RESUMO
We describe an in vitro selection procedure for oligodeoxynucleotide-directed mutagenesis, which produces mutants at frequencies of greater than 90%, facilitating the identification of mutants directly by nucleotide sequencing. The method is based on the selective methylation of the mutant strand by the incorporation of 5-methyl-dCTP. Restriction endonuclease digestion of the resulting hemimethylated DNA with MspI results in the nicking of only the nonmethylated-parental strand. The parental strand is removed by treatment with exonuclease III. The mutants are recovered by transformation of a mcrAB strain of Escherichia coli with the nascent strand.
Assuntos
Escherichia coli/genética , Mutação , Oligodesoxirribonucleotídeos , Sequência de Bases , Colífagos/genética , Enzimas de Restrição do DNA , DNA de Cadeia Simples/genética , MetilaçãoRESUMO
A clone coding for beta-lactoglobulin A has been isolated from a cDNA bank constructed from poly(A+)mRNA isolated from the bovine mammary gland. Its nucleotide sequence codes for the beta-lactoglobulin A, from amino acid residues Leu-11 to Ile-162, as based on the amino acid sequence reported by Braunitzer et al. [Z. Physiol. Chem. 354 (1973) 867-878]. In addition to the 455-bp coding sequence, our clone pB beta L4-10 contains a 3'-nontranslated region of approx. 270 bp.
Assuntos
Lactoglobulinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , DNA/genética , Genes , Dados de Sequência MolecularRESUMO
We describe 46 insertions of the Streptococcus faecalis transposon Tn917 into the chromosome of Bacillus subtilis. These insertion mutations were mapped genetically. Some caused auxotrophic requirements, and others were cryptic. These insertions were scattered around the B. subtilis chromosome. The mutant strains were useful in several ways for mapping and cloning B. subtilis genes and were added to the Bacillus Genetic Stock Center collection. Among the auxotrophic markers were a new serine auxotrophy and deletion-insertions that caused auxotrophy in one case for homoserine and threonine, in another case for uracil and either cysteine or methionine, and in a third case for leucine, isoleucine, and valine.
Assuntos
Bacillus subtilis/genética , Elementos de DNA Transponíveis , Enterococcus faecalis/genética , Mapeamento Cromossômico , DNA Bacteriano/genética , Genes Bacterianos , MutaçãoRESUMO
Two conserved histidine residues (His-101 and His-271) appear to be essential components in the active site of the enzyme xylose (glucose) isomerase (EC 5.3.1.5). These amino acid residues were targeted for mutagenesis on the basis of sequence homology among xylose isomerases isolated from Escherichia coli, Bacillus subtilis, Ampullariella sp. strain 3876, and Streptomyces violaceus-niger. Each residue was selectively replaced by site-directed mutagenesis and shown to be essential for activity. No measurable activity was observed for any mutations replacing either His-101 or His-271. Circular dichroism measurements revealed no significant change in the overall conformation of the mutant enzymes, and all formed dimers similar to the wild-type enzyme. Mutations at His-271 could be distinguished from those at His-101, since the former resulted in a thermolabile protein whereas no significant change in heat stability was observed for the latter. Based upon these results and structural data recently reported, we speculate that His-101 is the catalytic base mediating the reaction. Replacement of His-271 may render the enzyme thermolabile, since this residue appears to be a ligand for one of the metal ions in the active site of the enzyme.