Bacterial artificial chromosome cloning and mapping of a 630-kb human extrachromosomal structure.

Abstract

We have cloned and mapped a circular 630-kb human extrachromosomal structure {termed amplisome} using the bacterial artificial chromosome {BAC) cloning system. Twenty-one BACs were isolated from an amplisome-enriched library by colony hybridization. The insert sizes range from 25 to 143 kb, with an average size of 82 kb. The coverage of the amplisome in clones is -2.7-fold. To construct a physical map of the amplisome, we used three different but complementary methods: hybridization, STS content mapping, and fingerprinting. In addition, we compared the advantages and the drawbacks of these techniques in mapping the amplisomal BACs. The 21 BACs were grouped into two contigs and the two small gaps {3.5 and 26.5 kb) were filled by screening of a human genomic BAC library. The organization of the amplisome revealed by the BAC-based physical map is consistent with the long-range restriction map reported previously. Our results demonstrate that a 630-kb region can be rapidly cloned and mapped into contigs by use of the BAC system. Because of the low frequency {<0.1%) of chimerism and rearrangement, these BAC clones are ready for DNA sequencing and functional analysis.Human cell line HeLa-Bu25-10B3 was isolated by selection for growth in stepwise increases in methotrexate (MTX) concentration, and although it contains 300 copies of the dihydrofolate reductase (DHFR) gene, it lacks homogeneously staining regions (HSRs) and contains few (0-3 per cell) double minutes (DMs) (Masters et al. 1982). Further studies have shown that the DHFR gene is located on a 630-kb extrachromosomal element, termed an amplisome (Maurer et al. 1987). It has been found that the amplisome does not change in size and copy number during long-term culture with MTX selection, and its loss is much slower than that expected from simple dilution upon withdrawal of MTX (Pauletti et al. 1990). The amplisome was found to have one copy of the DHFR gene per molecule, and no repetitive structure was observed (Esnault et al. 1994). To characterize further the structure and organization of the DNA sequences on am- plisomes, we have cloned amplisomal DNA in a large insert vector system. Recently, bacterial vectors based on the bacteriophage P1 (Sternberg 1990) and on the Escherichia coli fertility plasmid IF-factor; bacterial artificial chromosomes (BAC)] (O'Conner et al. 1989;Hosoda et al. 1990;Shizuya et al. 1992) have been developed for the cloning of large DNA. These systems have a very low frequency of chimerism, and the inserts are very stable during propagation (Shizuya et al. 1992). BACs utilize the well-studied E. coli single-copy fertility plasmid and have been shown to be able to accept human DNA inserts as large as 300 kb. Very little or no rearrangement of the inserts has been observed even after >100 generations of serial growth (Shizuya et al. 1992). The transformation efficiency of the BAC can be as high as 107 clones/~g of DNA (Wang et al. 1994). Because of these advantages, we have used the BAC vector to construct an amplisome library and a p...