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S M Thomas

Publications and source records attributed to S M Thomas.

At least 127 records · Page 7Linked to original sources
PubMed1990

Enhanced conditions for DNA fingerprinting with biotinylated M13 bacteriophage.

Deoxyribonucleic acid (DNA) fingerprints are Southern blots which have a pattern resembling bar codes. The pattern is created by DNA probes that bind to variable-length repeated sequences of human genomic DNA digested with restriction endonucleases. To improve DNA fingerprints obtained with biotin-labeled M13mp8 replicative form (RF) bacteriophage as the gene probe, the conditions for hybridization and the subsequent washing steps of the filter were refined. Experiments were conducted varying the electrophoresis time, blotting membranes, hybridization solution, and posthybridization washes. The simplicity, sensitivity, and reliability of this nonistopic technique make possible its application for identification of individuals within a species, for parentage testing, and for monitoring bone marrow transplantation.

Bacteriophages↗
PubMed1989

Radical initiated alpha-tocopherol depletion and lipid peroxidation in mitochondrial membranes.

The relationships between antioxidant status, lipid peroxidation and membrane protein integrity have been studied in an isolated mitochondrial membrane system. Tocopherol was shown to be present in both the outer and inner membrane of normal rat liver mitochondria; 77.3 and 22.3% of the total alpha-tocopherol was present in the outer and inner membranes, respectively. The endogenous alpha-tocopherol was depleted in a time-dependent manner by low levels of ferrous iron and by irradiation in the presence or absence of ferrous iron. This antioxidant depletion was followed by the appearance of lipid hydroperoxides. Fragmentation of monoamine oxidase, an integral outer membrane protein, was observed at irradiation doses that caused by antioxidant depletion and peroxide generation.

Animals↗
PubMed1989

A continuous-flow automated assay for iodometric estimation of hydroperoxides.

An iodometric method for the analysis of hydroperoxides has been automated to allow analysis of aqueous biological samples (containing less than 20 mg/ml protein) and lipid hydroperoxide extracts. The evolution of triiodide ions is measured spectrophotometrically at 360 nm. Dependent on the type of sample, 30-60 samples can be analyzed per hour and the system allows detection of less than 100 pmol of peroxide. The assay is linear over a range of 100 pmol to 25 nmol. The sample volume used routinely was 80 microliters.

Animals↗
PubMed1989

Mutagenic DNA repair genes on plasmids from the 'pre-antibiotic era'.

Resistance transfer factors are natural conjugative plasmids encoding antibiotic resistance. Some also encode mutagenic DNA repair genes giving resistance to DNA damage and induced mutagenesis. It has been shown that antibiotic resistance has been acquired by recent transposition events; however, we show here that mutagenic repair genes existed much earlier on these types of plasmids. Conjugative plasmids from eight incompatibility groups from the Murray collection of 'pre-antibiotic era' enterobacteria were tested for complementation of mutagenic repair-deficient Escherichia coli umuC36. Although none of these plasmids carry transposon-encoded drug resistance genes, IncI1 and IncB plasmids were identified which restored ultraviolet resistance and induced mutability to umuC36 mutants. Furthermore they increased the UV resistance and induced mutability of wild-type E. coli, Klebsiella aerogenes and Citrobacter intermedius, thus showing that they could confer a general selective advantage to a variety of hosts. Like known mutagenic repair genes, complementation by these plasmid genes required the SOS response of the host cell. Nucleotide hybridisation showed that these plasmids harboured sequences similar to the impCAB locus, the mutagenic repair operon of modern-day IncI1 plasmids. The evolution of mutagenic repair genes is discussed.

DNA Repair↗
PubMed1989

Cloning of Salmonella typhimurium DNA encoding mutagenic DNA repair.

Mutagenic DNA repair in Escherichia coli is encoded by the umuDC operon. Salmonella typhimurium DNA which has homology with E. coli umuC and is able to complement E. coli umuC122::Tn5 and umuC36 mutations has been cloned. Complementation of umuD44 mutants and hybridization with E. coli umuD also occurred, but these activities were much weaker than with umuC. Restriction enzyme mapping indicated that the composition of the cloned fragment is different from the E. coli umuDC operon. Therefore, a umu-like function of S. typhimurium has been found; the phenotype of this function is weaker than that of its E. coli counterpart, which is consistent with the weak mutagenic response of S. typhimurium to UV compared with the response in E. coli.

Cloning, Molecular↗
PubMed1989

Gene encoding the alpha core subunit of Bacillus subtilis RNA polymerase is cotranscribed with the genes for initiation factor 1 and ribosomal proteins B, S13, S11, and L17.

We describe the genetic and transcriptional organization of the promoter-distal portion of the Bacillus subtilis alpha operon. By DNA sequence analysis of the region surrounding rpoA, the gene for the alpha core subunit of RNA polymerase, we identified six open reading frames by the similarity of their products to their counterparts in the Escherichia coli transcriptional and translational apparatus. Gene order in this region, given by gene product, was IF1-B-S13-S11-alpha-L17. Gene order in E. coli is similar but not identical: SecY-B-S13-S11-S4-alpha-L17. The B. subtilis alpha region differed most strikingly from E. coli in the presence of IF1 and the absence of ribosomal protein S4, which is the translational regulator of the E. coli alpha operon. In place of the gene for S4, B. subtilis had a 177-base-pair intercistronic region containing two possible promoter sequences. However, experiments with S1 mapping of in vivo transcripts, gene disruptions in the alpha region, and a single-copy transcriptional fusion vector all suggested that these possible promoters were largely inactive during logarithmic growth, that the major promoter for the alpha operon lay upstream from the region cloned, and that the genes in the IF1 to L17 interval were cotranscribed. Thus, the transcriptional organization of the region resembles that of E. coli, wherein the alpha operon is transcribed primarily from the upstream spc promoter, but the absence of the S4 gene suggests that the translational regulation of the region may differ more fundamentally.

Amino Acid Sequence↗
PubMed1988

Two mRNAs that differ by two nontemplated nucleotides encode the amino coterminal proteins P and V of the paramyxovirus SV5.

The "P" gene of the paramyxovirus SV5 encodes two known proteins, P (Mr approximately equal to 44,000) and V (Mr approximately equal to 24,000). The complete nucleotide sequence of the "P" gene has been obtained and is found to contain two open reading frames, neither of which is large enough to encode the P protein. We have shown that the P and V proteins are translated from two mRNAs that differ by the presence of two nontemplated G residues in the P mRNA. These two additional nucleotides convert the two open reading frames to one of 392 amino acids. The P and V proteins are amino coterminal and have 164 amino acids in common. The unique C terminus of V consists of a cysteine-rich region that resembles a cysteine-rich metal binding domain. An open reading frame that contains this cysteine-rich region exists in all other paramyxovirus "P" gene sequences examined, which suggests that it may have important biological significance.

Amino Acid Sequence↗
PubMed1987

RecA-independent mutagenesis in Escherichia coli may be subject to glucose repression.

The frameshift mutagen 9-aminoacridine (9AA) causes DNA damage via a recA+-independent mechanism in Escherichia coli. In this study we have exposed E. coli cells carrying the lacZ19124 frameshift marker to 9AA in defined minimal media, washed them, and plated to score for Lac+ revertants. Our results show that 9AA-induced reversion to Lac+ occurs in the absence of any exogenous carbon source and when cells are plated on media which do not allow much, if any, cell replication prior to expression of the revertant phenotype. When glycerol (1% w/v) was added to the liquid treatment medium, the number of Lac+ E. coli revertants was similar to that obtained when no carbon source was present. By contrast the addition of glucose (1% w/v) during the mutagenesis treatment caused a significant decrease in the number of revertants. Further experiments indicate that the repressing effects of glucose may be due to a reduction in cAMP concentration, since 9AA mutagenesis was abolished in a cya strain in which no adenylate cyclase is produced. These results are consistent with (but do not prove) the notion that at least one part of the process leading to 9AA mutagenesis is subject to catabolite repression.

Adenylyl Cyclases↗
PubMed1987

Frameshift mutagenesis by chloroquine in Escherichia coli and Salmonella typhimurium.

Chloroquine can be detected as a direct-acting mutagen in plate-incorporation assays using the excision-deficient Salmonella typhimurium strain TA97, but very much more effectively using the repair-proficient Escherichia coli strain DG1669 which carries the lacZ19124 marker. When tested at concentrations of 200-1000 micrograms/plate with strain DG1669, the mutagenicity of chloroquine is enhanced by the addition of Aroclor-induced rat-liver S9. Further experiments indicated that chloroquine-induced reversion frequencies were essentially identical in wild-type, recA, umuC and uvrC derivatives of DG1669, as well as in strains carrying the mutation enhancing plasmid pKM101, over a wide range of doses (0-1200 micrograms/plate). These results suggest that neither excision repair nor SOS-type repair are important in chloroquine-induced frameshift mutagenesis.

Animals↗
PubMed1987

Gene encoding the 37,000-dalton minor sigma factor of Bacillus subtilis RNA polymerase: isolation, nucleotide sequence, chromosomal locus, and cryptic function.

We began an analysis of rpoF, the gene encoding the cryptic, 37,000-dalton minor sigma factor (sigma-37) of Bacillus subtilis RNA polymerase. Using antibody raised against sigma-37 holoenzyme to probe a lambda gt11 expression vector library, we isolated a 901-base-pair EcoRI fragment that expressed the COOH-terminal half of sigma-37 fused to lacZ. We used this fragment as a hybridization probe to isolate the entire rpoF gene and additional flanking sequences. Identity of the cloned gene was confirmed by the size and immunological reaction of its product expressed in Escherichia coli and, after DNA sequencing, by the homology of its predicted product (264 residues; 30,143 daltons) with other sigma factors. The DNA sequence also suggested that rpoF may lie in a gene cluster. Upstream of rpoF was an open reading frame that would encode a protein of 17,992 daltons; this frame overlapped the rpoF-coding sequence by 41 base pairs. Immediately following rpoF was a reading frame that would encode a protein of at least 20,000 daltons; expression of this region may be translationally coupled to that of rpoF. By plasmid integration and PBS1 transduction, we found the chromosomal locus of rpoF linked to ddl and dal at 40 degrees on the B. subtilis map and near no known lesions affecting growth regulation or development. Further, an rpoF null mutation resulting from gene disruption had no effect on cell growth or sporulation in rich medium, suggesting that sigma-37 may partly control a regulon not directly involved in the sporulation process.

Amino Acid Sequence↗
PubMed1986

Free-radical-mediated fragmentation of monoamine oxidase in the mitochondrial membrane. Roles for lipid radicals.

A flux of hydroxyl radicals generated by gamma-irradiation can fragment monoamine oxidase in the membrane of submitochondrial particles. This fragmentation can be inhibited by mannitol and in addition is more extensive in monoamine oxidase preparations that have been depleted of lipid. This latter observation is consistent with the higher yields of fragmentation induced by hydroxyl radicals in soluble proteins in the absence of added lipids. In the absence of oxygen, gamma-irradiation of submitochondrial particles leads to cross-linking reactions. A flux of hydroperoxyl radicals also causes fragmentation, whereas one of superoxide is virtually inactive in this respect. The irradiation of submitochondrial particles leads in addition to the accumulation of products of lipid peroxidation. When these irradiated preparations are exposed to ferrous or cupric salts a further fragmentation of monoamine oxidase ensues, especially at acid pH. These transition-metal-catalysed reactions do not occur with irradiated preparations depleted of lipid, and the post-irradiation protein modifications are concomitant with further lipid peroxidation. The data indicate roles for lipid radicals in both fragmentation and cross-linking reactions of proteins in biological membranes. These reactions may have an important bearing on control of protein activity and of protein turnover in membranes.

Animals↗
PubMed1986

RecA-independent mutagenesis in Escherichia coli: effects of umuC and mucB mutations.

We have studied the effects of a umuC mutation on reversion of the lacZ19124 and lacZ19136 frameshift markers of Escherichia coli. Introduction of a umuC::Tn5 mutation into a strain carrying the lacZ19136 marker resulted in enhanced reversion by 9-aminoacridine and the acridine half-mustard ICR191, whereas reversion of the lacZ19124 marker was decreased (but not abolished) in a umuC strain. The reversion frequency of the lacZ19136 marker was decreased by the presence of plasmid pKM101, and further decreased by a derivative of pKM101 in which the mucB gene was inactivated by a Tn5 insertion. The reversion frequency of the lacZ19124 marker was relatively unchanged by either plasmid. Since both 9-aminoacridine and ICR191 mutagenesis of these strains is independent of the recA+ and lexA+ gene products, these results may suggest a broader role for the UmuC protein in regulating induced mutation frequencies than has previously been suspected. The contrasting effects of the umuC and mucB mutations on reversion of the lacZ19136 marker may suggest a copy number effect, or perhaps more likely that there are inherent (functional) differences between the UmuC and MucB proteins.

Aminacrine↗
PubMed1986

Oxidation induced proteolysis and its possible restriction by some secondary protein modifications.

The hydroxyl radical (.OH) in the presence of molecular oxygen (O2) fragmented BSA and formed distinct peptides as did hydrogen peroxide catalysed by copper. .OH in the absence of O2 caused protein crosslinking. The selective fragmentation was ascribed to cleavage at proline residues, a mechanism consistent with an increase in the proline/glutamate ratio. Other radicals, such as peroxyl and superoxide radicals caused no change in molecular weight of the protein. Peroxyl radicals and .OH were, however, able to cause some change in conformation of the protein which increased its susceptibility to hydrolysis and oxidation might thus be implicated in protein turnover. Mitochondrial monoamine oxidase (MAO) was also susceptible to free radical cleavage but lipids were able to compete with the protein for radical species. Peroxides also seemed to be involved in the cleavage of MAO and this was evidenced by a "post irradiation effect" and the ability of the hydroperoxy radical to cause degradation; in contrast to the case with BSA. In whole cells, depletion of the antioxidant GSH and inhibition of catalase increased endogenous protein degradation. Lens protein autolysis was increased under conditions where thiol autoxidation was permitted but glycosylation of protein caused a decrease in breakdown by extrinsic hydrolases. These results indicate that free radicals, possibly also associated with lipids may play a role in the regulation of protein turnover but that secondary modifications may promote the accumulation of damaged protein.

Animals↗
PubMed1985

Frameshift mutagenesis by 9-aminoacridine and ICR191 in Escherichia coli: effects of uvrB, recA and lexA mutations and of plasmid pKM101.

We have studied the effects of different repair capacities on reversion of two Escherichia coli strains (lacZ19124 and lacZ19136) by 9-aminoacridine (9AA) and the acridine half-mustard ICR191. Introduction of a uvrB mutation into these strains led to enhanced ICR191-induced reversion of lacZ19136 and reduced ICR191-induced reversion of lacZ19124. 9AA-induced reversion of lacZ19124 was essentially unchanged while reversion of lacZ19136 was reduced. Plasmid pKM101 reduced reversion of the two markers by each of the mutagens, except in the case of ICR191-induced reversion of the lacZ19124 marker where mutagenesis was slightly enhanced. Mutations in the recA and lexA genes had minimal effects on ICR191- and on 9AA-induced reversion of the lacZ markers; although 9AA-induced reversion of the lacZ19124 marker was somewhat reduced, most of the other results indicated that mutation yields were if anything higher in the recA or lexA backgrounds. Mutagenesis by 9AA and ICR191 would therefore appear to occur independently of the inducible error-prone repair process commonly referred to as SOS repair.

Aminacrine↗