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Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation in the human genome, accounting for more than 90% of all differences between individuals. Many complex phenotypes in humans have a significant genetic component and most of the variability is therefore likely to stem from differences in patterns of SNPs. Association studies involving the large-scale analysis of SNPs can help to identify genes affecting many human phenotype variations, including complex diseases and drug responses. SNPs therefore play a major role in all stages of the drug development process, from target identification through to clinical trials. SNPs are also the basis of pharmacogenomics, the tailoring of medicines to suit an individual's genome. Given the potential impact of SNPs on healthcare, the biotechnology industry has focussed urgently on the development of high-throughput methods for SNP genotyping. All genotyping methods are a mix and match of different allele discrimination and signal detection technologies and as such may represent the intellectual property of several individuals or organizations. In this review, we explore the patent issues surrounding SNP genotyping and how this is influencing large scale, commercially valuable projects involving SNPs.

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.

Escherichia coli strain SPA O converts methionine to ethylene by an inducible enzyme system. L-Cysteine, L-homocysteine, methionine derivatives and the sulphur-containing analogues of L-methionine also act as precursors of ethylene. Ethylene is produced by cell suspensions only in the presence of air; cell-free preparations can produce ethylene aerobically and anaerobically, but the extent to which they do so depends on the mode of culture growth. Light stimulates ethylene production by cell suspensions and its presence is essential for production by cell-free preparations. The kinetics of ethylene biogenesis and its pH and temperature optima suggest that ethylene is a secondary metabolite.

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).

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%.

The influence of the DNA synthesis inhibitor, nalidixic acid, on the properties of synchronous cultures of selected Rhodopseudomonas palustris swarmer cells was examined. There was little alteration in the changes in morphology, extinction, volume distribution and leucine incorporation up to bud development, and photosynthetic membrane lamellae were still synthesized de novo in the bud. However, there was no subsequent division, or flagellum or holdfast synthesis. Instead cells elongated by continued outgrowth of the abortive bud. Since DNA synthesis was also inhibited, this suggested a dependence of cell division, and flagellum and holdfast synthesis, on the completion of chromosome replication. By addition or removal of nalidixic acid at various times in the cell cycle, periods were demonstrated when the organism was insensitive to the antibiotic indicating that there was a pre-synthetic and post-synthetic gap in the pattern of DNA synthesis in R. palustris swarmers.

During growth of Escherichia coli strain SPA O in the presence of methionine, an intermediate accumulates in the medium. This intermediate reacts with 2,4-dinitrophenylhydrazine, and can be degraded to ethylene either enzymically or photochemically, the latter being stimulated by the addition of a flavin. The pH optimum for the photochemical degradation of this intermediate and 2-keto-4-methylthiobutyric acid (KMBA) is pH 3 whereas the optimum for methional is pH 6. The enzyme which converts the intermediate to ethylene also converts KMBA to ethylene and has many of the properties of a peroxidase including inhibition by catalase, cyanide, azide and anaerobiosis. The enzyme which synthesizes the intermediate is not known but requires oxygen and pyridoxal phosphate. A pathway for ethylene biosynthesis is proposed in which methionine is converted to KMBA which can be degraded either by peroxidase or in a flavin-mediated photochemical reaction. Its relevance to the properties of other ethylene-producing bacteria and to the proposed pathway of ethylene release by higher plants is discussed.

Rhodopseudomonas palustris was chosen as a model organism for studying bacterial differentiation. Synchronous populations selected by sucrose gradient centrifugation yielded more than 95% swarmer cells. The appearance and disappearance of cell morphological groupings and the doubling of cell numbers in cultures of such swarmer populations were very well defined. Cells were only motile for the first half of the division cycle, but motility was regained before division. Development gave rise to a distinct and characteristic pattern of extinction increase and particle volume distribution. The development of swarmers into mother cells and the dimorphic division of R. palustris are discussed as simple examples of differentiation.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A capsid mutant of phiX174 is capable of forming replicative form and synthesizing single strands at the restrictive temperature in a dnaB mutant of Escherichia coli. Under similar conditions, the wild-type bacteriophage is incapable of either step in viral synthesis.

The replication of M-13 in a strain of Escherichia coli with a thermosensitive lesion in deoxyribonucleic acid synthesis was studied. M-13 failed to replicate at the restrictive temperature, even when the parental replicative form was allowed to form at the permissive temperature. When cells which were actively producing phage at the permissive temperature were shifted to the restrictive temperature, phage production continued. The incorporation of radioactive label into phage particles at 42 C indicated that continued single-strand synthesis was unaffected by the lesion in the host cell.

Bacteriophage phiX174 is unable to replicate in Escherichia coli t3 at the restrictive temperature. However, if progeny phage synthesis is initiated at the permissive temperature, it will continue after a shift to the restrictive temperature.