-98 probably exist as fossils. A total of 21 BmPBLEs are flanked by ITRs and TTAA host dinucleotides, of which 5 contain a single ORF, implying that they may still be active. Interestingly, 16 BmPBLEs have CAC/GTG not CCC/GGG as the characteristic residues of ITRs, which is a surprising phenomenon first observed in the piggyBac families. Phylogenetic analysis indicates that many BmPBLEs have a close relation to mammals, especially to Homo sapiens, only a few being grouped with the T. ni piggyBac element. In addition, horizontal transfer was probably involved in the evolution of the piggyBac-like elements between B. mori and Daphnia pulicaria. The analysis of the BmPBLEs will contribute to our understanding of the characteristic of the piggyBac family and application of piggyBac in a wide range of insect species.

Amino Acid Sequence↗

PubMed1997

CM-gag, a transposable-like element reiterated in the genome of Culex pipiens mosquitoes, contains only a gag gene.

CM-gag elements constitute an homogeneous family of sequences that are reiterated in the genome of Culex pipiens strains from different continents. Apparently complete 1.75 kb CM-gag copies are flanked by target-site duplications and have a polyadenylation signal near their 3' end. They potentially contain a unique gene encoding a putative protein that displays homologies with nucleic acid binding proteins and the gag polypeptide of retroviruses and retrotransposons, but that does not encode a reverse transcriptase. CM-gag elements are similar in their genetic organization to the telomeric transposable sequences Het-A from Drosophila melanogaster, but Southern-hybridization patterns indicate that the former are more probably dispersed in various areas of the mosquito genome. The homogeneity of CM-gag copies that are distributed worldwide suggests that they have most probably been amplified recently. Furthermore, selective constraints against amino acid changes have been acting on these sequences, suggesting that they need to encode the gag-like protein to be incorporated into the chromosomes.

Amino Acid Sequence↗

PubMed2003

Phylogenetic analysis and possible function of bro-like genes, a multigene family widespread among large double-stranded DNA viruses of invertebrates and bacteria.

Baculovirus repeated open reading frame (bro) genes and their relatives constitute a multigene family, typically with multiple copies per genome, known to occur among certain insect dsDNA viruses and bacteriophages. Little is known about the evolutionary history and function of the proteins encoded by these genes. Here we have shown that bro and bro-like (bro-l) genes occur among viruses of two additional invertebrate viral families, Ascoviridae and Iridoviridae, and in prokaryotic class II transposons. Analysis of over 100 sequences showed that the N-terminal region, consisting of two subdomains, is the most conserved region and contains a DNA-binding motif that has been characterized previously. Phylogenetic analysis indicated that these proteins are distributed among eight groups, Groups 1-7 consisting of invertebrate virus proteins and Group 8 of proteins in bacteriophages and bacterial transposons. No bro genes were identified in databases of invertebrate or vertebrate genomes, vertebrate viruses and transposons, nor in prokaryotic genomes, except in prophages or transposons of the latter. The phylogenetic relationship between bro genes suggests that they have resulted from recombination of viral genomes that allowed the duplication and loss of genes, but also the acquisition of genes by horizontal transfer over evolutionary time. In addition, the maintenance and diversity of bro-l genes in different types of invertebrate dsDNA viruses, but not in vertebrate viruses, suggests that these proteins play an important role in invertebrate virus biology. Experiments with the unique orf2 bro gene of Autographa californica multicapsid nucleopolyhedrovirus showed that it is not required for replication, but may enhance replication during the occlusion phase of reproduction.

Amino Acid Sequence↗

PubMed2002

X-linked small GTPase and OXPHOS genes are candidates for the genetic basis of hybrid inviability in Drosophila.

Genetic studies on postmating reproductive isolation in Drosophila have suggested that the genetic basis of hybrid inviability is much less complex than the basis of hybrid sterility, and may be associated with defects affecting the cell cycle. Here I report the identification of a cluster of genes in the middle of the X chromosome of D. melanogaster, which may be responsible for the inviability of hybrids between Drosophila species. Genes from this cluster code for small Ras GTPases proteins, which are hypothesized here to interact with proteins involved in oxidative phosphorylation (OXPHOS), encoded by genes present within the same cluster. At least six genes influencing small Ras GTPases/OXPHOS activity are transcribed from the same strand across 35 kb genomic DNA. This interval is predicted to harbor genes which, when mutated, rescue otherwise inviable hybrids between D. melanogaster and its three most closely related species. Moreover, a total of 16 small GTPase/OXPHOS genes are found within 530 kb genomic DNA encompassing the above cluster. In D. melanogaster mutants which fully rescue lethal hybrids, major lesions have now been identified very near or within untranslated regions of two OXPHOS genes from the above cluster. These observations led to a hypothesis focusing on antagonistic co-evolution between biparentally inherited genes influencing putative GTPase/OXPHOS activity and mitochondrial genes encoding OXPHOS proteins. Alterations in some of these genes are postulated to override hybrid inviability, thus revealing a pathway which implicates mitotic genes as critical players in this barrier to reproduction.

Animals↗

PubMed1999

[Baculovirus: an example of an insect virus of use to humans].

Baculovirus is a viral pathogen of insects in general and lepidoptera in particular. The genome of this large virus consists of a circular, infectious bicatenary DNA molecule. At the end of its replication cycle in insects, baculovirus produces a large quantity of at least two proteins, i.e., polyedrine and polypeptide P10. These proteins are essential for transmission of the virus in nature, but are not necessary in cell cultures. Using molecular recombinant techniques, one or both of the genes coding for these proteins can be replaced by heterologous genes. In this way, baculovirus raised in vitro can be used to produce large quantities of the alien proteins at the end of the multiplication cycle. So far more than 3,000 different proteins have been expressed including several presenting interest as diagnostic tools (Puumala virus, Herpes simplex virus) or therapeutic treatment in man and animals (vaccinations against dengue, flu, malaria and production of anti-Rhesus immunoglobulins). Since it is based on the use of lepidoptera virus, this system would appear to be particularly safe. No vertebrate virus is able to replicate in the cell system used. Use of this genetic engineering tool will undoubtedly expand and holds great promise for the future.

Animals↗