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L C Waters

Publications and source records attributed to L C Waters.

At least 19 recordsLinked to original sources
PubMed2000

Integrated system for rapid PCR-based DNA analysis in microfluidic devices.

An integrated system for rapid PCR-based analysis on a microchip has been demonstrated. The system couples a compact thermal cycling assembly based on dual Peltier thermoelectric elements with a microchip gel electrophoresis platform. This configuration allows fast (approximately 1 min/ cycle) and efficient DNA amplification on-chip followed by electrophoretic sizing and detection on the same chip. An on-chip DNA concentration technique has been incorporated into the system to further reduce analysis time by decreasing the number of thermal cycles required. The concentration injection scheme enables detection of PCR products after performing as few as 10 thermal cycles, with a total analysis time of less than 20 min. The starting template copy number was less than 15 per injection volume.

DNA↗
PubMed2000

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.

Alleles↗
PubMed1999

Microfabricated porous membrane structure for sample concentration and electrophoretic analysis.

A microfabricated injection valve incorporating a porous membrane structure is reported that enables electrokinetic concentration of DNA samples using homogeneous buffer conditions followed by injection into a channel for electrophoretic analysis. The porous membrane was incorporated in the microchannel manifold by having two channels separated from each other by 3-12 microns and connected by a thin porous silicate layer. This design allows the passage of current to establish an electrical connection between the separated channels but prevents large molecules, e.g., DNA, from traversing the membrane. Concentrated DNA can be injected into the separation channel and electrophoretically analyzed. Experiments exhibit a nonlinear increase in concentration with time, and DNA fragments can be concentrated up to 2 orders of magnitude as shown by comparison of peak intensities for analysis performed with and without concentration.

DNA↗
PubMed1999

The sources, fate, and toxicity of chemical warfare agent degradation products.

We include in this review an assessment of the formation, environmental fate, and mammalian and ecotoxicity of CW agent degradation products relevant to environmental and occupational health. These parent CW agents include several vesicants: sulfur mustards [undistilled sulfur mustard (H), sulfur mustard (HD), and an HD/agent T mixture (HT)]; nitrogen mustards [ethylbis(2-chloroethyl)amine (HN1), methylbis(2-chloroethyl)amine (HN2), tris(2-chloroethyl)amine (HN3)], and Lewisite; four nerve agents (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX), tabun (GA), sarin (GB), and soman (GD)); and the blood agent cyanogen chloride. The degradation processes considered here include hydrolysis, microbial degradation, oxidation, and photolysis. We also briefly address decontamination but not combustion processes. Because CW agents are generally not considered very persistent, certain degradation products of significant persistence, even those that are not particularly toxic, may indicate previous CW agent presence or that degradation has occurred. Of those products for which there are data on both environmental fate and toxicity, only a few are both environmentally persistent and highly toxic. Major degradation products estimated to be of significant persistence (weeks to years) include thiodiglycol for HD; Lewisite oxide for Lewisite; and ethyl methyl phosphonic acid, methyl phosphonic acid, and possibly S-(2-diisopropylaminoethyl) methylphosphonothioic acid (EA 2192) for VX. Methyl phosphonic acid is also the ultimate hydrolysis product of both GB and GD. The GB product, isopropyl methylphosphonic acid, and a closely related contaminant of GB, diisopropyl methylphosphonate, are also persistent. Of all of these compounds, only Lewisite oxide and EA 2192 possess high mammalian toxicity. Unlike other CW agents, sulfur mustard agents (e.g., HD) are somewhat persistent; therefore, sites or conditions involving potential HD contamination should include an evaluation of both the agent and thiodiglycol.

Animals↗
PubMed1998

Multiple sample PCR amplification and electrophoretic analysis on a microchip.

Polymerase chain reactions (PCRs) were carried out on as many as four DNA samples at a time on a microchip device. The PCR products were then analyzed, either individually or together on the same device, by microchip gel electrophoresis. A standard PCR protocol was used to amplify 199- and 500-base pair (bp) regions of bacteriophage lambda DNA and 346- and 410-bp regions of E. coli genomic and plasmid DNAs, respectively. Thermal lysis of the bacteria was integrated into the PCR cycle. A product sizing medium, poly(dimethylacrylamide), and an intercalating dye for fluorescence detection were used in the electrophoretic analysis of the products. PCR product sizes were determined by coelectrophoresis with marker DNA.

DNA, Bacterial↗
PubMed1998

Constitutive and barbital-induced expression of the Cyp6a2 allele of a high producer strain of CYP6A2 in the genetic background of a low producer strain.

The levels of one or more cytochrome P450 (CYP) enzymes and the respective mRNAs are found to be higher in insecticide-resistant insects than in susceptible insects. To understand better how insects regulate the levels of CYPs, we examined the expression of the Cyp6a2 gene in various strains of Drosophila melanogaster. We also took a transgenic approach to understand the molecular mechanisms that are involved in strain variation of Cyp6a2 expression. RNA blot analysis showed that the constitutive expression of Cyp6a2 varies from strain to strain; the level of CYP6A2 mRNA is barely detectable in the underproducer ry506 strain, whereas it is very high in the overproducer 91-R and MHIII-D23 strains. The long terminal repeat (LTR) of mobile element 17.6 that is found in the 3' untranslated region (UTR) of the Cyp6a2 gene of some strains does not appear to have any role on the steady-state CYP6A2 mRNA level. We also found that the Cyp6a2 gene is inducible by barbital in 91-R, ry506 as well as 91-C, which carries an LTR insertion. To examine the genetic background of the underproducer ry506 strain with respect to Cyp6a2 expression, we transformed the ry506 strain with the Cyp6a2 allele of the overproducer 91-R strain (Cyp6a2-91 R) and measured the constitutive and barbital-induced expression of the Cyp6a2-91 R transgene in the transformed flies. The Cyp6a2-91 R transgene carrying 129 bp of DNA upstream of the ATG codon did not show any constitutive or barbital-induced expression in the ry506 host genome. However, transgenes with 1331 and 985 bp upstream DNA showed similar levels of constitutive expression that were higher than that of the endogenous Cyp6a2 gene of the ry506 host strain, but lower than the expression of the same gene in the 91-R strain. Both these transgenes, with 1331 and 985 bp upstream DNA, also showed induction with 0.1 M barbital. DNA sequence analysis revealed that in both 91-R and ry506, the upstream DNA between +1 and -985 bp contains a distal and a proximal group of three potential barbie boxes, i.e. cis-elements that are thought to be involved in barbiturate-mediated induction of CYP genes. Except for four bases located near the distal cluster of barbie boxes and two other bases, the base sequence of the upstream DNA is identical in ry506 and 91-R strains. These results suggest that the underproducer ry506 strain has the trans-regulatory factors to support constitutive and induced expression of the Cyp6a2-91 R allele carrying DNA between -129 and -1331 bp regions. Possible reasons for low constitutive expression of the endogenous Cyp6a2 gene and moderate level of expression of the Cyp6a2-91 R allele in the ry506 genetic background are discussed.

Alleles↗
PubMed1998

Degenerate oligonucleotide primed-polymerase chain reaction and capillary electrophoretic analysis of human DNA on microchip-based devices.

Random amplification of the human genome using the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) was performed in a silicon-glass chip. An aliquot of the DOP-PCR amplified genomic DNA was then introduced into another silicon-glass chip for a locus-specific, multiplex PCR of the dystrophin gene exons in order to detect deletions causing Duchenne/Becker muscular dystrophy. Amplicons were analyzed by both conventional capillary electrophoresis and microchip electrophoresis and results were compared to those obtained using standard non-chip-based PCR assays. Results from microchip electrophoresis were consistent with those from conventional capillary electrophoresis. Whole genome amplification products obtained by DOP-PCR proved to be a suitable template for multiplex PCR as long as amplicon size was < 250 bp. Successful detection and resolution of all PCR products from the multiplex PCR clearly shows the feasibility of performing complex PCR assays using microfabricated devices.

DNA↗
PubMed1998

Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing.

The steps of cell lysis, multiplex PCR amplification, and electrophoretic analysis are executed sequentially on a monolithic microchip device. The entire microchip is thermally cycled to lyse cells and to amplify DNA, and the products are then analyzed using a sieving medium for size separation and an intercalating dye for fluorescence detection. Using a standard PCR protocol, a 500-base pair (bp) region of bacteriophage lambda DNA and 154-, 264-, 346-, 410-, and 550-bp regions of E. coli genomic and plasmid DNAs are amplified. The electrophoretic analysis of the products is executed in <3 min following amplification using hydroxyethyl cellulose or poly(dimethylacrylamide) sieving gels. Product sizing is demonstrated by proportioning the amplified product with a DNA sizing ladder.

DNA, Bacterial↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1992

Possible involvement of the long terminal repeat of transposable element 17.6 in regulating expression of an insecticide resistance-associated P450 gene in Drosophila.

P450-A and P450-B are electrophoretically defined subsets of cytochrome P450 enzymes in Drosophila melanogaster. P450-A is present among all strains tested, whereas expression of P450-B is associated with resistance to insecticides. Monoclonal antibodies were used to obtain cDNA clones for an enzyme from each P450 subset (i.e., P450-A1 and P450-B1). The P450-B1 cDNA was sequenced and shown to code for a P450 of 507 amino acids. Its gene has been named CYP6A2. Comparative molecular analyses of a pair of susceptible, 91-C, and resistant, 91-R, Drosophila strains were made. There was 20-30 times more P450-B1 mRNA in 91-R than in 91-C, and the small amount of P450-B1 mRNA in 91-C was significantly larger in size than that in 91-R. The P450-B1 gene in 91-R was structurally different from that in 91-C but was not amplified. The P450-B1 gene in 91-C contained a solitary long terminal repeat of transposable element 17.6 in its 3' untranslated region. It was absent in the P450-B1 gene of 91-R. On the basis of features of the long terminal repeat and its location in the gene of the susceptible fly, we propose that a posttranscriptional mechanism involving mRNA stability could be involved in regulating P450-B1 gene expression.

Amino Acid Sequence↗
PubMed1991

Mutations induced by ionizing radiation in a plasmid replicated in human cells. I. Similar, nonrandom distribution of mutations in unirradiated and X-irradiated DNA.

The Escherichia coli supF gene encoding the suppressor tyrosine tRNA in a human shuttle plasmid, pZ189, was used as a target for molecular analysis of X-ray-induced mutations in human lymphoblastoid cells. Following replication of the in vitro-irradiated plasmid in human cells, the mutant supF-containing molecules were cloned by phenotypic screening in E. coli and the nature of the mutations was determined by direct sequencing of the tRNA gene. At 160 Gy the mutant frequency was 13 times (0.39%) that observed in unirradiated controls (0.031%). When control plasmid was replicated directly in E. coli, the mutant frequency was 16 times less than that of the plasmid passaged through the human cells. The distribution of mutations was highly nonrandom and remarkably similar in both irradiated and control DNAs. The majority of the mutations were transitions involving G.C pairs and occurred selectively at most 5'-TC (3'-AG) sequences. These mutations at C's were preferentially distributed in the nontranscribed strand. We propose that mutations in the control plasmid result from oxidative damages that occur during and/or after its incorporation into human cells and that these damages are similar to those induced by ionizing radiation. The hot spots for mutations suggest that the proximate nucleotide sequence and the overall conformation of the target DNA are important in the production and/or processing of these damages during repair and replication.

Base Composition↗
PubMed1991

Mutations induced by ionizing radiation in a plasmid replicated in human cells. II. Sequence analysis of alpha-particle-induced point mutations.

The human shuttle plasmid pZ189, containing the Escherichia coli supF gene as the mutational target, was irradiated in vitro with 210Po alpha particles and transfected into human lymphoblastoid cells. Plasmids which were replicated in human cells were recovered and those containing mutant supF genes were isolated by phenotypic screening in E. coli. The mutations were characterized by sequencing the tRNA gene. The mutant frequency increased linearly with the alpha-particle dose and, at 259 Gy, it was 16 times (0.29%) that observed in unirradiated controls (0.018%). The distribution of alpha-particle-induced point mutations was highly nonrandom and similar to that observed in the unirradiated or X-irradiated plasmid DNAs. The majority of the mutations were G.C----A.T transitions and occurred selectively at most 5'-TC (3'-AG) and 5'-CC (3'-GG) sequences. For the unirradiated control DNA, these mutations at C's (G's) were preferentially located in the nontranscribed strand, similar to the observation previously made for mutations in X-irradiated DNA. Such a strand bias was not observed for mutations in the alpha-particle-irradiated DNA. The data suggest that, although similar types of point mutations are induced in unirradiated, X-irradiated, and alpha-particle-irradiated DNAs, the mechanisms of their induction and the exact nature of the lesions involved may be quite different.

Alpha Particles↗
PubMed1990

Isolation of insecticide resistance-related forms of cytochrome P-450 from Drosophila melanogaster.

Significant purification of the ubiquitous cytochrome P-450-A and the strain-specific P-450-B from Drosophila melanogaster has been achieved by sequential chromatography on octylamino-agarose, DEAE-cellulose and hydroxyapatite. Preparations of P-450-A (specific contents of 7-9 nmol/mg) were homogeneous as determined by SDS/polyacrylamide-gel electrophoresis (PAGE) analysis. Preparations enriched for P-450-B (specific contents of 4-7 nmol/mg) contained significant amounts of P-450-A but were essentially free of other proteins as judged by SDS/PAGE. Partial reconstitution of 7-ethoxycoumarin de-ethylase activity was achieved using rabbit NADPH: cytochrome P450 reductase and purified preparations containing P450-B.

Animals↗
PubMed1990

N-methyl-N-nitrosourea-induced mutations in a shuttle plasmid replicated in human cells.

The supF gene of the recombinant shuttle plasmid pZ190 (modified pZ189) was used as a target to study the nature of mutations induced by N-methyl-N-nitrosourea (MNU) in human cells. Treatment of the intact plasmid with MNU followed by its replication in human lymphoblastoid cells led to extensive inactivation and no detectable mutations of the plasmid. However, exposure of the supF DNA fragment alone, followed by its ligation into the vector, caused a ten-fold increase in mutant frequency when replicated in O6-methylguanine-DNA methyltransferase-deficient cells (from 0.54 x 10(-3) to 5.8 x 10(-3)) and an 80-fold increase when replicated in cells containing normal levels of the enzyme (from 0.047 x 10(-3) to 3.8 x 10(-3)). About 45% of the mutant plasmid molecules recovered from human cells contained deletions and insertions. Sixty to 70% of the mutant molecules of wild-type size contained a single-base substitution. Most of these changes were of the G.C----A.T type, consistent with the hypothesis that O6-methylguanine is the primary mutagenic adduct induced by MNU. However, the distribution of mutation sites was highly nonrandom; more than half of all mutations were localized at the G.C position 123, and the rest were distributed in about a dozen sites. The high yield of mutations induced in the supF DNA in a host cell whose capacity for the removal of O6-methylguanine far exceeded the amount present in the supF suggests that the repair of damages in extrachromosomal DNA may be inefficient. This is supported by the observation that the yield of mutations in supF transfected into lymphoblastoid cells devoid of repair activity for O6-methylguanine was comparable to that observed with repair-proficient host cells. The present data, together with results of mutations induced in pZ189 by other agents, strongly suggest that one major determinant of mutational hot spots is the structure of the target DNA itself.

Bacterial Proteins↗
PubMed1989

Biotesting of wastewater: a comparative study using the Salmonella and CHO assay systems.

Means to assess the toxicity of wastewaters are essential to implementing the Federal Clean Water Act. Health risk assessment based on single chemicals is limited by the number of chemicals that can be identified and to those chemicals for which toxicity data are available. Long-term whole animal tests on large numbers of wastewater samples are not practical. In this study, two short-term tests, the Salmonella mutagenicity assay and the Chinese hamster ovary (CHO) cell assay for mutagenicity and cytotoxicity, were evaluated as potentially useful biomonitors of wastewaters. Standard assay protocols were modified to allow testing of up to 2.5 and 3.4 ml of unconcentrated water in the bacterial and mammalian cell tests, respectively. Cytotoxicity and mutagenicity were detected in some unconcentrated wastewater samples using these modifications. Data on eight wastewater samples, representing five different sites, indicated that the Salmonella test is the more sensitive indicator of mutagenic activity in those samples, whereas the CHO test is a sensitive indicator of the presence of cytotoxic components. Wastewater concentrates, prepared by adsorption onto XAD-2 and "blue cotton," were compared in the two bioassays. In a single concentrate, the two short-term tests detected distinctly different mutagens. Advantages of using the CHO-AS52 cell line instead of the CHO-K1BH4 line for detecting wastewater mutagens were indicated. This study illustrates the complementary use of multiple bioassays and concentration methods to detect and characterize toxic components in wastewater.

Animals↗
PubMed1984

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.

Animals↗