Automated system for capture and detection of nucleic acids.

A fully automated nucleic acid analysis system is described, which offers positive sample identification, improved sensitivity and reduced user interaction compared to conventional techniques. The system relies on the sequence-specific capture of DNA onto solid-phase particles, confirming product identity without the problems of interpretation and lack of sequence information inherent in gel-based analyses. The system can be used for sequence confirmation, mutation analysis and semiquantitative detection of PCR products.

Use of fortuitous in vitro mutations in a synthetic Cu-Zn superoxide dismutase gene to illuminate protein structure-function relationships.

A completely synthetic bovine copper-zinc superoxide dismutase gene (Cu-ZnSOD), designed using the most favoured codons for expression in yeast, was constructed. Fortuitous mutations introduced while cloning the synthetic gene permitted the additional construction of four altered-polypeptide products representing two single (Pro121-->Leu and Gly128-->Asp), one double (Pro100-->Leu, Arg113-->Lys) and one triple (Pro100-->Leu, Arg113-->Lys, Pro121-->Leu) mutant. All five versions of the gene were expressed in a SOD-deficient Escherichia coli strain. The 'wild-type' version of the gene and the two single-mutants were expressed to equal extents (approximately 8% of total soluble protein). However, compared with the 'wild-type' enzyme, one single-mutant (Gly128-->Asp) showed almost twice as much dismutase activity whilst the other (Pro121-->Leu) exhibited only 70% of the 'wild-type' level. In contrast, the double and triple mutants showed diminished expression of the gene (approximately 1 and 3% of total soluble protein, respectively) and almost no detectable SOD activity. Polyclonal antibovine SOD antibody bound all the recombinant proteins, although some of the products showed decreased size and probably altered conformations. The 'wild-type' superoxide dismutase recombinant was correctly dimerized and possessed dismutase activity, as did the Gly128-->Asp mutant despite the change in charge. Mutations in the other three versions affected enzyme folding and activity. The effect of the different mutations appeared to be additive, with the Pro121-->Leu substitution leading to the apparent proteolytic degradation of the enzyme in vivo.

Production of mutant dihydrofolate reductases of Lactobacillus casei for nuclear magnetic resonance spectroscopy.

Seven mutations (L4P, W21L, D26E, D26N, R57H, R57K and T63Q) affecting residues of dihydrofolate reductase of Lactobacillus casei, suspected of being important in substrate, inhibitor, or cofactor binding, were made by gapped-duplex site-directed mutagenesis. Expression of the L. casei dhfr gene required the removal of nucleotide sequences flanking the coding region. Temperature-inducible expression from the lambda pL promoter of plasmid pPLc28 allowed synthesis and subsequent affinity purification of five mutant proteins in amounts and purity sufficient for nuclear magnetic resonance (NMR) spectroscopic analysis (100 mg or more) from 10-liter cultures. W21L required the growth of 40-liter batches, and L4P was not found. Using a two-plasmid system with pcI857 providing lambda repressor and pMAC5-14 expressing the mutant gene, any auxotrophic strain of Escherichia coli can be used as a host, allowing isotopic labelling of each amino acid of any protein for rapid NMR peak assignment.

Cooperative DNA binding by lambda integration protein--a key component of specificity.

Quantitative analysis of nitrocellulose filter binding data by the method of Clore, Gronenborn and Davies [(1982) J. Mol. Biol. 155, 447-466] has been used to show that lambda integration protein (Int) exhibits cooperativity in binding to specific recognition sites within the attachment site region (lambda attP) of bacteriophage lambda DNA. Optimal values of the equilibrium constant obtained were 3.0(+/- 1.0) X 10(10) M-1 for the P' site using a model of three sites with equal affinity and 1.9(+/- 0.4) X 10(10) M-1 for the P1 site on a two-site model. The value of the cooperativity parameter alpha is 172(+106)(-66) in all cases. The occurrence of a consensus recognition sequence is necessary but not sufficient for strong binding; cooperative interaction between Int molecules binding to adjacent members of an array of binding sites is also essential. The occurrence of binding site arrays distinguishes lambda attP very clearly from other DNA sequences containing single recognition sites by chance.

A modified two primer approach to oligonucleotide-directed in vitro mutagenesis.

Using oligonucleotide-directed mutagenesis, we are trying to define the features of the protein structure that are important for the DNA and c-AMP binding by CAP from E. coli, the enzymic activity and putative DNA binding of dihydrofolate reductase of L. casei, and the functionally important regions of the self-splicing RNA of the r-RNA intron of Tetrahymena thermophila. We have used a modification of the method described by Norris et al. [1]. A mutagenic primer and an M13 universal sequencing primer are annealed simultaneously to a template from an M13 clone containing the DNA to be mutagenised and, after DNA strand extension, the fragment is cut out and recloned into either M13 or plasmid vectors. We have analysed the effect on the frequency of mutation of: the temperature used for strand extension; the class of base change attempted; the host mismatch repair system. A recently developed system for phenotypic detection of mutations in the Tetrahymena intron aided in determining mutation frequencies.

Differences in the cleavage of DNA by bleomycin under light (300-350 nm) and dark conditions.

Incubation of supercoiled plasmid DNA (both pBR 322 and pPLC-28) with increasing concentrations of bleomycin in the presence and absence of light (300-350 nm) showed extensive conversion of supercoiled to linear DNA which occurred at much lower levels of bleomycin (BLM) in the light than in the dark. Preliminary results indicate that superoxide dismutase (SOD) may offer DNA more protection against bleomycin in the dark than in the light conditions.