60. Direct Isolation of a Centromeric Region from a Human Mini-Chromosome by in Vivo Recombination in Yeast 

Natalay Kouprina, Motonobu Katoh, Mitsuo Oshimura, and Vladimir Larionov 
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC 27709 
kouprina@niehs.nih.gov 

Isolation of specific chromosomal regions and entire genes has typically involved cloning of random fragments as BACs or YACs followed by a long and laborious process to identify the region of interest. Using the recently developed TAR cloning technique in yeast¹, it has been possible to directly isolate specific chromosomal regions and genes from complex genomes as large linear or circular YACs. In this study we applied a modified version of this technique² for isolation of a centromeric region of the human mini-chromosome D1 containing 5 Mb of the human chromosome Y³. This mini-chromosome was generated by two rounds of telomere-directed chromosome breakage leading to a loss of sequences from both arms of the chromosome. Despite the small size and loss of a significant part of centromeric repeats (there is only 140 kb of alphoid DNA left), the D1 mini-chromosome segregates accurately in mitosis, suggesting that a 140 kb block of alphoid DNA alone or along with the short arm flanking sequences is sufficient for a centromere function. Taken in advantage that the first round of chromosome Y breakage resulted in truncation of the chromosome within a block of alphoid DNA (i.e. a new telomere and a block of alphoid DNA became physically linked), we developed a scheme to isolate a centromeric region from the mini-chromosome D1. Direct transformation of genomic DNA isolated from hybrid cells carrying the mini-chromosome into yeast spheroplasts resulted in a rescue a centromeric region as a set of linear YACs with sizes from 50 kb to 300 kb. To prevent YAC rearrangements due to the presence of multiple repeats, the isolates were maintained in the host strain with the conditional RAD52. Each YAC isolate containing an entire block of alphoid DNA (i.e. YACs bigger than 140 kb) was circularized, retrofitted into BAC with the NeoR mammalian selectable marker and accurately transferred into the E. coli cells. Since no detectable changes in YACs were observed after retrofitting to BACs, the BAC DNAs were transferred into human cells for further functional analysis. 

¹Kouprina et al. (1998) Proc. Natl. Acad. Sci. USA 95: 4469-4474. 
²Kouprina et al. (1998) Genome Research 8: 666-672. 
³Brown et al. (1996) Proc. Natl. Acad. Sci. USA 93: 7125-7130.