Cloning of genes encoding redox proteins of known amino acid sequence from a library of the Desulfovibrio vulgaris (Hildenborough) genome
1988; Elsevier BV; Volume: 67; Issue: 1 Linguagem: Inglês
10.1016/0378-1119(88)90010-8
ISSN1879-0038
Autores Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoA library of 900 recombinant phages has been constructed for the genome of Desulfovibrio vulgaris Hildenborough (1.7 × 106 bp) by cloning size-fractionated Sau3A fragments (15–20 kb) into the replacement vector λ-2001. When a hydrogenase gene probe, a 4.7-kb SalI-EcoRI fragment of known nucleotide sequence, was used to screen the plaque lifted library, 23 positive clones were found, which together span 31 kb of D. vulgaris DNA. To facilitate the cloning of genes with oligodeoxynucleotides as probes, DNA was purified for all clones in the library and spotted on a 16 × 16-cm grid of nitrocellulose. This grid was incubated sequentially to identify λ clones containing the gene for redox proteins of known amino acid sequence: cytochrome c3 (one 18-mer → four clones), flavodoxin (one 17-mer and one 26-mer → one clone) and rubredoxin (one 44-mer → 21 clones). The four eye-positive clones are also recognized by the rubredoxin oligodeoxynucleotide probe. Restriction mapping defines a 35-kb region of the D. vulgaris chromosome m which the rub and cyc loci are separated by 17.5 kb. The nucleotide sequence of the rubredoxin gene was determined and the deduced amino acid sequence found to agree with that determined by Bruschi [Biochim. Biophys. Acta 434 (1976) 4–17] with the exception of Thr-21 which is found to be encoded by GAC, an Asp codon. A plausible ribosome-binding site precedes the N-terminal initiator methionine residue. Rubredoxin does not have an N-terminal signal sequence which is in agreement with the cytoplasmic location of this redox protein.
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