Homologous recombination of exogenous DNA with the Sulfolobus acidocaldarius genome: properties and uses.
In order to quantify recombination between exogenous DNA and the Sulfolobus acidocaldarius chromosome, we electroporated pyrE (uracil-auxtotrophic) recipient strains with functional pyrE sequences and counted Pyr+ transformants by direct plating. Certain culture and post-electroporation conditions increased the yield of Pyr+ recombinants from non-replicating pyrE plasmid, whereas cognate methylation of SuaI restriction sites in the plasmid decreased it. Recombination of linear DNAs with the S. acidocaldarius genome was proportional to the length of a limiting overlap, but even synthetic oligonucleotides produced reasonable numbers of recombinants with appropriate recipient strains. To investigate uses of this latter property, we electroporated an 18-bp pyrE deletion mutant with mixtures of synthetic oligonucleotides altering glycine-55 of the orotate phosphoribosyl transferase encoded by pyrE. Pyr+ transformants were recovered in which this codon was converted to each of the alternatives encoded by the oligonucleotide mixtures, thereby identifying five amino acid substitutions tolerated at this position of the thermostable enzyme.
Kurosawa
Norio
N
Department of Environmental Engineering for Symbiosis, Soka University, Hachioji, Tokyo 192-8577, Japan.
Grogan
Dennis W
DW
D001483:Base Sequence; D019175:DNA Methylation; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics; D004274:DNA, Recombinant / Q000737:chemistry / Q000235:genetics; D018274:Electroporation; D020745:Genome, Archaeal; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D016945:Sulfolobus acidocaldarius / Q000235:genetics*
D019641:DNA, Archaeal; D004274:DNA, Recombinant