Catalogo Articoli (Spogli Riviste)

OPAC HELP

Titolo:
COMBINING LOCALIZED PCR MUTAGENESIS AND NATURAL TRANSFORMATION IN DIRECT GENETIC-ANALYSIS OF A TRANSCRIPTIONAL REGULATOR GENE, POBR
Autore:
KOK RG; DARGENIO DA; ORNSTON LN;
Indirizzi:
YALE UNIV,DEPT BIOL,POB 208103 NEW HAVEN CT 06520 YALE UNIV,DEPT BIOL NEW HAVEN CT 06520
Titolo Testata:
Journal of bacteriology
fascicolo: 13, volume: 179, anno: 1997,
pagine: 4270 - 4276
SICI:
0021-9193(1997)179:13<4270:CLPMAN>2.0.ZU;2-V
Fonte:
ISI
Lingua:
ENG
Soggetto:
P-HYDROXYBENZOATE HYDROXYLASE; ACINETOBACTER-CALCOACETICUS BD413; AQUATICUS DNA-POLYMERASE; PROTOCATECHUATE 3,4-DIOXYGENASE; STRUCTURAL GENE; EVOLUTIONARY DIVERGENCE; EXTRACELLULAR LIPASE; PSEUDOMONAS-PUTIDA; DEFICIENT MUTANTS; FIDELITY;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
41
Recensione:
Indirizzi per estratti:
Citazione:
R.G. Kok et al., "COMBINING LOCALIZED PCR MUTAGENESIS AND NATURAL TRANSFORMATION IN DIRECT GENETIC-ANALYSIS OF A TRANSCRIPTIONAL REGULATOR GENE, POBR", Journal of bacteriology, 179(13), 1997, pp. 4270-4276

Abstract

We present a procedure for efficient random mutagenesis of selected genes in a bacterial chromosome. The method combines PCR replication errors with the uptake of PCR-amplified DNA via natural transformation. Cloning of PCR fragments is not required, since mutations are transferred directly to the chromosome via homologous recombination. Random mutations were introduced into the Acinetobacter chromosomal pobR gene encoding the transcriptional activator of pobA, the structural gene for4-hydroxybenzoate 3-hydroxylase. Mutant strains with strongly reducedPobR activity were selected by demanding the inability to convert 4-hydroxybenzoate to a toxic metabolite. Of spontaneous pobR mutants, 80%carry the insertion element IS1236, rendering them inappropriate for structure-function studies. Transformation with Taq-amplified pobR DNAincreased the mutation frequency 240-fold and reduced the proportion of IS1236 inserts to undetectable levels. The relative fidelity of Pfupolymerase compared with Taq polymerase was illustrated by a reduced effect on the mutation frequency; a procedure for rapid assessment of relative polymerase fidelity in PCR follows from this observation. Over 150 independent mutations were localized by transformation with DNA fragments containing nested deletions of wild-type pobR. Sequence analysis of 89 of the mutant pobR alleles showed that the mutations were predominantly single-nucleotide substitutions broadly distributed within pobR. Promoter mutations were recovered, as were two mutations that are likely to block pobR translation, One third of the recovered mutations conferred a leaky or temperature-sensitive phenotype, whereas theremaining null mutations completely blocked growth with 4-hydroxybenzoate. Strains containing two different nonsense mutations in pobR weretransformed with PCR-amplified DNA to identify permissible codon substitutions. Independently, second-site suppressor mutations were recovered within pcaG, another member of the supraoperonic pca-qui-pob cluster on the Acinetobacter chromosome. This shows that combining PCR mutagenesis with natural transformation is of general utility.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 01/04/20 alle ore 11:03:15