Biomedical subjects

V K Walker

Publications and source records attributed to V K Walker.

51 records · Page 3Linked to original sources

PubMed1994

Isolation of an esterase conferring insecticide resistance in the mosquito Culex tarsalis.

Malathion resistance in a strain of Culex tarsalis mosquitoes is due primarily to the activity of a malathion carboxylesterase (MCE). The resistant strain was 150 times more resistant to malathion than the susceptible strain and was weakly resistant to malaoxon and carbaryl, but not to any other insecticide tested. The phenotype could be reversed with the carboxylesterase inhibitor triphenylphosphate, but no synergism was observed with either the phosphatase or polysubstrate monooxygenase inhibitors, NaF and piperonyl butoxide. MCE is expressed throughout development and is most concentrated in the gut tissues of the larvae. Subcellular fractionation indicated that MCE was localized primarily in the mitochondria of resistant insects and the cytoplasm of susceptible insects. The enzyme was purified to homogeneity from both strains, and has a molecular weight of 59,000. However, chromatofocusing indicated that resistant insects have two MCEs with pIs of 6.8 and 6.2, while susceptible insects possessed only one MCE with a pI of 6.8. The MCE unique to the resistant strain hydrolysed malathion 18 times faster than the MCE common to both strains, suggesting that malathion resistance in C. tarsalis is due to the presence of a qualitatively different esterase in the resistant strain.

Animals↗

PubMed1994

Purification of triosephosphate isomerase and isolation of its gene from the mosquito Culex tarsalis.

The enzyme triosephosphate isomerase (TPI) was purified to homogeneity from the mosquito Culex tarsalis. Anti-C. tarsalis TPI antibodies cross-reacted with TPIs from other organisms but bands on western blots were most intense with proteins from closely related Dipterans. Using a degenerate primer corresponding to the amino-terminal sequence of the protein in a polymerase chain reaction (PCR), a cDNA corresponding to the TPI gene (Tpi) was isolated and sequenced. Subsequently, a genomic sequence including 305 bp to the 5'-end of the coding sequence was obtained. Comparison of C. tarsalis Tpi to that of Drosophila melanogaster revealed that although the two genes had little similarity in the intron and 5' flanking sequences, they were highly similar (73% identity) in their coding sequence. The rate of synonymous substitution in insect genes may be slower than that of vertebrates, but the nonsynonymous substitution rate, and hence the rate of TPI evolution, appears to be faster in insects than in vertebrates.

Amino Acid Sequence↗

PubMed1993

A novel intron site in the triosephosphate isomerase gene from the mosquito Culex tarsalis.

The origin and function of introns in eukaryotic genes has provoked considerable debate since their discovery in 1977. Central to this issue are studies on the highly conserved enzyme, triosephosphate isomerase (TPI, EC 5.3.1.1). The 'introns early' argument suggests that introns are as old as the genes themselves and that the apparent correlation of many of the intron sites in plant, animal and fungal TPI genes with the boundaries of modules is evidence of the assembly of ancient proteins by exon shuffling. In contrast, the 'introns late' view holds that ancient genomes contained few if any introns; introns were inserted into pre-existing genes during the last billion years. We have found that the TPI gene from the mosquito, Culex tarsalis, contains an intron in a unique position that was predicted by W. Gilbert and the exon shuffling hypothesis.

Amino Acid Sequence↗

PubMed1993

Isolation and characterization of an antifreeze protein precursor from transgenic Drosophila: evidence for partial processing.

Transgenic Drosophila melanogaster that contain a winter flounder antifreeze protein (AFP) gene fused to the transcriptional and translational control sequences of the host heat shock protein 70 gene express the transgene under heat shock conditions. They secrete into the hemolymph small quantities of a protein that reacts with antisera to AFP and is of a similar size to the proAFP precursor. To facilitate purification and characterization of this precursor, transformed fly lines homozygous for inserts on the 2nd, 3rd and X chromosomes were crossed together to generate a line with five and six AFP genes present in males and females, respectively. AFP production in the multi-gene line was approximately equal to the sum of that observed in the three starting lines and was just sufficient to perturb the growth of ice crystals. The AFP component was purified from heat-denatured hemolymph of this line by cation- and anion-exchange chromatography, followed by reverse-phase HPLC. Edman degradation sequencing of the purified protein showed that its N-terminus began two amino acids in from the predicted signal peptide cleavage point. An additional amino acid sequence was present that began two amino acids further into the 'pro' sequence. These AFP products are consistent with processing of the proAFP in Drosophila by a type IV dipeptidyl aminopeptidase, as has been suggested for processing in flounder.

Amino Acid Sequence↗

PubMed1992

Differential translatability of antifreeze protein mRNAs in a transgenic host.

The expression of fusion gene constructs containing Drosophila regulatory sequences and the structural portions of fish antifreeze protein genes have been examined by transfer into Drosophila melanogaster using P elements. A fusion gene, containing the enhancer, promoter, and cap site of the yolk polypeptide 1 gene, joined in the 5'-untranslated region to the structural portion of the winter flounder type I antifreeze gene, was transcribed in mature female transformants to give an mRNA of the predicted size, but no antifreeze protein was detected by Western blotting. When the same antifreeze protein gene was fused to a Drosophila hsp 70 gene regulatory region and placed downstream of the yolk polypeptide gene enhancer, appropriate expression of mRNA was directed by both gene regulatory elements. However, a translation product from this mRNA was only observed under heat shock conditions and was present at low levels. It is suggested that type I antifreeze mRNA, with its high content of alanine codons and their grouping into clusters of up to seven in a row, is poorly translated when in competition with other host mRNAs. In agreement with this hypothesis, a fusion gene construct between the yolk protein gene regulatory region and two type III antifreeze protein genes produced sub-mmolar concentrations of antifreeze protein in mature females from each of several transgenic lines analysed. The type III antifreeze protein does not have an imbalanced amino acid composition or sequence irregularities, and may be an appropriate choice for conferring freeze protection to frost-susceptible hosts by gene transfer.

Animals↗

PubMed1991

Yolk polypeptide gene expression in cultured Drosophila cells.

The transfer of chimaeric plasmids to Drosophila melanogaster cell lines has been examined as a system for investigation of the hormonal regulation of the genes coding for D. melanogaster yolk polypeptide 1 (YP1) and Locusta migratoria vitellogenin B (VgB). Constructs containing promoters and putative 5'-regulatory sequences from these genes, ligated to bacterial chloramphenicol acetyltransferase (CAT) coding DNA, were transfected into Drosophila Kc (Kc-H) and S3 cells, and transient expression of CAT was assayed. Activity was expressed both from the homologous promoter of pYP1CAT and from the heterologous locust promoter of pVgCAT at comparable levels. In S3 cells, with calcium phosphate-mediated transfer of pYP1CAT there was a twofold induction of CAT activity after the addition of 10(-6) M ecdysterone, but no hormonal stimulation was noted when the polycation polybrene was used to achieve transfection. For Kc cells, calcium phosphate was ineffective for transfection, and after transfection with polybrene neither pYP1CAT nor pVgCAT was induced by the juvenile hormone (JH) analog methoprene. It is concluded that S3 cells may be useful for investigating the molecular basis of gene regulation by ecdysteroids, but conditions suitable for the analysis of JH action have not yet been established.

Animals↗

PubMed1990

The purification of dihydrofolate reductase from Drosophila melanogaster.

Dihydrofolate reductase (DHFR) has been purified over 30,000-fold from Drosophila adults with a yield of 35%, using a combination of low pH extraction, (NH4)2SO4 precipitation, Sephadex gel filtration, Affi-Gel blue affinity chromatography, ion exchange and gel filtration FPLC. The Drosophila enzyme is a soluble, 17-22 kDa monomeric protein displaying the two pH optima characteristic of eukaryotic DHFRs. The sequence of the first 23 amino acids from the amino-terminal end of the protein shows that Drosophila DHFR is more homologous to the mosquito and vertebrate DHFRs than to the prokaryotic enzymes. However, the percent similarity between the two insect enzymes is not as close as expected when compared to the virtually identical initial sequence conservation of mammalian DHFRs.

Amino Acid Sequence↗

PubMed1990

Wolffish antifreeze protein from transgenic Drosophila.

We have expressed two antifreeze protein genes from the Atlantic wolffish, Anarhichas lupus, in Drosophila melanogaster by placing them under the divergent transcriptional control of the host yolk polypeptide (1 and 2) gene promoters. Both genes were joined to the central promoter region by fusion within the DNA encoding the signal polypeptides. The chimeric genes were introduced into flightless mutant Drosophila through P-element transformation. Transformed adult females from individual lines accumulated 1.5-5 mg/ml of antifreeze protein in their hemolymph. The protein was purified to homogeneity from hemolymph following thermal denaturation, step elution from SP-Sephadex, and reverse-phase HPLC. It was recovered in high yield and retained full biological activity even though one of the two gene fusions gave rise to a seven amino acid N-terminal extension on its antifreeze protein product.

Amino Acid Sequence↗

PubMed1990

Kinetic characterization of dihydrofolate reductase from Drosophila melanogaster.

The kinetic characteristics of a purified insect dihydrofolate reductase (DHFR) have been described. The Km values for the substrate dihydrofolate and the cofactor NADPH have been estimated by primary and secondary Hanes plots to be 0.3 and 5.2 microM, respectively. Drosophila melanogaster DHFR can use folate and NADH at acidic pH values, but at a much lower rate than the preferred substrate and cofactor. Folic acid is a partial competitive inhibitor of Drosophila DHFR (Ki = 0.4 microM) and trimethoprim is a complete competitive inhibitor (Ki = 5.4 microM). Methotrexate binds less tightly to the Drosophila enzyme than to many other DHFRs (Kd = 0.9 nM). Drosophila DHFR is inhibited by KCl and organic mercurials and is slightly activated by urea. These data indicate that Drosophila DHFR has some characteristics which are typical of vertebrate DHFRs and others which are typical of prokaryotic DHFRs. The study of this enzyme, therefore, should aid in the definition of the structural features that are responsible for the kinetic characteristics in different DHFRs.

Animals↗

PubMed1987

Flounder antifreeze protein synthesis under heat shock control in transgenic Drosophila melanogaster.

The gene coding for the most abundant antifreeze protein (AFP) in the winter flounder was placed downstream of the Drosophila melanogaster hsp70 promoter and introduced into the D. melanogaster germ line by P-element-mediated transformation. In each of six transgenic strains tested, heat shock treatment induced the expression of two major AFP gene transcripts and one minor one. All three transcripts were spliced despite the lack of an obvious D. melanogaster internal intron-splicing sequence. The variation in transcript length was caused by selection of different polyadenylation sites. Western blots showed the presence of immunoreactive AFP in hemolymph from heat-shocked transformants. The immunoreactive material had a molecular weight of 6,200, which is consistent with the loss of the signal sequence from the primary translation product and the retention of the pro sequence. Thus, all the signals for flounder pre-mRNA and preprotein processing were recognized in D. melanogaster.

Animals↗

PubMed1985

Yolk polypeptide gene expression in Minute Drosophila females.

Minutes have been considered for some time to be mutant at the sites of synthesis of some components of the protein synthetic apparatus. To study the hypothetical relationship between Minutes and suboptimal translation, a group of abundant proteins, the yolk polypeptides, was assayed in outcrossed females bearing M(3)w, M(3)hy, or M(1)n mutations. Recently emerged Minute females contained a lower amount of yolk polypeptides, in both ovarian and nonovarian tissues, than their non-Minute sisters. This low level correlated with the lower abundance of cytoplasmic RNA in Minutes compared to control females. By 1 week of age, both M(3)w and their non-Minute sibs contained the same amount of yolk polypeptides and the corresponding mRNA. The double heterozygote, ap4/+;M(3)w/+, did not differ in yolk polypeptide content from control flies. M(3)w females demonstrated reduced fecundity during the period of low yolk polypeptide content but gradually increases egg deposition as yolk polypeptide levels rose. These results suggest that the low protein levels are due to the slower maturation of M(3)w, and not to less efficient translation machinery.

Animals↗

PubMed1984

Isolation of two Drosophila melanogaster genes abundantly expressed in the ovary during vitelline membrane synthesis.

Several genomic clones were isolated from a Drosophila library screened with cDNA prepared from abundant adult female mRNA. Cytoplasmic dot hybridizations have shown that the genes in all of these clones are expressed only in posteclosion (stages 8-14) follicles. One set of overlapping clones (lambda 20, lambda 28, and lambda 30) was localized by in situ hybridization to 66D, a previously described locus for chorion genes. Restriction mapping demonstrated that these clones contained chorion genes which had been isolated previously. Another clone, lambda 7, was mapped to chromosomal region 26A. This clone carries genes that hybridized to mRNA species similar or identical in size to the known chorion genes encompassed by lambda 28. Furthermore, one of these genes shows homology to the 66D chorion locus, apparently with the s18-1 gene. R-loop and S1-nuclease mapping indicated that lambda 7 contains two genes of 700-800 base pairs in length. Dot hybridization of cytoplasmic RNA from egg chambers demonstrated that these genes are expressed predominantly during stages 9 + 10, the time of vitelline membrane synthesis. Analysis of DNA extracted from embryos and various female tissues by dot hybridization showed that lambda 7 sequences are not amplified in the mature ovary. These results suggest that the two genes carried by lambda 7 and derived from region 26A may code for protein components of the vitelline membrane. In addition it appears that some evolutionary relatedness exists between one of these genes and a member of the chorion multigene family.

Animals↗

PubMed1981

Differential characterization of two leucine aminopeptidases in Drosophila melanogaster.

Two leucine aminopeptidases from Drosophila melanogaster larvae have been partially purified. The LAP A and D enzymes have similar biochemical characteristics including molecular weights of approximately equal to 280,000 daltons. Michaelis-Menten constants of approximately equal to 0.05 mM, associations with metal cofactors, and specificities toward natural and chromogenic substrates. They differ in their pH optima and spatial distributions. If the closely linked genes that code for these enzymes have resulted from a tandem gene duplication event, it is suggested that there has been subsequent evolutionary divergence. This would provide Drosophila larvae with two related, but functionally distinct enzymes.

Animals↗

PubMed1981

The control of ecdysterone-regulated puffs in Drosophila salivary glands.

The hormone ecdysterone induces a characteristic sequence of changes in puffing activity in the salivary gland chromosomes of Drosophila melanogaster. A few puffs are induced very rapidly by the hormone and a larger number are only active after a lag period of several hours. To study the interrelationship of the activities of these "early" and "late" puffs, genotypes aneuploid for two early puffs have been constructed. In the duplication genotype the early puffs are active for less time than in the euploids while in the deficient genotype they are active for a longer period. Under appropriate assay conditions duplication of the early puffs results in a greater and more rapid response of some, but not all, late puffs to the hormone. Deletion of the early puffs results in a delayed response of the same late puffs. These data support the idea that the early puffs are autoregulated and that their products control activity at some late puff sites.

Animals↗

PubMed1975

Genetic analysis of mitomycin C-induced interchange in Drosophila melanogaster females.

A genetic analysis of an array of mitomycin-induced rearrangements in immature Drosophila oocytes is reported. Induced aberrations were recovered representing detachments of the compound-X chromosome, Y chromosome fragments, X chromosome loss and mosaicism. The spectrum of rearrangements induced by mitomycin C was very similar to that induced by X-ray treatment of immature oocytes. This work suggests that mitomycin C has two models of action. The drug is radiomimetic for it induces the types of aberrations recovered after X-irradiation. Mitomycin C also seems to have a delayed effect which is reflected in the relatively high recovery of mosaics.

Animals↗