Centromeric and Noncentromeric ADE2-Selectable Fragmentation Vectors for Yeast Artificial Chromosomes in AB1380

Abstract

We have constructed a set of fragmentation vectors for the truncation of either the centromeric or the noncentromeric end of YACs containing a human DNA insert. These vectors carry ADE2 or HIS5 as the selectable marker, enabling direct use in AB1380, the host strain of most publicly available YAC libraries. Centromeric fragmentation vectors for AB1380 have not been reported previously; the noncentromeric vectors show high frequencies of fragmentation.Yeast artificial chromosomes (YACs) consist of a large (50-2500 kb) DNA insert, flanked on both sides by a yeast selectable marker and a telomere, and on one side by a yeast centromere. An important application of YACs lies in the mapping of the human genome and the isolation of human disease genes. YAC contigs now cover nearly the entire human genome (Chumakov et al. 1995), but the depth of these contigs is often not sufficient to allow highresolution ordering of markers. Because many candidate regions for disease genes have been allocated to YAC contigs still comprising several megabases, improvement of the resolution of these contigs is highly desirable. More recent, complementary resources in contig generation are bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) libraries. Although valuable in the ultimate generation of sequence-ready genomic regions, they preclude the delineation of the available YAC contig information in the megabase range, as the assembly of BAC/PAC contigs is a de novo endeavor. YAC fragmentation, that is, the creation of YACs with deletions from one end (Pavan et al. 1990), is an attractive method for obtaining more refined mapping while optimally using existing knowledge. Compared to the common route of constructing cosmid contigs, YAC fragmentation is a simple and quick alternative. Furthermore, YAC fragmentation provides the possibility of discarding the noninteresting parts of a YAC, which is useful for subsequent studies like the production of welldefined restriction maps, subcloning, gene searches, and gene characterizations, and as an attractive tool to focus YAC-derived transgene studies.YAC fragmentation is based on the presence of sequences in the YAC insert homologous to a part of the fragmentation vector, which further contains a selectable marker, a telomere, and a centromere if the centromeric arm is to be targeted. Common repeats like the long interspersed repetitive element (LINE) and, in particular, Alu are attractive candidates for this purpose, as they occur frequently in YACs with a human insert. The series of fragmentation vectors described by Pavan et al. (1991) (Albertsen et al. 1990;Anand et al. 1990;Larin et al. 1991), have been constructed in yeast strain AB1380, which is not his3 ‫מ‬ . Therefore, YACs must first be transferred to a his3 ‫מ‬ background, through meiosis or kar1 transfer (Spencer et al. 1994), before these vectors can be used. To bypass this problem, we have adapted existing fragmentation vectors with the ADE2 gene and/or CEN6, thereby creating a set of vectors that ca...