Larry C. Waters

Three second chromosome-linked clustered Cyp6 genes show differential constitutive and barbital-induced expression in DDT-resistant and susceptible strains of Drosophila melanogaster

The level of expression of the Cyp6a2 gene is much higher in the DDT-resistant 91-R strain than in the susceptible 91-C strain of Drosophila melanogaster (Waters et al. (1992b) Proc. Natl. Acad. Sci. USA, 89, 4855-4859). To understand the role of Cyp6a2 and related genes in insecticide resistance, we have isolated and characterized two new Cyp6 genes from the 91-R strain. The polypeptides encoded by these two genes, Cyp6a8 and Cyp6a9, show 77 and 75% amino acid sequence similarity, and 60 and 55% identity with Cyp6a2 of D. melanogaster, respectively. In the genome, Cyp6a8 and Cyp6a9 genes are closely clustered within 4 kb and map at region 51C of the second chromosome. In between them another Cyp gene is present which is more related to Cyp6a9 than to Cyp6a8. The Cyp6a8 gene which is transcriptionally highly active in 91-R, moderately active in ry506 and silent in the 91-C strain hybridizes with 2.0- and 1.8-kb RNAs. Two different-sized RNAs, 2.1 and 1.8 kb, also hybridize with the Cyp6a9 and/or Cyp6a9-related genes. While the level of 2.1-kb RNA is similar in all three strains, the level of 1.8-kb RNA is highest in the 91-R strain and barely detectable in 91-C strain. Transgenic experiments showed that a 8.3-kb BamHI fragment contains the cis-regulatory elements for the expression of both Cyp6a8 and Cyp6a9-related genes. Barbital induces all these genes in all three strains and increases the levels of the two Cyp6a8 transcripts and the 1.8-kb RNA produced by the Cyp6a9 and/or Cyp6a9-related genes. Expression of the Cyp6a8 gene is down-regulated in the hybrids of 91-R and 91-C strains despite the fact that the hybrids carry one copy of the highly active allele of the Cyp6a8 gene of the 91-R strain. Based on these results we propose that the Cyp6a8 gene in 91-C strain may be turned off by an active repressor which might be inhibited by barbital treatment. In the 91-R strain, the putative repressor may be defective, allowing high level of constitutive expression of the Cyp6a8 gene.

Factors on the third chromosome affect the level of Cyp6a2 and Cyp6a8 expression in Drosophila melanogaster

The expression of two second chromosome-linked cytochrome P450 genes, Cyp6a2 and Cyp6a8, of Drosophila melanogaster was measured in various strains. Six different strains, including ry(506) and 91-C, showed low or undetectable levels of CYP6A2 and CYP6A8 mRNAs, suggesting that low expression is the wild-type phenotype of Cyp6a2 and Cyp6a8 genes. In the 91-R and MHIII-D23 strains, however, both these genes are overexpressed. In order to examine the genetic basis of Cyp6a2 and Cyp6a8 expression, CYP6A2 and CYP6A8 RNA levels were measured in the F1 hybrids of overproducer (91-R and MHIII-D23) and underproducer (ry(506) and 91-C) strains. Results showed that the total amounts of CYP6A2 and CYP6A8 mRNAs in the F1 hybrids were lower than half the amounts of these RNAs found in the overproducer parental strains. This suggested that the underproducer strains carry loci which downregulate Cyp6a2 and Cyp6a8 gene expression. To determine the chromosome linkage of these loci, several stocks homozygous for the second chromosome of overproducer 91-R strain and, therefore, homozygous for the Cyp6a2-91R and Cyp6a8-91R alleles were synthesized. The third chromosomes in all these stocks were from the underproducer ry(506) strain. The levels of expression of both Cyp6a2-91R and Cyp6a8-91R genes in these three stocks were significantly lower than that observed in the 91-R strain. One of these stocks, named iso-2, showing reduced expression, was used to synthesize two new isogenic stocks by resubstituting the third chromosome of ry(506) origin with third chromosomes of the 91-R strain. Expression of both Cyp6a2-91R and Cyp6a8-91R alleles was found to be much higher in these two resubstituted isogenic stocks than in the progenitor iso-2 stock. Taken together, these results suggest that the second chromosome-linked Cyp6a2 and Cypa8 genes are regulated by loci present on the third chromosome, and the wild-type function of these loci is to repress these two Cyp genes. The data also suggest that Cyp6a2 and Cyp6a8 overexpression in the 91-R and MHIII-D23 strains is more likely due to mutation in the repressor locus (or loci) rather than in the cis-regulatory sequences of the Cyp6a2 and Cyp6a8 genes.

Regulation of insecticide resistance-related cytochrome P-450 expression in Drosophila melanogaster

Abstract Two protein bands, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, account for most of the cytochrome P -450 in Drosophila melanogaster . P -450-A is ubiquitous among strains tested; whereas P -450-B is unique to certain strains. Dimethylnitrosamine demethylase activity is associated with P -450-B. Biochemical and genetic analyses showed that genes located on chromosome II were required for P -450-B expression. These genes were mapped within an interval that includes a major insecticide-resistance locus. Regulatory loci on chromosome III were required for maximum expression of P -450-B. One of these regulatory loci was mapped at another major resistance locus on chromosome III. There was a good correlation between P -450-B expression and resistance to phenylurea among the different strains tested. These results indicate that Drosophila can be a useful model to study the molecular mechanisms of insecticide resistance.

Monoclonal antibodies to resistance-related forms of cytochrome P450 in Drosophila melanogaster

Abstract Drosophila cytochrome P450 is resolved by sodium dodecylsulfate-polyacrylamide gel electrophoresis into two subsets. P450-A 4 , ∼59 kDa, is ubiquitous among strains whereas P450-B, ∼56 kDa, is expressed in strains resistant to insecticides. Monoclonal antibodies (MoAbs) to each of these subsets were produced. MoAbs 13-2e and 8-1d were specific for epitopes in the P450-A and P450-B subsets, respectively. Although P450-B is virtually undetectable by protein staining in insecticide-susceptible strains, immunoreactive P450-B was detected in these strains. However, immunoreactive P450-B was 10 to 20 times higher in strains selected for DDT resistance than in was shown to be 10 to 100 times higher than in the unselected controls. These and previous data show that P450-B expression is regulated by resistance genes on the second and third chromosomes of Drosophila and is correlated with resistance to phenylurea, DDT, and malathion, with N -nitrosodimethylamine demethylase activity and with immunoreactivity with MoAb 8-1d.

Some physical and biological properties of 4-thiouridine- and dihydrouridine-deficient tRNA from chloramphenicol-treated Escherichia coli

Abstract The tRNA synthesized by Escherichia coli during chloramphenicol treatment has altered chromatographic properties, as analyzed by reversed-phase chromatography. The data suggest that most of the tRNAs synthesized in the presence of chloramphenicol are immature, chromatographically distinct forms, capable of being converted to mature forms upon removal of chloramphenicol. Methylation of tRNA during chloramphenicol treatment appears to be quantitatively and qualitatively normal. The tRNA synthesized during chloramphenicol treatment differs from normal tRNA mainly in that it contains 60–70% less 4-thiouridine and dihydrouridine. Preliminary experiments indicate that these two minor bases are not required for normal aminoacylation. Phenylalanyl-, valyl-, isoleucyl- and lysyl-tRNAs from untreated and chloramphenicol-treated cells are equally functional in an in vitro hemoglobin-synthesizing system.

Altered chromatographic properties of tRNA from chloramphenicol-treated Escherichia coli☆

Abstract Several tRNAs from E. coli which had been treated with chloramphenicol (CAP) are shown to have altered chromatographic properties. The tRNA synthesized after CAP treatment is altered, whereas that already present in normal cells is not. Preliminary experiments indicate that the new aminoacyl-tRNAs which we observe are not results of undermethylation nor degradation.

Experimental evaluation of two field test kits for the detection of PAHs by immunoassay

Immunoassay-based field analytical methods are rapidly gaining acceptance by the U.S. Environmental Protection Agency for use in site characterization and remediation. Many analysts, accustomed to using traditional laboratory methods, are reluctant to use immunochemical technology until they are convinced of its effectiveness. Therefore, it is important that experiences with using alternative technologies be shared with the analytical community. In this study, two immunoassay-based field test kits for polyaromatic hydrocarbons (PAHs) were evaluated. One was used in a quantitative format, the other in a semiquantitative format. Samples spiked with a commercial PAH mixture or creosote and field samples including soil and coal-derived liquids were analyzed. For the most effective use of the method, it is important to know the relative response factors (RRFs) for test ana-lytes relative to the kit standards. RRFs for creosote-spiked soil were about 2.3 and 0.11 for the quantitative and semiquantitative tests, respectively. This 20-fold difference in RRF between the two kits held for all the different samples tested and reflects the different antibodies and reference compounds used in the two tests. Potential problems with the integrity of the kit standards, which could significantly affect the interpretation of the test results, are discussed. Recovery of the PAH mixture and creosote from soil ranged from 75 to 90%. The specificity of a third test kit for carcinogenic PAHs was verified with the coal-derived liquids. Overall, both kits gave accurate and reproducible results and were judged to be effective tests for the analysis of PAH contaminated samples.

Transfer rnas associated with the 70s rna of akr murine leukemia virus.

Summary The specificity of the aminoacyl-tRNA synthetase and tRNA interaction was used to identify the amino acid tRNAs most tenaciously bound to the 70S RNA of the AKR murine leukemia virus. Of the 17 amino acid tRNAs tested for, proline tRNA was the major one which was dissociated from the viral RNA at temperatures above 60°.

Natural variation in the expression of cytochrome P-450 and dimethylnitrosamine demethylase in Drosophila

Electrophoresis of Drosophila microsomes resolves two major heme-containing protein bands with apparent molecular weights of 59,290 (band a) and 55,750 (band b). The hemoproteins in these two bands can account for most of the cytochrome P-450 in the organism. Band a is present in all strains examined: band b is not. Dimethylnitrosamine demethylase, a P-450 enzyme, is a component of band b.

Lysine tRNA is the predominant tRNA in murine mammary tumor virus.

Abstract The method of aminoacylation and subsequent identification of the esterified amino acids was used to characterize the transfer RNAs in murine mammary tumor virus. Lysine tRNA was the major tRNA in both “free” 4S RNA and “7OS-associated” 4S RNA in virus derived from either tissue culture or mouse milk.