Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences.
AUTOR(ES)
Cai, Y P
RESUMO
Use of the sacB gene (J. L. Ried and A. Collmer, Gene 57:239-246, 1987) provides a simple, effective, positive selection for double recombinants in Anabaena sp. strain PCC 7120, a filamentous cyanobacterium. This gene, which encodes the secretory levansucrase of Bacillus subtilis, was inserted into the vector portion of a suicide plasmid bearing a mutant version of a chromosomal gene. Cells of colonies in which such a plasmid had integrated into the Anabaena chromosome through single recombination were plated on solid medium containing 5% sucrose. Under this condition, the presence of the sacB gene is lethal. A small fraction of the cells from initially sucrose-sensitive colonies became sucrose resistant; the majority of these sucrose-resistant derivatives had undergone a second recombinational event in which the sacB-containing vector had been lost and the wild-type form of the chromosomal gene had been replaced by the mutant form. By the use of this technique, we mutated two selected genes in the chromosome of Anabaena sp. strain PCC 7120. The conditionally lethal nature of the sacB gene was also used to detect insertion sequences from this Anabaena strain. Sucrose-resistant colonies derived from cells bearing a sacB-containing autonomously replicating plasmid were analyzed. Five different, presumed insertion sequences were found to have inserted into the sacB gene of the plasmids in these colonies. One of them, denoted IS892, was characterized by physical mapping. It is 1.7 kilobases in size and is present in at least five copies in the genome of Anabaena sp. strain PCC 7120.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=209118Documentos Relacionados
- Characterization of the IS895 family of insertion sequences from the cyanobacterium Anabaena sp. strain PCC 7120.
- Regulation of an Osmoticum-Responsive Gene in Anabaena sp. Strain PCC 7120
- Anabaena sp. Strain PCC 7120 hetY Gene Influences Heterocyst Development
- Regulation of Anabaena sp. strain PCC 7120 glutamine synthetase activity in a Synechocystis sp. strain PCC 6803 derivative strain bearing the Anabaena glnA gene and a mutated host glnA gene.
- Anabaena sp. strain PCC 7120 ntcA gene required for growth on nitrate and heterocyst development.