Biochemical construction and selection of hybrid plasmids containing specific segments of the Escherichia coli genome.
AUTOR(ES)
Clarke, L
RESUMO
Using a poly(dA-dT) "connector" method, a population of annealed hybrid circular DNAs was constructed in vitro; each hybrid DNA circle containing one full-length molecule of poly(dT)-tailed DNA from E1 colicinogenic factor (Col E1) fragmented by EcoRI endonuclease annealed to any one of a collection of poly(dA)-tailed linear DNA fragments of the entire E. coli genome. This annealed, but unligated, hybrid DNA was used to transform several different auxotrophic mutants of E. coli, and by direct selection, bacterial clones were isolated which contained specific hybrid plasmids. In this manner, bacterial strains containing Col E1 hybrid plasmids carrying the entire tryptophan operon or the arabinsoe and leucine operons were isolated. The methods described should allow the molecular cloning of any portion of the E. coli genome by selection from a pool of DNA molecules containing at least several hundred different hybrids representing the entire bacterial genome.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=388721Documentos Relacionados
- Construction and characterization of hybrid plasmids containing the Escherichia coli nrd region.
- Construction and expression of hybrid plasmids containing Escherichia coli K-12 uxu genes.
- In vivo excision and amplification of large segments of the Escherichia coli genome.
- Construction of hybrid plasmids containing the Escherichia coli uxaB gene: analysis of its regulation and direction of transcription.
- Biochemical construction of specific chimeric plasmids from ColE1 DNA and unfractionated Escherichia coli DNA.