Construction and Characterization of a Human Chromosome 2-Specific BAC Library

Wang, Min; Chen, Xiao Ning; Shouse, Stephanie; Manson, Jim; Wu, Qiang; Li, Rumei; Wrestler, Janet; Noya, David; Sun, Z.; Korenberg, Julie R.; Lai, Eric

doi:10.1006/geno.1994.1662

Cited by 23 publications

(24 citation statements)

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“…We have consistently observed very high background of white colonies containing no insert when BamHI was used as the cloning site. This problem is not observed when HindIII was used as the cloning site in our human chromosome-specific libraries (chromosomes 1 and 2; Wang et al 1994). …”

Section: Amplisomal Bac Library Constructionmentioning

confidence: 93%

“…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 physical map with hybridization, STS mapping, and fingerprinting.…”

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confidence: 99%

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

Wang

Shouse²,

Lipes³

et al. 1996

Genome Res.

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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...

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Section: Amplisomal Bac Library Constructionmentioning

confidence: 93%

mentioning

confidence: 99%

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

Wang

Shouse²,

Lipes³

et al. 1996

Genome Res.

View full text Add to dashboard Cite

show abstract

“…An initial experimental strategy for producing a high-resolution physical map of the T. cruzi 87 Mb genome should be based on large genomic fragments cloned in yeast artificial chromosomes (YACs) (Burke et al 1987, Smith 1994) and bacterial artificial chromosomes (BACs) (Shizuya et al 1992, Wang 1994, which can be assigned to genomic locations by hibridization with different markers and chromosome specific probes.…”

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confidence: 99%

“…The BAC system is based on the single copy plasmid F factor of Escherichia coli (Shizuya 1992, Wang 1994, that is capable of maintaining foreign DNA fragments of more than 200 kb, with a high degree of structural stability. In fact, the F factor not only codes for genes that are essential to regulate its own replication but also controls its copy number.…”

mentioning

confidence: 99%

“…In fact, the F factor not only codes for genes that are essential to regulate its own replication but also controls its copy number. The pBAC vector incorporates these essential genes as well as a chloramphenicol resistance marker and a multiple-cloning site (Shizuya 1992, Wang 1994. As discussed below, this system has been assigned a primary role in the T. cruzi project.…”

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