Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector.

Abstract

A bacterial coning system for mapping and analysis of complex genomes has been developed. The BAC system (for bacterial artificial chromosome) is based on Escherichia colt and its single-copy plasmid F factor. It is capable of maintaining human genomic DNA fragments of >300 kilobase pai-. Individual clones of human DNA appear to be maintained with a high degree ofstructural stability in the host, even after 100 generations of serial growth. Because of high cloning efficiency, easy manipulation of the cloned DNA, and stable maintenance of inserted DNA, the BAC system may facilitate construction of DNA libraries of complex genomes with fuller representation and subsequent rapid analysis of complex genomic structure.There is currently underway an intense effort to construct a high-resolution physical map of each of the human chromosomes. Eventually, these maps will be composed of overlapping fragments of human DNA and will allow the direct acquisition of DNA fragments that correspond to specific genes. Completion of the physical map requires the availability of comprehensive libraries of DNA clones in appropriate vectors. Furthermore, the accuracy and efficiency of physical mapping increase progressively with the size of the clone firgments in these libraries. Thus, the construction of libraries using yeast artificial chromosomes (YACs), which permit cloning of fragments of ;500 kilobase pairs (kb), represents a fundamental advance in our ability to generate physical maps that order DNA over multi-megabase distances (1). However, some difficulties have been encountered with the manipulation of YAC libraries (2-4). Thus, for example, in various libraries a fraction of the clones result from co-cloning events; i.e., they include in a single clone noncontiguous DNA fiagments. We describe here a bacterial cloning system that we refer to as BACs, bacterial artificial chromosomes. This system may provide a supplement and alternative to the YAC system for some applications requiring cloning of large fragments. The BAC system is based on the well-studied Escherichia coli F factor. Replication of the F factor in E. coli is strictly controlled (5). The F plasmid is maintained in low copy number (one or two copies per cell), thus reducing the potential for recombination between DNA fragments carried by the plasmid. Furthermore, F factors carrying inserted bacterial DNA are capable of maintaining fragments as large as 1 megabase pair, suggesting that the F factor is suitable for cloning of large DNA fragments (6). Other bacterial systems for cloning large DNA have been developed. For example, the system based on bacteriophage P1 is in use (7). However, the P1 vector has a maximum cloning capacity of 100 kb. A bacterial system based on F factors has been reported (8). However, in this system, human DNA inserts >120 kb have not been cloned and characterized. The BAC system allows us to clone large DNA from a variety of complex genomic sources into bacteria, where the DNA is stable, easy to manipulate, and represents a si...

Construction and characterization of a half million clone BAC library of durum wheat (Triticum turgidum ssp. durum)

“…Yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs) are valuable tools that allow the cloning of large fragments of DNA and are used to construct representative libraries of entire genomes (Burke et al 1987;Shizuya et al 1992). BAC libraries are generally preferred over YAC libraries because of their relatively simple production process, greater clone stability and low frequency of chimerism (Woo et al 1994).…”

Construction and characterization of a bacterial artificial chromosome (BAC) library for the A genome of wheat

“…Bacterial artificial chromosome (BAC) libraries (Shizuya et al 1992) have been recently developed from many different crop plants (Frijters et al 1997;Marek and Shoemaker 1997;Vinatzer et al 1998;Woo et al 1994;Zhang et al 1996). These large insert libraries have been essential for physically mapping and for cloning valuable genes (Brommonschenkel and Tanksley 1997;Caetano-Anolles and Gresshoff 1996;Chen et al 1997;Danesh et al 1998;Wang et al 1995).…”

Proteomics of marine bacteria

“…Fosmid vectors allow for a size selection of the cloned DNA fragments from 32 to 43 kb by packaging the DNA in l-phage heads. Shizuya et al [26] developed the bacterial cloning system BAC for mapping and analysis of complex genomes. Because of its high cloning efficiency and the stable maintenance of inserted DNA, the BAC system is able to facilitate the construction of DNA libraries of complex environmental genomic samples but also provides a comprehensive representation of the genome sequence of one organism.…”