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PubMed1996

Geographic structure of insect populations: gene flow, phylogeography, and their uses.

Geographic structure of populations is a fundamental component of ecology and evolution that combines both demographic and genetic processes, such as gene flow and migration, genetic drift, selection, and population extinction. Recent advances in both molecular biology and theory have revolutionized the field and have not only expanded the availability of data but also facilitated the accessibility and interpretation of current data. These new techniques allow analysis of genetic similarity among populations to be coupled with phylogeography and the distribution of genotypes within and among populations relative to the history of those genotypes. The numerous case studies described herein illustrate the growing impact of geographic structure on insect science, as well as the importance of insect model systems for understanding general concepts in ecology and evolution.

Journal Article↗
PubMed1985

The promoter of the late p10 gene in the insect nuclear polyhedrosis virus Autographa californica: activation by viral gene products and sensitivity to DNA methylation.

In lepidopteran insect cells infected with the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), two major late viral gene products are expressed: the polyhedrin, a 28 000 mol. wt. protein which makes up the mass of the nuclear inclusion bodies, and a 10 000 mol. wt. protein (p10) whose function is unknown. The nucleotide sequences of these strong promoters conform to those of other eukaryotic promoters and are rich in AT base pairs. We used the pSVO-CAT construct containing the prokaryotic gene chloramphenicol acetyl transferase (CAT) to study the function of the p10 gene promoter in insect and mammalian cells. Upon transfection of the pAcp10-CAT construct, which contained 402 bp of the p10 gene of AcNPV DNA in the HindIII site of pSVO-CAT, CAT activity was determined. The p10 gene promoter was inactive in human HeLa cells and in uninfected Spodoptera frugiperda insect cells. The same promoter was active, however, in AcNPV-infected S. frugiperda cells and exhibited optimal activity when cells were transfected 18 h after infection with the insect virus. This finding demonstrated directly that the p10 gene promoter required other viral gene products for its activity in insect cells. The nature of these products was unknown. The p10 gene promoter sequence contained one 5'-CCGG-3' site 40 bp upstream from the cap site of the gene and two such sites 178 and 192 bp downstream from the ATG initiation codon of the gene. Since Drosophila DNA or S. frugiperda DNA contained no 5-methylcytosine or extremely small amounts of it, we were interested in determining the effect of site-specific methylations on the p10 gene insect virus promoter. Methylation at the 5'-CCGG-3' sites led to a block of this promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyltransferases↗
PubMedDate not supplied

The closely related neuropeptide genes encoding adipokinetic hormones I and II have very different 5'-flanking regions.

Adipokinetic hormones I and II are 10- and 8-amino-acid grasshopper neuropeptides that are derived from 63- and 61-amino-acid peptide precursors, respectively. Each precursor is encoded by a separate gene consisting of three very small exons separated by two large introns. The identical exon structure of the two genes suggests that they evolved through duplication of a common ancestral gene. Despite the precise conservation of exon structure and the similarity of the coding sequences, the two genes have very different 5'-flanking regions, suggesting that they are differentially regulated. For example, sequences similar to the vertebrate insulin enhancer elements NIR and FAR are present upstream of the promoter region of the adipokinetic hormone II gene, but not in the adipokinetic hormone I gene. Both of these insect genes contain short interspersed repetitive DNA sequences in their introns that may have facilitated a gene duplication event.

Amino Acid Sequence↗
PubMed2004

Analysis of the insect os-d-like gene family.

Insect OS-D-like proteins, also known as chemosensory (CSP) or sensory appendage proteins (SAP), are broadly expressed in various insect tissues, where they are thought to bind short to medium chain length fatty acids and their derivatives. Although their specific function remains uncertain, OS-D-like members have been isolated from sensory organs (including the sensillum lymph in some cases), and a role in olfaction similar to that of the insect odorant binding proteins (OBP) has been suggested for some. We have identified 15 new OS-D-like sequences: four from cDNA clones described herein and 11 from sequence databases. The os-d-like genes from the Anopheles gambiae, Apis mellifera, Drosophila melanogaster, and Drosophila pseudoobscura genomes typically have single, small introns with a conserved splice site. Together with all family members entered on GenBank, a total of 70 OS-D-like proteins, representing the insect orders Diptera, Dictyoptera, Hymenoptera, Lepidoptera, Orthoptera, and Phasmatodea, were analyzed. A neighbor joining distance phenogram identified several protein similarity classes that were characterized by highly conserved sequence motifs, including (A) N-terminal YTTKYDN(V/I)(N/D)(L/V)DEIL, (B) central DGKELKXX(I/L)PDAL, and (C) C-terminal KYDP. In contrast, three similarity classes were characterized by their diversion from these conserved motifs. The functional importance of conserved amino acid residues is discussed in relation to the crystal and NMR structures of MbraCSPA6.

Amino Acid Sequence↗
PubMed2004

[Transfection and expression of recombinant human beta-defensin-2 gene in insect cells].

OBJECTIVE: This study was conducted to determine the feasibility and relevant technique itinerary for the production of hBD-2 with Baculovirus Expression Vector System (BEVS). METHODS: The full hBD-2/His cDNA was amplified from rpcDNA3.1/hBD-2/myc-His by using PCR with a pair of primers (hBD2p10 and hBD2p11) and was inserted into the MCS of transfer vector: pAcGHLT-A. AcNPV DNA and rpAcGHLT-A/hBD-2/His were co-transfected into Sf21 cells. Recombination would take place within the Sf21 cells between the homologous regions in the transfer vector and AcNPV DNA. After 5 days of co-transfer, both supernatant of the experimental cells and positive control cells were collected. Sf21 cells were infected with virus rAcNPVhBD-2/His and then determined by end-point dilution assay. The expression of hBD-2/His in both cell lysate and supernatant was analyzed by western blot with specific 6 poly-histamines antibody. RESULTS: Both enzyme cutting result and sequence analysis showed that recombinant hBD-2 with C terminal of bi-tags of myc and 6xHis had been inserted into the transfer vector of BEVS system correctly, and recombinant transfer vector rpAcGHLT-A/hBD-2/His had been constructed successfully. End-point dilution assay proved that recombinant virus rAcNPVhBD-2/His had been acquired. Western blot revealed that lysate of Sf21 cells transfected by rAcNPVhBD-2/His showed a band of relative moleculal mass about 47.5 x 10(3) which implied that a fuse peptide of hBD-2/His with up-stream of GST tag, 6xHistag, protein kinase A site and thrombin cleavage had expressed. The culture supernatant showed two bands of relative moleculal mass about 40 x 10(3) and 30 x 10(3), which were inferred to be the proceeded products of the fuse peptide during secretion process from cell into culture supernatant. CONCLUSION: These results suggested that it may be feasible to use BEVS system as a high efficient biologic reactor for producing recombinant hBD-2.

Animals↗
PubMed1994

Conservation of a functional hierarchy between mammalian and insect Hox/HOM genes.

We have generated several transgenic Drosophila strains containing different mouse Hox genes under heat shock control and studied how their generalized expression affects Drosophila larval patterns. We find that they have spatially restricted effects which correlate with their genetic order and expression pattern in the mouse; as they are expressed more posteriorly in the mouse, they have more extensive effects in Drosophila. The generalized expressions of Hoxd-8 and d-9 modify Drosophila anterior head segment(s), but have no effect in the rest of the body. Hoxd-10 expression affects head and thorax, but not the abdomen. Finally, Hoxd-11 alters head, thorax not the abdomen. Finally, Hoxd-11 alters head, thorax and abdomen. The developmental effect of the Hox genes consists of a homeotic transformation of the affected segment(s), which exhibit a 'ground' pattern similar to that obtained in the absence of homeotic information, suggesting that Hox genes are able to inactivate Drosophila homeotic genes, but do not specify a pattern of their own. A partial exception is Hoxd-11 which, even though it has a general suppressing effect, can also activate the resident Abdominal-B and empty spiracles genes in ectopic positions. Our results strongly suggest a general conservation of the functional hierarchy of homeotic genes that correlates with genetic order and expression patterns.

Abdomen↗
PubMed1993

Expression of chick and yeast beta-tubulin-encoding genes in insect cells.

A chick cDNA encoding the beta 2 isotype of tubulin (beta 2Tub) was cloned into a baculovirus expression vector designed to produce unfused proteins, and several recombinant viruses (re-viruses) were isolated. Immunoblotting studies of homogenates of insect cells infected with re-virus showed a 50-kDa protein that reacted with antibodies specific for beta Tub. Cells infected with the re-virus appeared to contain much higher levels of beta Tub than uninfected control cells, perhaps as much as five- to tenfold higher. Isotype-specific antibody for beta 2Tub showed little reaction in uninfected cells or cells infected with wild-type virus; strong reaction was found with cells infected with re-virus. Analysis by gel filtration of extracts of cells infected with re-virus showed that almost all beta Tub eluted in the column void volume, suggesting that it was aggregated or associated with other cell proteins. Recombinant baculoviruses producing Saccharomyces cerevisiae beta Tub were also isolated. Immunoblotting studies using antibodies specific for yeast beta Tub showed a 50-kDa protein which was absent in uninfected cells or cells infected with wt virus. Immunofluorescence studies suggest that yeast beta Tub is incorporated poorly, if at all, into the insect cell cytoskeleton.

Animals↗
PubMed2000

Insect globin gene polymorphisms: intronic minisatellites and a retroposon interrupting exon 1 of homologous globin genes in Chironomus (Diptera).

Exon 1 of globin gene ct-13RT in clone lambdagb2-1 from Chironomus thummi contains a 444nt SINE (CTRT1). Based on in situ hybridization to polytene salivary gland chromosomes, C. thummi (ct), C. piger (cp) and C. tentans (ctn) contain copies of CTRT1 at multiple chromosomal loci. Genomic PCR amplifications reveal interrupted (ct-13RT) and uninterrupted (ct-13) alleles of the globin gene in the German population of C. thummi maintained in our laboratory, and only uninterrupted alleles or their homologs in different populations of C. thummi, C. piger and C. tentans. PCR amplification did generate different length fragments from cp-13 gene homologs in natural and laboratory C. piger populations that were due to variation in the length of minisatellite expansions of the central introns of the genes rather than a CTRT1-like SINE. Within minisatellite arrays, aligned homologs were more similar than paralogs in a single population, indicating that a tandem cluster of these repeats predated separation of the C. piger populations. The ct-13 genes of several C. thummi populations lack the minisatellites, suggesting their origin in C. piger only after the thummi/piger split. CTRT1 transposition into a ct-13 allele is even more recent, occurring after separation of German and other European C. thummi populations. The nearly intact ct-13RT and comparison with its intact ct-13 allele support a very recent transposition of the CTRT1 SINE into one of at least two already diverged ct-13 globin gene alleles. PCR analysis of DNA from individual adults in C. thummi shows a 1:2:1 distribution of ct-13/ct-13:ct-13/ct-13RT:ct-13RT/ct-13RT genotypes, consistent with a neutral spread of the ct-13RT allele since transposition, and indicating that the hemoglobin encoded by ct-13 is not necessary for survival, at least in a laboratory population of C. thummi.

Alleles↗
PubMed1991

Modification of the coding sequence enhances plant expression of insect control protein genes.

Increased expression of the insect control protein genes of Bacillus thuringiensis in plants has been critical to the development of genetically improved plants with agronomically acceptable levels of insect resistance. The expression of the cryIA(b) gene was compared to partially modified (3% nucleotide difference) and to fully modified (21% nucleotide difference) cryIA(b) and cryIA(c) genes in tobacco and tomato. The modified genes increased the frequency of plants that produced the proteins at quantities sufficient to control insects and dramatically increased the levels of these proteins. Among the most highly expressing transformed plants for each gene, the plants with the partially modified cryIA(b) gene had a 10-fold higher level of insect control protein and plants with the fully modified cryIA(b) had a 100-fold higher level of CryIA(b) protein compared with the wild-type gene. Similar results were obtained with the fully modified cryIA(c) gene in plants. Specific sequences of the partially modified cryIA(b) gene were analyzed for their ability to affect cryIA(b) gene expression in tobacco. The DNA sequence of a single region was identified as important to the improvement of plant expression of the cryIA(b) gene. The increased levels of cryIA(b) mRNA were not directly proportional to the increased levels of CryIA(b) protein in plants transformed with the modified cryIA(b) genes, indicating that the nucleotide sequence of these genes had an effect in improving their translational efficiency in plants.

Bacillus thuringiensis↗
PubMed1999

Gene transfer into insect brain and cell-specific expression of bombyxin gene.

A transgene reporter consisting of the bombyxin gene promoter and the green fluorescent protein coding region was introduced into intact brains of the silkworm Bombyx mori by in vitro electroporation. After in vitro culture of the brains, the fluorescence derived from the introduced reporter gene was observed in all cases in eight neurosecretory cells that had previously been identified as bombyxin-producing cells (BPCs). Although the fluorescence was not always observed in all cells, it was specific to BPCs, indicating that the reporter was under the control of the bombyxin gene promoter in a BPC-specific manner. Electroporatical introduction of a reporter gene was therefore found to be a suitable method for analyzing cell-specific expression in intact tissues and to be substitute for germ-line transmission of reporters in the transgenic system. Application of this technique enables us to analyze the cell-specific expression of transgene reporters within a few days and treat more than several dozens of the reporters within 1 month, which is difficult to do with the transgenic system.

Amino Acid Sequence↗
PubMed2002

fushi tarazu: a Hox gene changes its role.

The Hox genes play a role in anteroposterior axis specification of bilaterian animals that has been conserved for more than 600 million years. However, some of these genes have occasionally changed their roles in evolution. For example, the insect gene fushi tarazu (ftz), although localised in the Hox cluster, no longer acts as a Hox gene, but is involved in segmentation and nervous system development. Recent data of Mouchel-Vielh et al., and Hughes and Kaufman on ftz homologues in a crustacean and a myriapod, respectively, shed new light onto the evolution of this gene.

Animals↗
PubMed1999

Identification of a cDNA encoding a retinoid X receptor homologue from Schistosoma mansoni. Evidence for a role in female-specific gene expression.

Schistosoma mansoni, a multicelluar eukaryotic blood fluke, is a major cause of morbidity worldwide in humans. The study of female parasite growth, development, and gene regulation is important because the eggs produced are responsible for the pathogenesis observed in schistosomiasis. p14, an eggshell precursor gene expressed only in sexually mature females in response to a male stimulus, is a model for female-specific gene regulation. The upstream region of the p14 gene shares sequences present in insect genes known to be regulated in a sex-, temporal-, and tissue-specific manner by members of the steroid receptor superfamily. Herein, we report the identification and characterization of a cDNA that encodes the S. mansoni (Sm) RXR homologue. Sequence analysis predicts and Western blot analysis confirms the synthesis of a 74-kDa protein, the largest member of the RXR family reported to date. We show by electrophoretic mobility shift assay analysis that SmRXR binds to cis-elements of the p14 gene including a direct repeat that follows the "3-4-5" rule of binding elements recognized by members of the steroid receptor superfamily. Furthermore, we demonstrate that SmRXR can act as a transcription activator in the yeast one-hybrid system. Through quantitative reverse transcriptase-polymerase chain reaction, we show that the SmRXR gene is constitutively expressed and thus must play multiple roles throughout the schistosome life cycle.

Amino Acid Sequence↗
PubMed1988

Primary structure of apolipophorin-III from the migratory locust, Locusta migratoria. Potential amphipathic structures and molecular evolution of an insect apolipoprotein.

The amino acid sequence of an insect apolipoprotein, apolipophorin-III from Locusta migratoria, has been deduced from the sequence of its cloned cDNA. The mature hemolymph protein consists of 161 amino acids. Optimized alignments of this protein with apolipophorin-III from the tobacco hornworm, Manduca sexta, disclosed an overall sequence identity of only 29%, even though the two proteins are functionally equivalent. The L. migratoria sequence is composed of 12 repeating peptides that are variable in length. Six amphipathic helical segments of varying length were identified in each protein using a newly described algorithm for detecting such secondary structures. The degree of sequence identity between the two insect apoproteins is considerably less than that observed among orthologous mammalian apolipoproteins. However, calculation of the rates of synonymous and nonsynonymous nucleotide substitutions indicates that the insect genes may be evolving at rates similar to the mammalian apolipoprotein genes. Further comparative analyses of insect and mammalian apolipoproteins should provide insights about the limits of sequence diversity tolerated by their predicted amphipathic helical domains.

Amino Acid Sequence↗
PubMed2003

Post-integration behavior of a Mos1 mariner gene vector in Aedes aegypti.

The post-integration behavior of insect gene vectors will determine the types of applications for which they can be used. Transposon mutagenesis, enhancer trapping, and the use of transposable elements as genetic drive systems in insects requires transposable elements with high rates of remobilization in the presence of transposase. We investigated the post-integration behavior of the Mos1 mariner element in transgenic Aedes aegypti by examining both germ-line and somatic transpositions of a non-autonomous element in the presence of Mos1 transposase. Somatic transpositions were occasionally detected while germ-line transposition was only rarely observed. Only a single germ-line transposition event was recovered after screening 14,000 progeny. The observed patterns of transposition suggest that Mos1 movement takes place between the S phase and anaphase. The data reported here indicate that Mos1 will be a useful vector in Ae. aegypti for applications requiring a very high degree of vector stability but will have limited use in the construction of genetic drive, enhancer trap, or transposon tagging systems in this species.

Aedes↗