Alexander Graham

Isolation of cDNA clones using yeast artificial chromosome probes

The cloning of large DNA fragments of hundreds of kilobases in Yeast artificial chromosomes, has simplified the analysis of regions of the genome previously cloned by cosmid walking. The mapping of expressed sequences within cosmid contigs has relied on the association of genes with sequence motifs defined by rare-cutting endonucleases, and the identification of sequence conservation between species. We reasoned that if the contribution of repetitive sequences to filter hybridizations could be minimised, then the use of large cloned DNAs as hybridisation probes to screen cDNA libraries would greatly simplify the characterisation of hitherto unidentified genes. In this paper we demonstrate the use of this approach by using a YAC, containing 180 kb of human genomic DNA including the aldose reductase gene, as a probe to isolate an aldose reductase cDNA from a lambda gt11 human foetal liver cDNA library.

Immunochemical analysis of the membrane-bound hydrogenase of Escherichia coli

The membrane-bound hydrogenase [EC 1.12.-.-l ofEscherichia coli is involved in the energy-conserving oxidation of hydrogen [l-4] via fumarate reductase [EC 1.3.99.11 and also in the formate hydrogenlyase pathway which converts formate to COZ and Hz [5]. E. coli hydrogenase from aerobically grown cells has been isolated and characterised [6] and the enzyme from anaerobically grown cells has been partially characterised [7]. Antibodies specific for Bacillus subtilis membrane-bound succinate dehydrogenase [EC 1.3.99.11 have been raised from activity-stained precipitin arcs located after analysis of crude fractions by crossed immunoelectrophoresis using antisera raised to detergent-solubilised membranes [8]. We have used a similar approach to prepare antibodies specific for E. coli hydrogenase and its use has enabled the subunit MW of the enzyme from anaerobically grown cells to be determined. We also report the isolation and immunological characterisation of two new E. coli mutants which specifically lack hydrogenase activity.

Arrangement of respiratory nitrate reductase in the cytoplasmic membrane of escherichia coli: Location of β subunit

The membrane-bound nitrate reductase (EC 1.7.99.4) from Escherichia coli functions as the terminal enzyme of the respiratory chain of the organism, when grown anaerobically in the presence of nitrate as electron acceptor. The transfer of reducing equivalents from reduced ubiquinol, through cytochrome b-556No; and nitrate reductase, to NO; is accompanied by the net translocation of protons across the cytoplasmic membrane [I]. The topography of the cytochrome b-556Noinitrate reductase complex has been studied both functionally and structurally. The use of artificial permeant and non-permeant reductants [2] has established that the cytochrome b-556No; can accept electrons at the periplasmic surface and that at least part of nitrate reductase is able to donate electrons to oxidised dyes at the internal surface of the cytoplasmic membrane. Lactoperoxidase-catalysed radio-iodination [3] has located the cytochrome b-556No; at the periplasmic face and the a-subunit ofnitrate reductase (M, 150 000) at the cytoplasmic face of the membrane. Thislocation of the a-subunit has been confirmed by transglutaminase-catalysed covalent labelling [4] and by immunofluorescence studies using antibodies specific for the a-subunit [ 51. The location of the P-subunit (Mr 59 000) of nitrate reductase has not been established. Here we have investigated the topography of nitrate reductase using two non-membrane permeant reagents,