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.

Sequence similarities exist between terminal inverted repeats (TIRs) of some miniature inverted-repeat transposable element (MITE) families isolated from a wide range of organisms, including plants, insects, and humans, and TIRs of DNA transposons from the pogo family. We present here evidence that one of these MITE families, previously described for Arabidopsis thaliana, is derived from a larger element encoding a putative transposase. We have named this novel class II transposon Lemi1. We show that its putative product is related to transposases of the Tc1/mariner superfamily, being closer to the pogo family. A similar truncated element was found in a tomato DNA sequence, indicating an ancient origin and/or horizontal transfer for this family of elements. These results are reminiscent of those recently reported for the human genome, where other members of the pogo family, named Tiggers, are believed to be responsible for the generation of abundant MITE-like elements in an early primate ancestor. These results further suggest that some MITE families, which are highly reiterated in plant, insect, and human genomes, could have arisen from a similar mechanism, implicating pogo-like elements.

Organophosphorus insecticide (OP) resistance in several Culex species is associated with increased esterase activity resulting from amplification of the corresponding structural gene. In Culex pipiens quinquefasciatus, high levels of OP resistance (=800 times) are due to the esterase Bi gene, which is amplified at least 250-fold. This gene has now been sequenced, and the structure of the amplification unit (amplicon) encompassing the structural gene has been partially characterized. The inferred amino acid sequence of the enzyme revealed regions of strong homology with other eukaryotic serine-esterases, such as cholinesterases, which are the target of OPs. The amplicon covers at least 30 kilobases and contains a constant and highly conserved "core" of 25 kilobases. This core carries a single copy of the esterase gene (2.8 kilobases) as well as other sequences that are present as single or low number copies in the genomes of mosquitoes lacking overproduction of the esterase Bi protein. In the amplicon, the esterase gene is framed by two DNA sequences that are repeated in other parts of the genome of resistant mosquitoes and found in the genome of susceptible mosquitoes but not near the esterase Bi gene. It is suggested that these repetitive sequences may have a role in the amplification process.Gene amplification seems to be a fundamental and widely occurring mechanism of overproduction ofproteins for counteracting environmental stress. Amplification has been shown to result in high levels ofresistance in insects that have been under selection with organophosphorus insecticides (OPs) (1-3), in alfalfa controlled with the herbicide Lphosphinothricin (4), and in Leishmania (5) and cultured mammalian cells (6) grown in the presence ofantiproliferative drugs. Populations of several species of mosquitoes of the genus Culex have become resistant to a variety of OPs due to increased production of detoxifying esterases (7). The three esterases B studied thus far-i.e., B1, B2, and B3-were shown to be amplified (1, 2). There is evidence indicating that amplification of each esterase B gene occurred as a single and independent event (2), suggesting that it may be induced by a specific mechanism. In order to explore the nature of this mechanism, we have undertaken the characterization of the amplified DNA structures. We report here the nucleotide sequencett and intron and exon organization of the esterase B1 gene, which, in Californian Culex pipiens quinquefasciatus, has been shown to be amplified at least 250-fold (1). In addition, we show that large DNA sequences are coamplified with the structural gene and characterize part of these sequences. (2)]. MATERIALS AND METHODSCloning Genomic DNA. Genomic DNA of Tem-R adults was partially digested by restriction enzyme Sau3AI. Sizeselected 15-to 22-kilobase (kb) DNA fragments thus generated were ligated to the BamHI site of the arms of phage vector A-EMBL3. After packaging of the concatenated DNA molecules, the recombinant phage library was plated on Escherichia coli C6...

Energy charge, adenine nucleotide levels, and protein synthesis were studied during the transfer of rice seedlngs from air to anoxia. Within minutes, the energy charge value dropped from 0.90 in air to 0.50 in the seed and 0.60 in the coleoptile after the transfer to a nitrogen atmosphere, and then increased to a value of 0.80 during the subsequent hours. The sum of nucleotides also dropped to 60% of the value in air in the seeds and to 30% in the coleoptiles. However, during the anaerobic growth of coleoptiles, a considerable increase in the nucleotide pool occurred.The incorporation of amino acids into proteins was measured at different stages in anoxic treatment. In rice embryo, we observed a considerable protein synthesis correlated with a high value of energy charge under anoxia. The analysis oflabeled proteins by two-dimensional polyacrylamide gel electrophoresis showed a modified pattern of polypeptides synthesized during anoxic treatment. Some of these proteins were intensively labeled and appeared to be induced by anaerobic treatment.Our data indicate that high metabolic activity occurs in rice embryo under anoxia, which can be correlated with a high energy charge value. These phenomena may be part of the mechanisms which permit the adaptation of rice embryos to anaerobiosis.In nonchlorophyllous plant tissues and in animals, it is generally assumed that ATP synthesis is drastically reduced when oxidative phosphorylation is inhibited by a lack of 02. The knowledge of the balance between ATP regeneration and utilization for metabolic work should permit a better understanding of the mechanisms used by these organisms to survive under anoxic conditions.In animal tissues, there is a rapid inhibition of protein synthesis after a few min of anoxia (10,12 Preliminary data obtained in our laboratory indicate that rice embryos or coleoptiles germinated in anoxia or transferred from air to nitrogen have a high energy charge (20, 21) and an active RNA (2) and DNA synthesis (16).The purpose of this paper is to study the effect of anoxia on adenine nucleotide levels, energy charge, and incorporation of labeled amino acids into proteins in rice embryos. The correlation between energy charge and protein synthesis in anoxia is discussed, and anoxic protein patterns sustain the hypothesis of an adaptation of rice embryo to anaerobic conditions. MATERIALS AND METHODSPlant Materials. Rice seeds (Oryza sativa L., var. Cigalon) cultivated by the Station d'Amelioration des Plantes (INRA, Montpellier, France) were mechanically husked and sterilized with commercial (NaOCl (150 g Cl/l), as described previously (16).Germination of Rice Seeds. Seeds were placed in glass flasks with water and shaken at 26 C in darkness. They remained under aerobic conditions for 40 to 48 h until coleoptiles reached 4 to 5 mm.For anoxia, samples were placed in a nitrogen flow (100 ml/ min), as previously described (16). Oxygen content was checked with an 02 analyzer (WOM, Mecanalyse, France) or by gas chromatography. PO2 in N2 was lower than...

No abstract