High expression of Cyp6g1, a cytochrome P450 gene, does not necessarily confer DDT resistance in Drosophila melanogaster

Cytochrome P450 monooxygenases, a family of detoxifying enzymes, are thought to confer resistance to various insecticides including DDT. Daborn et al. [Daborn, P., Yen, J.L., Bogwitz, M., Le Goff, G., Feil, et al. 2002. A single p450 allele associated with insecticide resistance in Drosophila. Science 297, 2253-2256.] suggested that the Accord transposable element causes overexpression of a Cyp6g1 allele, which has spread globally and is the basis of DDT resistance in Drosophila melanogaster populations. To determine whether the same phenomenon also operates in other Drosophila strains, we investigated 91-R, 91-C, ry(506), Wisconsin, Canton-SH and Hikone-RH strains. While the LC(50) values for the 91-R and Wisconsin strains are 8348 microg and 447 microg of DDT, respectively, values for the other four strains range between 0.74 to 20.9 microg. As expected, the susceptible ry(506) and 91-C strains have about 16-33-fold lower levels of CYP6G1 mRNA than the resistant 91-R and Wisconsin strains. Surprisingly, CYP6G1 mRNA and protein levels in the Canton-SH and Hikone-RH strains are as high as in the two resistant strains, yet they are as susceptible as the 91-C strain. The susceptible phenotype of the Canton-SH and Hikone-RH strains is not due to mutation in the Cyp6g1 gene; sequence analysis showed that Cyp6g1 alleles of resistant and susceptible strains are very similar and cannot be classified into resistant and susceptible alleles. As observed by others, we also found that only the 5'-upstream DNA of overexpressing alleles of Cyp6g1 has an insertional DNA, which is similar to Accord and Ninja elements. To examine the role of Cyp6g1 in DDT resistance, we substituted the Cyp6g1 allele of the 91-R strain with the allele from the susceptible 91-C strain via recombination and synthesized three recombinant lines. All three lines lacked Accord insertion and showed low expression of Cyp6g1 like the 91-C strain, yet they were as highly resistant as the 91-R strain. We conclude a strain may not have to have Accord insertion in the Cyp6g1 gene and the Cyp6g1 itself may not have to be overexpressed for DDT resistance to occur.     

DDT resistance in Drosophila correlates with Cyp6g1 over-expression and confers cross-resistance to the neonicotinoid imidacloprid

Mutagenesis can be used as a means of predicting likely mechanisms of resistance to novel classes of insecticides. We used chemical mutagenesis in Drosophila to screen for mutants that had become resistant to imidacloprid, a neonicotinoid insecticide. Here we report the isolation of two new dominant imidacloprid-resistant mutants. By recombinational mapping we show that these map to the same location as Rst(2)DDT. Furthermore, we show that pre-existing Rst(2)DDT alleles in turn confer cross-resistance to imidacloprid. In order to localize the Rst(2)DDT gene more precisely, we mapped resistance to both DDT and imidacloprid with respect to P-element markers whose genomic location is known. By screening for recombinants between these P-elements and resistance we localized the gene between 48D5-6 and 48F3-6 on the polytene chromosome map. The genomic sequence in this interval shows a cluster of cytochrome P450 genes, one of which, Cyp6g1, is over-expressed in all resistant strains examined. We are now testing the hypothesis that resistance to both compounds is associated with over-expression of this P450 gene.     

Regulatory elements involved in the tissue-specific expression of the yellow gene of Drosophila

Summary We have assessed the DNA sequence requirements for the correct spatial pattern and phenotypic expression of y in the late embryo/larvae. The wild-type larval phenotype requires both the regions between-294 bp and-92 bp and a portion of the intron; the sequence element(s) located within the intron can act in a position independent manner to effect the wild-type larval phenotype. The larval expression pattern was examined by tissue experiments in situ and by staining germline transformants derived from various y/lacZ fusion constructs. The larval expression of y is restricted to the mouthparts, microsetae and anal plates. While the-495 bp to+194 bp region alone cannot effect a wild-type larval expression pattern, this region in conjunction with the intron appears to be sufficient to drive -gal expression in an essentially wild-type pattern. Our data further suggest that the-294 bp to-92 bp region contains elements which specify the larval pattern and that the element(s) in the intron normally act to enhance the level of expression necessary for the wild-type larval phenotype. We also present a phenotypic analysis of the adult cuticle structures of germline transformants derived from a variety of deletion and rearrangement constructs of the y gene. This analysis has revealed several new features associated with the regulation of y expression.     

Cis-regulatory elements used to control gene expression in plants

Plant biotechnology is a dynamically developing science, which comprises many fields of knowledge. Novel plant genetic engineering findings highly influence the improvement of industrial production. These findings mostly concern cis-regulatory elements, which are sequences controlling gene expression at all developmental stages. They comprise of promoters, enhancers, insulators and silencers, which are used to construct synthetic expression cassettes. Examples of most important cis-regulatory elements are reviewed in the present paper. Variability among core promoters content and distal promoter regions impedes evaluation of interactions between them during the artificial promoters construction. Synthetic promoters and artificial expression cassettes trigger a significant increase in gene expression level, better properties and quality of a product. Accumulating knowledge about gene promoters, cis sequences and their cooperating factors allows uniform expression systems and highly predictable results.     

Point mutations associated with insecticide resistance in the Drosophila cytochrome P450 Cyp6a2 enable DDT metabolism.

Three point mutations R335S, L336V and V476L, distinguish the sequence of a cytochrome P450 CYP6A2 variant assumed to be responsible for 1,1,1-trichloro-2,2-bis-(4'-chlorophenyl)ethane (DDT) resistance in the RDDT(R) strain of Drosophila melanogaster. To determine the impact of each mutation on the function of CYP6A2, the wild-type enzyme (CYP6A2wt) of Cyp6a2 was expressed in Escherichia coli as well as three variants carrying a single mutation, the double mutant CYP6A2vSV and the triple mutant CYP6A2vSVL. All CYP6A2 variants were less stable than the CYP6A2wt protein. Two activities enhanced in the RDDT(R) strain were measured with all recombinant proteins, namely testosterone hydroxylation and DDT metabolism. Testosterone was hydroxylated at the 2beta position with little quantitative variation among the variants. In contrast, metabolism of DDT was strongly affected by the mutations. The CYP6A2vSVL enzyme had an enhanced metabolism of DDT, producing dicofol, dichlorodiphenyldichloroethane and dichlorodiphenyl acetic acid. The apparent affinity of the enzymes CYP6A2wt and CYP6A2vSVL for DDT and testosterone was not significantly different as revealed by the type I difference spectra. Sequence alignments with CYP102A1 provided clues to the positions of the amino acids mutated in CYP6A2. These mutations were found spatially clustered in the vicinity of the distal end of helix I relative to the substrate recognition valley. Thus this area, including helix J, is important for the structure and activity of CYP6A2. Furthermore, we show here that point mutations in a cytochrome P450 can have a prominent role in insecticide resistance.     

Cis-regulatory sequences leading to female-specific expression of yolk protein genes 1 and 2 in the fat body of Drosophila melanogaster

We determined the physical linkage of six mouse Hox3 homeobox sequences, including a new homeobox sequence (Hox3.5), by analysis of overlapping genomic clones. Additionally, we defined the locations of Hox 1.7 and Hox 1.8 in the Hox 1 cluster. Analysis of the expression patterns of Hox 3.6 and Hox 3.5 during embryogenesis revealed that the relationship between relative position in the Hox 3 cluster and expression domain along antero-posterior axis appears similar to that seen for members of the other Hox clusters.     

Age dependence of the level of copia retrotransposon expression in the testes of Drosophila melanogaster

Expression of the lacZ reporter gene controlled by various deletion derivatives of the regulatory region of the copia retrotransposon was studied in the testes of adult transgenic males of the Drosophila melanogaster y1W67c23(2) strain at the age of 3, 6-9, 12-15, 18-21, and 24-27 days. When the construct contained the full-length regulatory region, which included the 5'-long terminal repeat (LTR) and the 5'-untranslated region (UTR), expression was the lowest in males aged 12-15 days and the highest in males aged 3 or 24-27 days. A similar V-shaped age dependence was previously observed for the copia transposition rate and RNA content in a strain with a high rate of copia transposition. Thus, the V-shaped age dependence of expression, which is unusual for Drosophila, proved to be characteristic of copia regardless of its transposition rate. Deletion of the 5' or 3' end of the LTR, but not of the UTR, changed the age dependence of the level of reporter gene expression. In this case, expression was the highest in 3-day-old males and gradually decreased with age, as typical for many Drosophila genes. It was assumed that the 5'- and 3'-terminal regions of the copia LTR contain regulatory elements responsible for the V-shaped age dependence of expression, while the expression level depends to a greater extent on the regulatory elements of UTR.     

Molecular cloning and tissue-specific expression of the mutator2 gene (mu2) in Drosophila melanogaster.

We present here the molecular cloning and characterization of the mutator2 (mu2) gene of Drosophila melanogaster together with further genetic analyses of its mutant phenotype. mu2 functions in oogenesis during meiotic recombination, during repair of radiation damage in mature oocytes, and in proliferating somatic cells, where mu2 mutations cause an increase in somatic recombination. Our data show that mu2 represents a novel component in the processing of double strand breaks (DSBs) in female meiosis. mu2 does not code for a DNA repair enzyme because mu2 mutants are not hypersensitive to DSB-inducing agents. We have mapped and cloned the mu2 gene and rescued the mu2 phenotype by germ-line transformation with genomic DNA fragments containing the mu2 gene. Sequencing its cDNA demonstrates that mu2 encodes a novel 139-kD protein, which is highly basic in the carboxy half and carries three nuclear localization signals and a helix-loop-helix domain. Consistent with the sex-specific mutant phenotype, the gene is expressed in ovaries but not in testes. During oogenesis its RNA is rapidly transported from the nurse cells into the oocyte where it accumulates specifically at the anterior margin. Expression is also prominent in diploid proliferating cells of larval somatic tissues. Our genetic and molecular data are consistent with the model that mu2 encodes a structural component of the oocyte nucleus. The MU2 protein may be involved in controlling chromatin structure and thus may influence the processing of DNA DSBs.     

Mobilization of retrotransposon copia in the Drosophila melanogaster genome]

Considerable heterogeneity of retrotransposon copia sites of location on polytene chromosomes was revealed in one of the substocks of the inbred Drosophila melanogaster stock. Heterogeneity of copia sites of location was found in no other substocks analyzed. The heterogeneity was shown to be caused by copia insertions in new sites. The frequency of insertions is about 12% per haploid genome per generation. The retrotransposon excisions and somatic transpositions were not observed. The location of retrotransposons mdg1, mdg2, mdg3, mdg4, 297 and H.M.S. Beagle appeared to be stable in all the stocks analyzed. Thus, a model system allowing to study mechanisms of retrotransposon copia transpositions in D. melanogaster tissues as well as phenotypic effects of copia mobilization is described.     

World-wide survey of an Accord insertion and its association with DDT resistance in Drosophila melanogaster

Previous work showed that insecticide resistance in Drosophila melanogaster is correlated with the insertion of an Accord-like element into the 5' region of the cytochrome P450 gene, Cyp6g1. Here, we study the distribution of the Accord-like element in 673 recently collected D. melanogaster lines from 34 world-wide populations. We also examine the extent of microsatellite variability along a 180-kilobase (kb) genomic region of chromosome II encompassing the resistance gene. We confirm a 100% correlation of the Accord insertion with insecticide resistance and a significant reduction in variability extending at least 20 kb downstream of the Cyp6g1 gene. The frequency of the Accord insertion differs significantly between East African (32-55%) and nonAfrican (85-100%) populations. This pattern is consistent with a selective sweep driving the Accord insertion close to fixation in nonAfrican populations as a result of the insecticide resistance phenotype it confers. This study confirms that hitchhiking mapping can be used to identify beneficial mutations in natural populations.     

Cloning of murine G1RP, a novel gene related to Drosophila melanogaster g1

To study the nature of genes that are induced during the apoptotic death of myeloid precursor cells, we performed representational difference analysis (RDA) using 32Dcl3 myeloblastic cells that were deprived of IL-3 for 24h. We have isolated a novel cDNA (g1-related protein, G1RP) that is homologous to g1, a Drosophila melanogaster zinc-finger protein that is expressed in the mesoderm. Northern blot analysis using RNAs derived from 32Dcl3 cells that have been grown in the absence of IL-3 demonstrates that the G1RP message is upregulated in these cells following the removal of IL-3, suggesting that this gene may regulate growth factor withdrawal-induced apoptosis of myeloid precursor cells.