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Biomedical subjects

S B Primrose

Publications and source records attributed to S B Primrose.

At least 19 recordsLinked to original sources
PubMed1986

The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K12. Comparison of the primary structures of the aspartate aminotransferase and aromatic aminotransferase of E. coli with those of the pig aspartate aminotransferase isoenzymes.

In this paper we describe the cloning and sequence analysis of the tyrB and aspC genes from Escherichia coli K12, which encode the aromatic aminotransferase and aspartate aminotransferase respectively. The tyrB gene was isolated from a cosmid carrying the nearby dnaB gene, identified by its ability to complement a dnaB lesion. Deletion and linker insertion analysis located the tyrB gene to a 1.7-kilobase NruI-HindIII-digest fragment. Sequence analysis revealed a gene encoding a 43 000 Da polypeptide. The gene starts with a GTG codon and is closely followed by a structure resembling a rho independent terminator. The aspC gene was cloned by screening gene banks, prepared from a prototrophic E. coli K12 strain, for plasmids able to complement the aspC tyrB lesions in the aminotransferase-deficient strain HW225. Sub-cloning and deletion analysis located the aspC gene on a 1.8-kilobase HincII-StuI-digest fragment. Sequence analysis revealed the presence of a gene encoding a 43 000 Da protein, the sequence of which is identical with that previously obtained for the aspartate aminotransferase from E. coli B. Considerable overproduction of the two enzymes was demonstrated. We compared the deduced protein sequences with those of the pig mitochondrial and cytoplasmic aspartate aminotransferases. From the extensive homology observed we are able to propose that the two E. coli enzymes possess subunit structures, subunit interactions and coenzyme-binding and substrate-binding sites that are very similar both to each other and to those of the mammalian enzymes and therefore must also have very similar catalytic mechanisms. Comparison of the aspC and tyrB gene sequences reveals that they appear to have diverged as much as is possible within the constraints of functionality and codon usage.

Animals↗
PubMed1983

Properties of thyP3-containing plasmids in Bacillus subtilis.

A series of hybrid plasmid molecules which contain both antibiotic resistance genes and the thyP3 gene of the Bacillus subtilis bacteriophage phi 3T have been constructed. Monomeric or restriction enzyme-cleaved plasmid DNA is capable of transforming competent cells to thymine prototrophy only. However, multimeric plasmid DNA can transform competent cells to both thymine prototrophy and antibiotic resistance. Cells which have been transformed to thymine prototrophy only do not contain extrachromosomal plasmid DNA but instead contain the thyP3 gene integrated into the host chromosome; the antibiotic resistance genes, however, do not become integrated into the chromosome. Although the thyP3-containing plasmids have extensive DNA sequence homology with the B. subtilis chromosome, they can be stably maintained, extrachromosomally, even in recE4+ hosts, in complex broth, and in the absence of antibiotics.

Acetyltransferases↗
PubMed1982

Recombination between short direct repeats in a recA host.

Spontaneous deletion derivatives of plasmid pHV14, a recombinant of pBR322, have been isolated in the recA strain HB101. About 2.5 kilobases (Kb) of DNA has been lost in each of the four plasmids described including, in two cases the region involved in control of copy number and the initiation of replication. Sequence analysis of each deletion junction has revealed that in 3 out of 4 cases the deletion event occurred by recombination between short direct repeats of as little as 7 base pairs (bp).

Adenosine Triphosphatases↗
PubMed1982

Isolation of a bisegmented double-stranded RNA virus from Thirlmere reservoir.

A novel bisegmented double-stranded RNA virus has been isolated from water processed from Thirlmere reservoir. The virus is icosahedral, 58 nm in diam., has a buoyant density of 1.32 g/ml in CsCl, has an S value of 400 and a RNA/protein ratio of 0.087. The two linear segments of RNA have approx. mol. wt. of 2.26 X 10(6) and 2.09 X 10(6). The virus contains six polypeptides. The virus was isolated in Drosophila melanogaster cells and fails to replicate in other insect, amphibian, avian, piscine, mammalian and plant cells tested. The virus is biochemically different from infectious pancreatic necrosis virus (IPNV) and Drosophila X virus (DXV). The virus is also serologically unrelated to IPNV (strain Sp) and another invertebrate pathogenic virus, Tellina virus 1. The virus shares common antigens with DXV but is not completely identical.

Animals↗
PubMed1982

Methods for studying aquatic bacteriophage ecology.

Methods are described for enumerating the different kinds of bacteriophage present in virus concentrates prepared from 120 liters of water. Although developed specifically for use with coliphages, they should be applicable to viruses infecting other hosts. These methods involve mixed indicators, equilibrium buoyant-density centrifugation, use of enzymes or inhibitors or both, and specific hybridization probes, either alone or in combination. With these methods, it was possible to specifically enumerate filamentous and isometric male-specific (F-specific) phages, the different classes of P-group plasmid-specific phages, phiX174-like phages, and lambda-like phages. Some applications of these methods, including measurement of virus inactivation in the environment and the extent of fecal pollution, are discussed.

Journal Article↗
PubMed1981

A portable device for the rapid concentration of viruses from large volumes of natural freshwater.

A portable device for the rapid concentration of viruses from natural freshwaters described and its performance in field use is evaluated. The system handled up to 500 litres of water in less than 90 min at a cost of only 2 pounds per sample. Where the samples contained sufficient bacteriophages for detection by direct plating the apparent phage recoveries were greater than 75%. Plant and animal viruses were also concentrated from waters with this system.

Bacteriophages↗
PubMed1981

Effect of growth rate and nutrient limitation on the transformability of Escherichia coli with plasmid deoxyribonucleic acid.

The observed transformation frequency by plasmid deoxyribonucleic acid of Escherichia coli grown in continuous culture was found to depend on both the steady-state growth rate and the type of nutrient used to limit growth. With carbon, nitrogen, or phosphorus limitation, the faster the growth rate, the higher the transformation frequency. The increase in transformation frequency associated with higher rates was shown to be due to more transformable cells in the population rather than an increased efficiency of deoxyribonucleic acid uptake. Growth rate had relatively little effect on the transformability of cells from sulfate- and Mg2+-limited chemostats, indicating that some factor other than the growth rate must influence the frequency of transformation. Regardless of the nutrient limitation or the growth rate, no transformants were obtained in the absence of CaCl2.

Calcium Chloride↗
PubMed1980

Maintenance of some ColE1-type plasmids in chemostat culture.

When cells carrying the plasmids RP1, pDS4101 (a ColK derivative) or pDS1109 (a ColE1 derivative) were maintained in chemostat culture in the absence of antibiotic selection, plasmid-free segregants were not detected after 120 generations of nutrient-limited growth. By contrast, plasmid-free segregants of pMB9- and pBR322-containing cells arose after approximately 30 generations, irrespective of the host genetic background. However, even though pDS1109 was maintained its copy-number fell five-fold during 80 generations of limited growth. It is suggested that loss of pBR322 occurs following a similar copy-number decrease which results in defective segregation of the plasmid to daughter host cells. This defective segregation was not complemented in trans by either RP1 or pDS4101.

Cell Division↗
PubMed1980

Rapid concentration of bacteriophages from large volumes of freshwater: evaluation of positively charged, microporous filters.

Microporous, positively charged Zeta Plus 60 S filters were evaluated for bacteriophage recovery from large volumes of natural water. A variety of phages adsorbed efficiently to the filter medium at water pH levels below neutrality, but adsorption was reduced above pH 7. Adsorbed phages were easily eluted with an arginine/1% beef extract solution, pH 9.0. A concentration procedure suitable for the field isolation of bacteriophages from large volumes of river water was devised. The procedure involves, (1) prefiltration through 10" cartridge depth filters, (2) adjustment of water pH to pH 5.5--6.0, (3) adsorption of the phages on to Zeta Plus 60 S filters, (4) elution of bound phage in a small volume of eluent, (5) secondary concentration by ultrafiltration of the resulting eluates. Using this procedure, bacteriophages in 65 1 of river water was concentrated to 35 ml with recoveries in the range 50--60%.

Adsorption↗
PubMed1980

Polyol metabolism by Rhizobium trifolii.

In Rhizobium trifolii 7000, the polyols myo-inositol, xylitol, ribitol, D-arabitol, D-mannitol, D-sorbital, and dulcitol are metabolized by inducible nicotinamide adenine dinucleotide-dependent polyol dehydrogenases. Five different polyol dehydrogenases were recognized: inositol dehydrogenase, specific for inositil; ribitol dehydrogenase, specific for ribitol; D-arabitol dehydrogenase, which oxidized D-arabitol, D-mannitol, and D-sorbitol; xylitol dehydrogenase, which oxidized xylitol and D-sorbitol; and dulcitol dehydrogenase, which oxidized dulcitol, ribitol, xylitol, and sorbitol. Apart from inositil and xylitol, all of the polyols induced more than one polyol dehydrogenase and polyol transport system, but the heterologous polyol dehydrogenases and polyol transport systems were not coordinately induced by a particular polyol. With the exception of xylitol, all of the polyols tested served as growth substrates. A mutant of trifolii 7000, which was constitutive for dulcitol dehydrogenase, could also grow on xylitol.

Aldehyde Reductase↗
PubMed1979

Biosynthesis of ethylene from methionine. Isolation of the putative intermediate 4-methylthio-2-oxobutanoate from culture fluids of bacteria and fungi.

Methods are described for identifying the 2,4-dinitrophenylhydrazones of 4-methylthio-2-oxobutanoate by means of t.l.c., n.m.r. and mass spectroscopy. By using these methods 4-methylthio-2-oxobutanoate, a putative intermediate in the biosynthesis of ethylene from methionine, has been identified in culture fluids of Aeromonas hydrophila B12E and a coryneform bacterium D7F grown in the presence of methionine. Relative to 4-methylthio-2-oxobutanoate, the yield of 3-(methylthio)propanal (methional) from the same cultures was less than 1%. Because 4-[2H]methylthio-2-oxobutanoate was obtained from cultures grown on [Me-2H]methionine, the 4-methylthio-2-oxobutanoate must be derived from methionine. By means of t.l.c. alone, 4-methylthio-2-oxobutanoate was identified in the culture fluids of a range of bacteria, the yeast Saccharomyces cerevisiae and the fungus Penicillium digitatum. A photochemical assay developed for 4-methylthio-2-oxobutanoate shows it to be a product of the metabolism of methionine by Escherichia, Pseudomonas, Bacillus, Acinetobacter, Aeromonas, Rhizobium and Corynebacterium species.

Bacteria↗
PubMed1979

Effect of glucose on polyol metabolism by Rhizobium trifolii.

The effect of glucose on polyol metabolism by Rhizobium trifolii was studied. Phenomena similar to catabolite repression and catabolite inhibition were observed. The catabolism of glucose to at least glucose 6-phosphate was required for the effects to be exerted.

Biological Transport↗
PubMed1978

Rapid purification of plasmid DNAs by hydroxyapatite chromatography.

A method is described for the rapid preparation of plasmid DNAs of molecular weight up to 14 X 10(6). This method involves the chromatography, at room temperature, of bacterial cleared lysates on hydroxyapatite in the presence of high concentrations of phosphate and urea. All detectable protein and RNA contamination of plasmid DNA is removed by this procedure and the conformation of the plasmid DNA is unaffected. Less than 0.5% chromosomal DNA is present in the purified preparation and even this can be removed if necessary by a simple extention of the procedure to include a heat-denaturation step. The method is extremely rapid and amenable to large-scale plasmid preparation; 5 mg ColE1 DNA have been purified within 40 min. The yield of plasmid DNA is similar to that obtained with the conventional dye-centrifugation technique, however the purity is greater.

Chromatography↗