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1.
Insect Biochem Mol Biol ; 33(6): 563-77, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12770575

ABSTRACT

The term "knockdown resistance" is used to describe cases of resistance to diphenylethane (e.g. DDT) and pyrethroid insecticides in insects and other arthropods that result from reduced sensitivity of the nervous system. Knockdown resistance, first identified and characterized in the house fly (Musca domestica) in the 1950's, remains a threat to the continued usefulness of pyrethroids in the control of many pest species. Research since 1990 has provided a wealth of new information on the molecular basis of knockdown resistance. This paper reviews these recent developments with emphasis on the results of genetic linkage analyses, the identification of gene mutations associated with knockdown resistance, and the functional characterization of resistance-associated mutations. Results of these studies identify voltage-sensitive sodium channel genes orthologous to the para gene of Drosophila melanogaster as the site of multiple knockdown resistance mutations and define the molecular mechanisms by which these mutations cause pyrethroid resistance. These results also provide new insight into the mechanisms by which pyrethroids modify the function of voltage-sensitive sodium channels.


Subject(s)
Insecta/genetics , Insecticide Resistance/genetics , Pyrethrins , Amino Acid Substitution , Animals , Genes, Insect , Genetic Linkage , Mutation , Polymorphism, Genetic , Polymorphism, Single Nucleotide , Protein Structure, Tertiary , Sodium Channels/chemistry , Sodium Channels/genetics , Sodium Channels/metabolism
2.
Insect Biochem Mol Biol ; 31(10): 949-64, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11483431

ABSTRACT

In this report, we describe the structural and functional analyses of four acyl-CoA desaturase-encoding cDNAs that we isolated from RNA expressed in the pheromone gland of the corn earworm, Helicoverpa zea. We deduced the homology relationships of the encoded proteins, designated HzPGDs1, HzPGDs2, HzPGDs3 and HzFBDs, to each other and to previously described desaturases of the cabbage looper moth, Trichoplusia ni, the fly, Drosophila melanogaster, and other more distantly related organisms. We also isolated genomic DNA fragments of the four H. zea desaturase-encoding genes, determined the locations of introns present in them, and compared them to conserved intron positions in reported desaturase genes of other species. We measured the levels of the four desaturase mRNAs in H. zea pheromone glands and larval fat bodies by RT-PCR. We established the functional identities of the deduced proteins HzPGDs1 and HzPGDs2, encoded by the two desaturase mRNAs that are differentially and abundantly expressed in pheromone glands of sexually mature adult H. zea females, by functional expression of their encoding cDNAs in a desaturase-deficient mutant, ole1, of the yeast Saccharomyces cerevisiae. We compared the unique unsaturated fatty acid profiles of HzPGDs1- and HzPGDs2-expressing transformants to those of strains expressing previously described Delta11 and Delta9 desaturases of T. ni.


Subject(s)
Fatty Acid Desaturases/genetics , Moths/enzymology , Amino Acid Sequence , Animals , Base Sequence , DNA Probes , DNA, Complementary , Fat Body/metabolism , Fatty Acid Desaturases/classification , Genes, Insect , Humans , Larva/metabolism , Molecular Sequence Data , Moths/genetics , Pheromones , Polymerase Chain Reaction/methods , RNA, Messenger/metabolism , Sequence Analysis, DNA
3.
J Exp Biol ; 204(Pt 22): 3935-42, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11807111

ABSTRACT

We investigated the effects of sap of the common oleander Nerium indicum (Apocyanaceae) on male fertility and spermatogenesis in the oriental tobacco budworm Helicoverpa assulta. We found that continuous feeding of oleander sap during the larval period significantly affects fertility in males but not in females. This effect was also induced by direct injection of oleander sap into the hemocoel of 2-day-old pupae. Histological analyses of developing testes following oleander injection revealed a developmental delay and progressively more severe morphological abnormalities in the later stages of development. The effects of oleander sap on spermatogenesis in H. assulta were associated with greatly reduced levels of the two major polyamines, spermidine and spermine, in testis compared with saline-injected controls. In contrast, levels of putrescine, which is a precursor of both spermidine and spermine, and the activities of the enzymes ornithine decarboxylase and arginine decarboxylase, which are involved in the biosynthesis of putrescine, were initially elevated following oleander injection, but subsequently failed to undergo the induction that normally occurs during late pupal development. The effects of oleander sap on spermidine and spermine levels could be the result of direct inhibition by chemical constituents of the oleander sap of one of the enzymes used in common in the conversions of putrescine to spermidine and spermidine to spermine; alternatively, these effects on polyamine metabolism could be secondary to the disruption of a more fundamental process in the developmental program guiding spermatogenesis in H. assulta.


Subject(s)
Lepidoptera/physiology , Nerium/metabolism , Animals , Carboxy-Lyases/metabolism , Fertility/drug effects , Male , Ornithine Decarboxylase/metabolism , Plant Extracts/pharmacology , Putrescine/metabolism , Spermatogenesis/drug effects , Spermidine/metabolism , Spermine/metabolism , Testis/drug effects , Testis/growth & development , Testis/metabolism
4.
Proc Natl Acad Sci U S A ; 97(17): 9449-54, 2000 Aug 15.
Article in English | MEDLINE | ID: mdl-10920187

ABSTRACT

Drosophila melanogaster cuticular pheromones consist of unsaturated hydrocarbons with at least one double bond in position 7: 7 tricosene (T) in males and 7,11 heptacosadiene (HD) in females. However, in many African populations like the Tai strain, females possess low levels of 7,11 HD and high levels of its positional isomer 5,9 HD. We have previously isolated a desaturase gene, desat1, from the Canton-S strain (CS), a 7,11 HD-2-rich morph of D. melanogaster. This desaturase is located in 87C, a locus that has been involved in the difference between 7,11 HD and 5,9 HD morphs. Therefore, we have searched for different desaturase isoforms in both strains. We first cloned desat1 in the Tai strain and report here functional expression of desat1 in CS and Tai. In both strains, the Desat1 enzymes have the same Delta9 specificity and preferentially use palmitate as a substrate, leading to the synthesis of omega7 fatty acids. Also found was a desaturase sequence, named desat2, with a homologous catalytic domain and a markedly different N-terminal domain compared with desat1. In CS genome, it lies 3.8 kb upstream of desat1 and is not transcribed in either sex. In the Tai strain, it is expressed only in females and acts preferentially on myristate, leading to the synthesis of omega5 fatty acids. We suggest, therefore, that desat2 might play a control role in the biosynthesis of 5,9 HD hydrocarbons in Tai females and could explain the dienic hydrocarbon polymorphism in D. melanogaster.


Subject(s)
Drosophila melanogaster/enzymology , Genes, Insect/genetics , Hydrocarbons/metabolism , Sex Characteristics , Stearoyl-CoA Desaturase/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , Drosophila melanogaster/classification , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Exons/genetics , Fatty Acids/analysis , Fatty Acids/chemistry , Fatty Acids/metabolism , Female , Genetic Complementation Test , Hydrocarbons/chemistry , Introns/genetics , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Male , Molecular Sequence Data , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Sequence Alignment , Stearoyl-CoA Desaturase/chemistry , Stearoyl-CoA Desaturase/genetics , Substrate Specificity
5.
Insect Biochem Mol Biol ; 29(5): 435-43, 1999 May.
Article in English | MEDLINE | ID: mdl-10380655

ABSTRACT

Acyl-CoA delta 9-desaturases play essential roles in fatty acid metabolism and the regulation of cell membrane fluidity. In this research, a cDNA sequence was obtained from Trichoplusia ni adult fat body mRNA by using RT-PCR with degenerate primers based on other characterized delta 9-desaturase sequences. The remainder of the sequence was amplified using 3'- and 5'-RACE. A 1439 bp cDNA reconstructed from three overlapping PCR products contains an ORF encoding a 353-amino acids (aa) protein that shows clear homology (greater than 50% aa identity and greater than 65% aa similarity to characterized insect and vertebrate desaturases). The ORF of this cDNA was subcloned into an expression vector, which relieved the unsaturated fatty acid (UFA) auxotrophy of a desaturase-deficient yeast strain following genetic transformation. The newly characterized desaturase from T. ni produced fatty acids delta 9-16 and delta 9-18 in a 1:6 ratio, compared to a 5:1 ratio, respectively, with the yeast delta 9 desaturase. A Northern blot hybridization and a RT-PCR experiment showed that temporal and tissue-specific patterns of expression of the corresponding mRNA are distinct from those of the delta 11-desaturase mRNA present in the pheromone glands of adult females. Based on its homology to other desaturases, the widespread distribution of its corresponding mRNA in various tissues, and its functional assay, we conclude that this cDNA encodes the apoprotein corresponding to the desaturase component of the metabolic delta 9-desaturase complex of T. ni.


Subject(s)
Fatty Acid Desaturases/genetics , Moths/enzymology , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary , Fatty Acid Desaturases/metabolism , Fatty Acid Desaturases/physiology , Molecular Sequence Data , Moths/genetics , RNA, Messenger , Rats , Sequence Homology, Amino Acid , Stearoyl-CoA Desaturase , Tissue Distribution
6.
Insect Biochem Mol Biol ; 29(2): 185-94, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10196741

ABSTRACT

The super-kdr insecticide resistance trait of the house fly confers resistance to pyrethroids and DDT by reducing the sensitivity of the fly nervous system. The super-kdr genetic locus is tightly linked to the Vssc1 gene, which encodes a voltage-sensitive sodium channel alpha subunit that is the principal site of pyrethroid action. DNA sequence analysis of Vssc1 alleles from several independent super-kdr fly strains identified two amino acid substitutions associated with the super-kdr trait: replacement of leucine at position 1014 with phenylalanine (L1014F), which has been shown to cause the kdr resistance trait in this species, and replacement of methionine at position 918 with threonine (M918T). We examined the functional significance of these mutations by expressing house fly sodium channels containing them in Xenopus laevis oocytes and by characterizing the biophysical properties and pyrethroid sensitivities of the expressed channels using two-electrode voltage clamp. House fly sodium channels that were specifically modified by site-directed mutagenesis to contain the M918T/L1014F double mutation gave reduced levels of sodium current expression in oocytes but otherwise exhibited functional properties similar to those of wildtype channels and channels containing the L1014F substitution. However, M918T/L1014F channels were completely insensitive to high concentrations of the pyrethroids cismethrin and cypermethrin. House fly sodium channels specifically modified to contain the M918T single mutation, which is not known to exist in nature except in association with the L1014F mutation, gave very small sodium currents in oocytes. Assays of these currents in the presence of high concentrations of cismethrin suggest that this mutation alone is sufficient to abolish the pyrethroid sensitivity of house fly sodium channels. These results define the functional significance of the Vssc1 mutations associated with the super-kdr trait of the house fly and are consistent with the hypothesis that the super-kdr trait arose by selection of a second-site mutation (M918T) that confers to flies possessing it even greater resistance than the kdr allele containing the L1014F mutation.


Subject(s)
Genes, Insect/genetics , Houseflies/genetics , Insecticide Resistance/genetics , Insecticides/pharmacology , Point Mutation/genetics , Sodium Channels/genetics , Animals , Mutagenesis, Site-Directed , Oocytes , Patch-Clamp Techniques , Pyrethrins/pharmacology , Sequence Analysis , Xenopus/genetics
7.
Proc Natl Acad Sci U S A ; 95(26): 15287-92, 1998 Dec 22.
Article in English | MEDLINE | ID: mdl-9860961

ABSTRACT

Desaturation of coenzyme-A esters of saturated fatty acids is a common feature of sex pheromone biosynthetic pathways in the Lepidoptera. The enzymes that catalyze this step share several biochemical properties with the ubiquitous acyl-CoA Delta9-desaturases of animals and fungi, suggesting a common ancestral origin. Unlike metabolic acyl-CoA Delta9-desaturases, pheromone desaturases have evolved unusual regio- and stereoselective activities that contribute to the remarkable diversity of chemical structures used as pheromones in this large taxonomic group. In this report, we describe the isolation of a cDNA encoding a pheromone gland desaturase from the cabbage looper moth, Trichoplusia ni, a species in which all unsaturated pheromone products are produced via a Delta11Z-desaturation mechanism. The largest ORF of the approximately 1,250-bp cDNA encodes a 349-aa apoprotein (PDesat-Tn Delta11Z) with a predicted molecular mass of 40,240 Da. Its hydrophobicity profile is similar overall to those of rat and yeast Delta9-desaturases, suggesting conserved transmembrane topology. A 182-aa core domain delimited by conserved histidine-rich motifs implicated in iron-binding and catalysis has 72 and 58% similarity (including conservative substitutions) to acyl-CoA Delta9Z-desaturases of rat and yeast, respectively. Northern blot analysis revealed an approximately 1,250-nt PDesat-Tn Delta11Z mRNA that is consistent with the spatial and temporal distribution of Delta11-desaturase enzyme activity. Genetic transformation of a desaturase-deficient strain of the yeast Saccharomyces cerevisiae with an expression plasmid encoding PDesat-Tn Delta11Z resulted in complementation of the strain's fatty acid auxotrophy and the production of Delta11Z-unsaturated fatty acids.


Subject(s)
Fatty Acid Desaturases/genetics , Moths/enzymology , Amino Acid Sequence , Animals , Cloning, Molecular , Conserved Sequence , DNA, Complementary , Endocrine Glands/enzymology , Fatty Acid Desaturases/biosynthesis , Fatty Acid Desaturases/chemistry , Genes, Insect , Molecular Sequence Data , Molecular Weight , Moths/genetics , Open Reading Frames , Pheromones/metabolism , Rats , Saccharomyces cerevisiae/enzymology , Sequence Alignment , Sequence Homology, Amino Acid
8.
Insect Biochem Mol Biol ; 28(4): 239-49, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9684332

ABSTRACT

Sex pheromone production in the corn earworm, Helicoverpa zea, is regulated by a 33-amino-acid neuropeptide named Hez-PBAN (pheromone biosynthesis activating neuropeptide). Hez-PBAN is encoded in a preprohormone that also contains four other structurally related peptides. Two recombinant baculoviruses that contain two different sequences of Hez-PBAN cDNA under the control of a strong polyhedrin promotor were constructed. The first virus, AcWT-PBAN, contains the entire prepro-Hez-PBAN coding sequence. The second virus, AcBX-PBAN, contains a synthetic chimera gene encoding a bombyxin signal peptide sequence fused to a pro-Hez-PBAN sequence. Cell extracts, culture medium of BTI-TN-5B1-4 cells, and hemolymph from 4th instar Trichoplusia ni larvae, all infected with AcBX-PBAN, showed a high level of pheromonotropic activity. Pheromonotropic activity was not detected in the cells infected with AcWT-PBAN. Results of chromatographic and immunochemical studies showed that some of the potential processing sites in the expressed pro-Hez-PBAN sequence were not used during posttranslational processing in the AcBX-PBAN-4-infected BTI-TN-5B1-4 cells and 4th instar T. ni larvae. However, the processing pattern of the recombinant pro-Hez-PBAN in AcBX-PBAN-infected 4th instar T. ni larvae was similar to that exhibited in the central nervous system of H. zea adult females, since a PBAN-like immunoreactive-peptide-band was found in the hemolymph of Ac-BX-PBAN-4-infected 4th instar T. ni larvae. In a droplet feeding assay, neonate and 3rd instar T. ni larvae infected with AcBX-PBAN-4 showed a significant reduction in survival time (26% and 19%, respectively) when compared to control larvae that were infected with a polyhedrin-deficient virus, Ac-E10.


Subject(s)
Baculoviridae/genetics , Gene Expression , Insecta/genetics , Neuropeptides/genetics , Protein Processing, Post-Translational , Sex Attractants/genetics , Amino Acid Sequence , Animals , Insect Control/methods , Insecta/physiology , Larva , Molecular Sequence Data , Neuropeptides/metabolism , Sex Attractants/metabolism
9.
Insect Biochem Mol Biol ; 27(10): 807-12, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9474777

ABSTRACT

Voltage-sensitive sodium channels encoded by a full-length cDNA corresponding to the Vssc1 gene of the house fly (Musca domestica) were expressed in Xenopus laevis oocytes either alone or in combination with the tipE gene product of Drosophila melanogaster and were characterized by two-electrode voltage clamp. Vssc1 cRNA alone produced very small (50-150 nA) sodium currents, whereas the combination of Vssc1 and tipE cRNAs produced robust (0.5-3 microA), rapidly inactivating sodium currents. The pyrethroid insecticide cismethrin prolonged the sodium current carried by Vssc1/tipE sodium channels during a depolarizing pulse and induced a tail current after repolarization. The Vssc1 cDNA was specifically mutated to substitute phenylalanine for leucine at position 1014 of the inferred amino acid sequence (L1014F), a polymorphism shown previously to be associated with the kdr (knockdown resistance) trait of the house fly. The L1014F substitution reduced the sensitivity of expressed house fly sodium channels to cismethrin at least 10-fold and increased the rate of decay of pyrethroid-induced sodium tail currents. These results demonstrate that the resistance-associated L1014F mutation confers a reduction in the sensitivity of house fly sodium channels to pyrethroids that is sufficient to account for the kdr resistance trait.


Subject(s)
Houseflies/genetics , Insecticide Resistance/genetics , Insecticides/pharmacology , Point Mutation , Pyrethrins/pharmacology , Sodium Channels/genetics , Animals , DNA, Complementary/genetics , Electrophysiology , Houseflies/physiology , In Vitro Techniques , Sodium Channels/physiology , Xenopus laevis
10.
Insect Biochem Mol Biol ; 26(4): 319-26, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8814780

ABSTRACT

The kdr insecticide resistance trait of the house fly (Musca domestica .L.), which confers reduced neuronal sensitivity to DDT and pyrethroid insecticides, was previously shown to exhibit tight genetic linkage to restriction fragment length polymorphism markers lying within a voltage-sensitive sodium channel gene that is homologous to the para gene of Drosophila melanogaster. In the present study, the 6315 nucleotide coding sequences of this voltage-sensitive sodium channel gene from insecticide-susceptible (NAIDM strain) and kdr (538ge strain) house flies were determined by automated direct DNA sequencing of PCR fragments obtained by amplification on first strand cDNA from adult heads. The deduced 2105-residue amino acid sequence from each strain exhibited overall structure and organization typical of sodium channel alpha subunit genes and was 90.0% identical to that of the D. melanogaster para gene product. We did not detect any splice variants among voltage-sensitive sodium channel cDNAs obtained from adult house fly head preparations. Comparison of the coding sequence of the voltage-sensitive sodium channel gene of the kdr house fly strain to that of the NAIDM strain revealed 12 amino acid differences in the 538ge strain. The significance of these polymorphisms as candidate resistance-conferring mutations is discussed.


Subject(s)
Genes, Insect , Houseflies/genetics , Insecticide Resistance/genetics , Sodium Channels/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA Primers , Genetic Variation , Houseflies/drug effects , Molecular Sequence Data , Sequence Homology, Amino Acid
11.
Proc Natl Acad Sci U S A ; 91(14): 6506-10, 1994 Jul 05.
Article in English | MEDLINE | ID: mdl-8022813

ABSTRACT

Sex pheromone biosynthesis in a number of moth species is induced by a conserved 33-amino acid amidated neuropeptide PBAN (pheromone biosynthesis-activating neuropeptide). We have isolated and characterized the Helicoverpa zea PBAN cDNA corresponding to a 766-nucleotide mRNA that is expressed in the subesophageal ganglion of adult moths. This mRNA is encoded on a transcription unit comprising 6 exons. The longest open reading frame of the cDNA encodes a 194-amino acid precursor protein that contains the PBAN peptide sequence. Proteolytic processing of this protein, which has structural features consistent with its being a preprohormone, is predicted to generate Hez-PBAN and four additional neuropeptides having a common C-terminal pentapeptide motif, Phe-Xaa-Pro-(Arg or Lys)-Leu (Xaa = Gly, Ser, or Thr), which is also found in insect pyrokinin and myotropin peptide families.


Subject(s)
Lepidoptera/genetics , Lepidoptera/metabolism , Neuropeptides/biosynthesis , Protein Precursors/biosynthesis , Sex Attractants/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Bombyx/genetics , Bombyx/metabolism , DNA/genetics , DNA Primers , DNA, Complementary/analysis , Exons , Female , Male , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Homology, Amino Acid
12.
Insect Biochem Mol Biol ; 24(4): 363-71, 1994 Apr.
Article in English | MEDLINE | ID: mdl-8025558

ABSTRACT

A polymerase chain reaction (PCR)-based homology probing strategy was employed to screen Drosophila melanogaster genomic DNA for sequences encoding a conserved amino acid 'signature motif' known to be present in vertebrate GABA receptor and glycine receptor subunit genes. This approach yielded three discrete amplified sequence elements (designated LCCH1, LCCH2, and LCCH3) that contained open reading frames and > 40% amino acid sequence identity to the corresponding regions of vertebrate ligand-gated chloride channel genes. Genomic DNA clones corresponding to each element were isolated and sequenced, and predicted amino acid sequences corresponding to the second (M2) and third (M3) transmembrane domains of vertebrate genes were analyzed for identity or similarity to known sequences. LCCH1 was identical to the Rdl gene, a known GABA receptor subunit gene from D. melanogaster, whereas LCCH2 and LCCH3 were novel D. melanogaster sequences that exhibited structural similarity to other members of the ligand-gated chloride channel gene family. LCCH2 was equally divergent in M2 and M3 (46-49% amino acid identity) from all other known members of this family and may therefore represent a new subunit or receptor class within this family. LCCH2 was localized by in situ hybridization to cytogenetic region 75A on the left arm of chromosome 3. LCCH3 was closely related to mammalian (79% amino acid identity) and snail (96% amino acid identity) GABA receptor beta subunits and may therefore be the homologue in D. melanogaster of this subunit class. LCCH3 was localized by in situ hybridization to cytogenetic region 13F on the X chromosome.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Chloride Channels/genetics , Drosophila melanogaster/genetics , Genes, Insect/genetics , Receptors, GABA/genetics , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Conserved Sequence , DNA Primers/chemical synthesis , DNA Primers/genetics , Genomic Library , Models, Genetic , Molecular Sequence Data , Polymerase Chain Reaction , Receptors, Glycine/genetics , Sequence Analysis, DNA , Sequence Homology, Amino Acid
13.
Proc Natl Acad Sci U S A ; 91(7): 2483-7, 1994 Mar 29.
Article in English | MEDLINE | ID: mdl-8146143

ABSTRACT

The kdr insecticide resistance trait in the house fly, Musca domestica, confers resistance to the rapid paralysis (knockdown) and lethal effects of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and pyrethroids. Flies with the kdr trait exhibit reduced neuronal sensitivity to these compounds, which are known to act at voltage-sensitive sodium channels of nerve membranes. To test the hypothesis that a mutation in a voltage-sensitive sodium channel gene confers the kdr phenotype, we have cloned genomic DNA corresponding to a segment of the house fly homologue of the para sodium channel gene of Drosophila melanogaster, identified restriction-site polymorphisms within this segment between the kdr strain 538ge and an inbred insecticide-susceptible lab stain, and developed a sensitive polymerase chain reaction-based diagnostic procedure to determine the sodium channel genotype of individual flies. A genetic linkage analysis performed with these molecular markers shows that the kdr trait is tightly linked (within about 1 map unit) to the voltage-sensitive sodium channel gene segment exhibiting the DNA sequence polymorphism. These findings provide genetic evidence for a mutation at or near a voltage-sensitive sodium channel gene as the basis for kdr resistance.


Subject(s)
DDT/pharmacology , Genes, Insect/genetics , Houseflies/genetics , Insecticide Resistance/genetics , Sodium Channels/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA Primers , Female , Genetic Linkage , Male , Models, Molecular , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Homology, Amino Acid
14.
Biochem Biophys Res Commun ; 193(2): 474-82, 1993 Jun 15.
Article in English | MEDLINE | ID: mdl-7685594

ABSTRACT

A cDNA encoding a novel member of the ligand-gated chloride channel gene family of insects has been isolated from Drosophila melanogaster and characterized. The 1488 nucleotide open reading frame of this cDNA encodes an amino acid sequence having structural features conserved among ligand-gated chloride channel subunit proteins, including four hydrophobic domains capable of forming transmembrane helices (M1-M4), an octapeptide "signature motif" occurring in M2, a large intracellular domain between M3 and M4, and cysteine residues postulated to form a disulfide-bridged loop structure in the extracellular domain. Among characterized members of this gene family, this gene exhibits the greatest similarity to GABA receptor beta subunit genes. This high level of structural similarity suggests that its encoded protein may be a functional homologue of the beta subunit family in the D. melanogaster nervous system.


Subject(s)
Drosophila melanogaster/genetics , Multigene Family , Receptors, GABA-A/genetics , Amino Acid Sequence , Animals , Base Sequence , Chloride Channels , Cloning, Molecular , DNA , Drosophila melanogaster/metabolism , Gene Library , Ion Channels/genetics , Macromolecular Substances , Membrane Proteins/genetics , Molecular Sequence Data , Oligodeoxyribonucleotides , Oligonucleotides, Antisense , Polymerase Chain Reaction , Protein Structure, Secondary , Receptors, GABA-A/chemistry , Sequence Homology, Amino Acid
15.
Dev Biol ; 155(2): 558-68, 1993 Feb.
Article in English | MEDLINE | ID: mdl-8432405

ABSTRACT

Genomic and cDNA clones of a gene expressed after a blood meal in the mosquito, Aedes aegypti, were identified as having significant similarity to the vitelline membrane protein genes of Drosophila melanogaster. The predicted protein had unusually high contents of alanine, histidine, and proline and contained a region of hydrophobic amino acids that was highly conserved in the predicted protein of the D. melanogaster vitelline membrane protein genes. The 15a gene was expressed from 5 to 40 hr after a blood meal. It was expressed only in the follicle cells of the ovary, particularly in the cells surrounding the oocyte. The 15a gene was expressed in ovaries of the blood-fed, decapitated female in response to an injection of 20-hydroxyecdysone, and in ovaries from non-blood-fed females incubated with the hormone, even in the presence of cycloheximide. A second gene, with weaker homology to 15a, is presumably another member of a family of related genes, as is the case with D. melanogaster vitelline membrane protein genes. This second gene contained a coding sequence similar to a decapeptide recently isolated from mosquito ovaries as an "oostatic factor" (Borovsky et al., FASEB J. 4, 3015-3020, 1990).


Subject(s)
Aedes/genetics , Drosophila melanogaster/genetics , Egg Proteins/genetics , Gene Expression Regulation , Insect Proteins , Membrane Proteins/genetics , Vitelline Membrane/metabolism , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , DNA , Ecdysterone/physiology , Egg Proteins/metabolism , Female , In Situ Hybridization , Membrane Proteins/metabolism , Molecular Sequence Data , Organ Specificity/genetics , Ovary/metabolism , Restriction Mapping , Sequence Homology, Amino Acid , Species Specificity
16.
Gene ; 116(2): 165-72, 1992 Jul 15.
Article in English | MEDLINE | ID: mdl-1634114

ABSTRACT

The nucleotide sequence encoding the scorpion insectotoxin I5A was chemically synthesized and expressed in yeast, bacteria and tobacco. The I5A peptides produced in these organisms were purified using an immunoaffinity chromatography procedure. I5A produced using the bacterial secretion system was efficiently secreted and released into the culture medium. In contrast, only a trace amount of I5A was detected in bacterial cytosols when expressed from a direct expression vector, suggesting that I5A was unstable in bacterial cells. I5A secreted from yeast using an alpha-factor signal sequence was shown to have an N-terminal (Glu-Ala)2 extension, indicating incomplete processing of the secreted peptide by dipeptidyl aminopeptidase A. In tobacco, a nonsecreted form of the protein was produced. No measurable insect toxicity was observed when insect larvae were assayed, regardless of whether I5A was produced in yeast, bacteria or tobacco. The lack of toxicity is almost certainly the result of improper folding due to incorrect disulfide bond formation. The inability to produce a biologically active peptide must be overcome before scorpion toxins might be used for the genetic engineering of plants for insect resistance. The yeast and bacterial expression systems described here may be useful for further studies on the problem of expressing a biologically active peptide.


Subject(s)
Escherichia coli/genetics , Nicotiana/genetics , Plants, Toxic , Saccharomyces cerevisiae/genetics , Scorpion Venoms/genetics , Amino Acid Sequence , Base Sequence , Biological Assay , Escherichia coli/metabolism , Gene Expression , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Scorpion Venoms/biosynthesis , Scorpion Venoms/metabolism , Nicotiana/metabolism
17.
Mol Gen Genet ; 226(1-2): 241-9, 1991 Apr.
Article in English | MEDLINE | ID: mdl-1903503

ABSTRACT

Region 26A of the second chromosome of Drosophila melanogaster has been extensively characterized at the genetic level. We report here the cloning of virtually the entire 26A region via a bidirectional chromosome walk. Deletion and translocation breakpoints in the 26A interval have been localized at the molecular level by both chromosomal in situ hybridization and Southern analysis. The locations of the genetically defined loci in this chromosomal region have also been correlated with transcriptional units mapped onto the DNA of the proximal region of the chromosomal walk. The position of the alpha-glycerophosphate dehydrogenase (alpha-Gpdh) gene in 26A5-7 has been confirmed and a putative transcriptional unit for the beta-galactosidase-1 (beta-Gal-1) gene has been identified in the 26A7-9 interval.


Subject(s)
Drosophila melanogaster/genetics , Glycerolphosphate Dehydrogenase/genetics , Animals , Blotting, Northern , Blotting, Southern , Chromosome Walking , Cloning, Molecular , Drosophila melanogaster/enzymology , Nucleic Acid Hybridization , Transcription, Genetic , beta-Galactosidase/genetics
18.
Article in English | MEDLINE | ID: mdl-1685414

ABSTRACT

1. In assays of 36Cl- uptake into mouse brain vesicles, 100 microM GABA markedly increased both the initial rate of 36Cl- uptake and the total amount of chloride taken up over a 120-sec incubation period. Specific GABA-dependent 36Cl- uptake (the difference between total and background uptake) was essentially complete within 15 sec of incubation. 2. Incubation with GABA following preincubation with 10 microM endrin, a polychlorocycloalkane insecticide and established blocker of GABA-gated chloride channels, showed a stimulation of uptake over background levels that was much slower in onset than that observed with GABA alone but nevertheless achieved virtually the same level of stimulation above background levels after 90 sec of incubation with GABA. 3. In electrophysiological assays of GABA receptors expressed in Xenopus oocytes following injection with rat brain mRNA, endrin (20 microM) effectively blocked the transient currents elicited by brief exposure of oocytes to GABA (200 microM). However, prolonged exposure to GABA in the absence of perfusion produced a large, slowly-developing inward current. 4. The actions of several known GABA antagonists were also compared as inhibitors of GABA-dependent 36Cl- uptake into mouse brain vesicles at short (4 sec) and long (120 sec) incubation times using concentrations of inhibitors known to produce approximately 70-90% inhibition of GABA-dependent chloride uptake in 4-sec incubations. Picrotoxinin and TBPS, like endrin, were completely ineffective as inhibitors in 120-sec incubations. In contrast, bicuculline was almost as effective at 120 sec as at 4 sec, and avermectin Bla produced approximately 50% inhibition of the GABA response after 120 sec.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Bridged Bicyclo Compounds, Heterocyclic , Convulsants/pharmacology , Membrane Proteins/drug effects , gamma-Aminobutyric Acid/pharmacology , Animals , Brain/drug effects , Brain/physiology , Brain/ultrastructure , Bridged Bicyclo Compounds/pharmacology , Chloride Channels , Chlorides/metabolism , Chlorides/pharmacokinetics , Endrin/pharmacology , GABA Antagonists , Ion Channel Gating/drug effects , Kinetics , Male , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/physiology , Mice , Mice, Inbred ICR , Picrotoxin/analogs & derivatives , Picrotoxin/pharmacology , Rats , Rats, Inbred Strains , Receptors, GABA-A/physiology , Sesterterpenes , Xenopus , gamma-Aminobutyric Acid/physiology
19.
Arch Insect Biochem Physiol ; 16(1): 45-53, 1991.
Article in English | MEDLINE | ID: mdl-1665994

ABSTRACT

A segment of the house fly (Musca domestica) homologue of the para (paralytic) sodium channel gene of Drosophila melanogaster was isolated by using mixed sequence oligonucleotide primers in the polymerase chain reaction (PCR). The specificity of the procedure was demonstrated by genomic Southern analysis using the housefly PCR amplification product as a probe and by DNA sequence analysis. The latter showed structural homology to the para gene, but not to the corresponding region of DSC1, another D. melanogaster gene with structural similarity to vertebrate sodium channel genes.


Subject(s)
Houseflies/genetics , Sodium Channels/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , DNA/genetics , DNA/isolation & purification , Drosophila/genetics , Houseflies/physiology , Molecular Sequence Data , Oligodeoxyribonucleotides , Polymerase Chain Reaction/methods , Rats , Sequence Homology, Nucleic Acid
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