Construction of an ordered cosmid collection of the Escherichia coli K-12 W3110 chromosome

Abstract

A cosmid library of the Escherichia coli K-12 W3110 chromosome was constructed in which clones were assigned to locations on the chromosome map by hybridization and genetic marker complementation tests. Approximately 70% of the genome was represented by this library. The identified clones can be maintained in the homologous system and would facilitate genetic studies of E. coli.Escherichia coli is one of the best-investigated microorganism in terms of gene structure and function, and more than 1,000 genes have been identified and localized on the circular chromosome map (2). Nevertheless, the number of genes characterized corresponds to only about one-third of the entire number estimated to be present on the E. coli chromosome. To understand the whole genetic system of E. coli, further isolation of genes with known genetic markers as well as a systematic search for new genes would be necessary. The first gene library of the E. coli chromosome was constructed by Clarke and Carbon in 1975 (10). Since then, the library has been screened to isolate clones which complement a variety of mutations at known locations on the genetic map. So far, about 300 clones have been mapped and analyzed by using this library (2, 36). In the work described in this paper, we attempted to construct an ordered cosmid library of the E. coli chromosome in which clones are assigned to their specific locations. The which appeared on ampicillin-containing agar plates were subjected to screening.For screening of clones, the -dot hybridization procedure involving isolated plasmid DNA was adopted to reduce the background level in hybridization. Although the colony

FhuF, an iron‐regulated protein of Escherichia coli with a new type of [2Fe‐2S] center

“…The fhuF gene region was cloned from λ phage 8D1 (672) of the Kohara collection [12] resulting in plasmid pKF7572. The cloned gene was sequenced, and agreed with the respective part of the published genome sequence [11].…”

“…DNA was sequenced by the dideoxy chain-termination method using the AutoRead sequencing kit and the A. L. F. sequencer (Pharmacia Biotech). A DNA Thermal Cycler (Perkin Elmer Cetus) was used for PCR.All E. coli chromosomal DNA positions and section numbers are those of the E. coli genome sequence [10,11].The fhuF gene region was cloned on a 3-kb EcoRV DNA fragment from λ phage 8D1 (672) of the Kohara collection [12] in the high-copy-number vector pSU18 and recloned into vector pT7-5, resulting in plasmid pKF7572. To obtain an insert coding…”

“…The fhuF gene region was cloned on a 3-kb EcoRV DNA fragment from λ phage 8D1 (672) of the Kohara collection [12] in the high-copy-number vector pSU18 and recloned into vector pT7-5, resulting in plasmid pKF7572. To obtain an insert coding only for FhuF, the fhuF gene was amplified by PCR and cloned into pSU18, resulting in plasmid pKF181.…”

A combined genetic and physical map of the Streptomyces coelicolor A3(2) chromosome

“…Later, physical maps, in some cases at least partially related to a genetic map, have been obtained by similar approaches for several other bacteria, including species ofAnabaena, Campylobacter, Caulobacter, Haemophilus, Lactococcus, Mycoplasma, Myxococcus, Pseudomonas, Rhodobacter, and Shigella (6,16,19,24,55,59,60,70,(75)(76)(77)85). A quite different procedure, construction of an ordered collection of overlapping clones, has also been used to generate a physicalgenetic map of E. coli (54,86).…”

“…Myxococcus, Pseudomonas, Rhodobacter, and Shigella (6,16,19,24,55,59,60,70,[75][76][77]85). A quite different procedure, construction of an ordered collection of overlapping clones, has also been used to generate a physicalgenetic map of E. coli (54,86 small segments of the chromosome that carry gene clusters of interest. S. coelicolor A3(2) produces at least five secondary metabolites, four of them antibiotics, whose genetic study is a major preoccupation, together with analysis of the genetic control of sporulation and its correlation with antibiotic production (17,41).…”