Claude Mouches

Amplification of an esterase gene is responsible for insecticide resistance in a California Culex mosquito

An esterase gene from the mosquito Culex quinquefasciatus that is responsible for resistance to a variety of organophosphorus (OP) insecticides was cloned in lambda gt11 phage. This gene was used to investigate the genetic mechanism of the high production of the esterase B1 it encodes in OP-resistant Culex quinquefasciatus Say (Tem-R strain) from California. Adults of the Tem-R strain were found to possess at least 250 times more copies of the gene than adults of a susceptible strain (S-Lab). The finding that selection by pesticides may result in the amplification of genes encoding detoxifying enzymes in whole, normally developed, reproducing insects emphasizes the biological importance of this mechanism and opens new areas of investigation in pesticide resistance management.

Amplification of various esterase B's responsible for organophosphate resistance in Culex mosquitoes.

Increased detoxification by esterases is a common mechanism of resistance to organophosphate (OP) insecticides in insects. Utilizing a partial cDNA of this gene, we probed genomic fragments generated by EcoR1 restriction enzyme in various laboratory and natural populations of Culex that are OP resistant

Biochemical characterization of the esterases A1 and B1 associated with organophosphate resistance in the Culex pipiens L. Complex

Abstract Two esterases, A1 and B1, displaying a high activity in organophosphate (OP) resistant Culex pipiens L. from southern France and in C. quinquefasciatus Say from California, respectively, have been analyzed. Both enzymes are shown to be soluble and to constitute a large proportion of the proteins (1–3% for esterase A1 and 6–12% for esterase B1). The size of native esterase A1 was estimated between 118 and 134 kDa, that of esterase B1 67 kDa. Upon SDS denaturation, esterase B1 leads a single polypeptide of 67 kDa which suggests that it is a monomeric protein; esterase A1 leads also a single polypeptide of 60 kDa suggesting a homodimeric structure of the protein. These observations are discussed with regards to esterase E4 of Myzus persicae Sultz.

Turnip yellow mosaic virus-RNA replicase: Partial purification of the enzyme from the solubilized enzyme-template complex

Abstract The turnip yellow mosaic virus (TYMV) RNA-replicase bound to the chloroplast fraction of a participate cell-free preparation isolated from TYMV-infected Chinese cabbage leaves catalyzes the synthesis of RNA of the viral type, “plus” RNA, in the absence of added “minus” RNA template. Addition of TYMV-RNA does not stimulate RNA synthesis. The complex formed by the replicase bound to “minus” RNA template can be removed from the chloroplast fraction by solubilization with a non-ionic detergent, Lubrol W, and separated from insoluble material by centrifugation. The resulting supernatant contains the template-bound replicase as well as a small proportion of template-free replicase molecules. Treatment of this preparation with a polyethylene glycol-dextran two-phase aqueous system at high salt concentration removes the replicase from the template and yields, after centrifugation, a polyethylene glycol upper phase containing the template-free replicase molecules (PEG-enzyme) and a lower dextran phase containing a small proportion of replicase molecules still bound to their template. The soluble PEG-enzyme is markedly stimulated by added RNA. With TYMV-RNA as the added RNA, the product synthesized by the enzyme is RNA complementary to TYMV-RNA, i.e., “minus” RNA, indicating the enzyme uses the viral RNA as template. The “minus”-RNA synthesized remains hydrogen bonded to the viral RNA template and forms a RNase resistant, double-stranded structure. Chromatography on cellulose CF 11 column reveals two different double-stranded structures, one, fully double-stranded, in which the synthesized “minus” RNA strand is hydrogen bonded to template RNA of equal length, and one partly double-stranded and partly single-stranded in which probably the template RNA is very much longer than the synthesized “minus” RNA. The PEG-enzyme seems to be sensitive to high ionic strength since its activity can be completely suppressed by various salts; removal of the salts restores the activity.

Characterization and genetic organization of full-length copies of a LINE retroposon family dispersed in the genome of Culex pipiens mosquitoes

Many full-length copies of a long interspersed repetitive element family, designated Juan-C, are reiterated in the genome of Culex pipiens mosquitoes. The complete Juan-C elements have a length of 4.48 kb. They are terminated at one end with an adenosine-rich sequence preceded with an AATAAA polyadenylation signal, lack terminal repeats and cause duplication of the host DNA at the site of their integration. Full-length Juan-C copies display two long open reading frames potentially encoding two proteins. The first one includes a domain typical of nucleic-acid-binding proteins, while the second resembles reverse transcriptases. Therefore, Juan-C elements are similar to LINE retroposons in their overall genetic organization and can probably be transposed by reverse transcription of an RNA intermediate. Juan-C elements are most similar in their sequence and coding potential to the Juan-A elements which are reiterated in mosquito species belonging to the genus Aedes. They also display homologies with some Drosophila LINEs such as Jockey, suggesting that all these elements have arisen from a common precursor. Nearly identical full-length Juan-C copies are amplified in C. pipiens strains from different continents. This finding that Juan-C retroposons reiterated in different strains form an homogeneous family is interpreted to indicate that these elements have spread recently in the C. pipiens species.

Characterization of the unlinked 16S rDNA and 23S-5S rRNA operon of Wolbachia pipientis, a prokaryotic parasite of insect gonads

The rRNA-encoding genes (rDNAs) have been cloned and characterized from Wolbachia pipientis (Wp), the gonadial bacteria-like parasite of the mosquito Culex pipiens (Cp) and the moth Ephestia cautella (Ec). In Wp from both insect species the rDNAs are organized in a way which appears to be very unusual. The rRNAs are encoded by two unlinked transcription units, each present in a single copy per genome. One contains the 16S rDNA only, while the other is an operon encoding both the 23S and 5S rDNAs. Each transcription unit contains two putative upstream promoters, and downstream a Rho-independent terminator. The 16S rDNA, as well as the 23S-5S rRNA operon are not linked to any tRNA-encoding sequence and lack the antitermination boxes which are usually present immediately downstream from eubacterial promoters of rDNAs. Wp infecting Ec and Cp are highly similar taking as criteria the rDNAs and their flanking sequences. However, it clearly appears that each insect species harbours a different and specific Wp strain, or even subspecies. Phylogenetic relationships deduced from the complete sequences of their rDNAs undoubtedly confirm that Wp from Cp and Ec belong to the alpha-group of Proteobacteria, and are closely related to the Rickettsia.

Characterization of the virus encoded subunit of turnip yellow mosaic virus rna replicase

An antiserum raised against TYMV-RNA encoded protein P115 partially inhibits TYMV RNA replicase activity, demonstrating that this protein is involved in TYMV RNA synthesis. The detection of protein P115 by an antibody linked polymerase assay demonstrates that protein P115 is indeed a subunit of the TYMV RNA replicase, the enzyme known to synthesize viral RNA in infected Chinese cabbage. The use of translation products of other tymoviruses indicates that the serological relationship between the virus-encoded replicase subunits of these viruses and protein P115 is very weak at the best.

Sequence of a truncated LINE-like retroposon dispersed in the genome of Culex mosquitoes

The Juan elements belong to a family of reiterated sequences scattered through the genome of Culex mosquitoes. The nucleotide sequence of three Juan copies has been determined. The results show that the Juan elements are truncated retroposon-like elements belonging to LINEs.

Messenger properties of TYMV-RNA.

Abstract Turnip yellow mosaic virus (TYMV) RNA extracted from virions (“virion” TYMV-RNA) contains two types of molecules of molecular weight 2 × 10 6 : one which resists heat denaturation (“intact” TYMV-RNA) and one which yields smaller fragments upon heat denaturation (TYMV-RNA containing hidden breaks). A small RNA molecule of 0.2 × 10 6 MW is also present in virions. “Virion” TYMV-RNA can therefore be separated into “intact” RNA and smaller molecules by heat denaturation followed by sucrose gradient centrifugation. The RNA from the sucrose gradient fractions were analyzed by formamide PAGE: The small RNA with a molecular weight of 0.2 × 10 6 was separated from “intact” TYMV-RNA. The amount of coat protein synthesized by the RNA fractions when added to the wheat germ cell-free system was quite proportional to the amount of small RNA present. We conclude that coat protein synthesis is not directed by “intact” TYMV-RNA but by the small RNA. In addition, infectivity studies show that “intact” TYMV-RNA is fully infectious and thus it also contains the coat protein cistron.

Temporal genetic variation in European eel Anguilla anguilla (Linnaeus, 1748): A fine scale investigation in the Adour estuary

Abstract The genetic structure of arrival cohorts of European eels in the continental basin of Adour, France, was analysed. A regular sampling was carried out during three successive seasons of migration to ensure a representative sampling of complete recruitment in the estuary. Genetic variability within this sample was evaluated by analysis of highly polymorphic microsatellite markers. A high level of polymorphism was highlighted but a lack of genetic differentiation was observed between cohorts collected during the three successive seasonal migrations.