We have generated a nested series of interstitial deletions in a fragment of human X chromosome-derived DNA cloned into a yeast artificial chromosome (YAC) vector. A yeast strain carrying the YAC was transformed with a linear recombination substrate containing at one end a sequence that is uniquely represented on the YAC and at the other end a truncated long interspersed repetitive element (LINE 1, or Li). Homologous recombination between the YAC and the input DNA resulted in a nested series of interstitial deletions, the largest of which was 500 kilobases. In combination with terminal deletions that can be generated through homologous recombination, the interstitial deletions are useful for mapping and studying gene structure-function relationships.The ability to clone large fragments of DNA into yeast artificial chromosomes (YACs) is providing a new method for the analysis of complex genomes (1). Large fragments of DNA (as long as 1 megabase) can be introduced into YAC vectors to yield artificial chromosomes.The open-ended size capacity of YAC cloning technology suggests that virtually any mammalian gene or gene complex can be isolated as a single contiguous segment of DNA. Because of the large insert size, methods to facilitate the mapping and functional analysis of genes within the YACs are needed. In addition, the development of methods for targeted modification and transfer of the modified YACs back into cultured cells and experimental organisms will provide powerful tools for the study of gene structure and function.The long-range structure-function relationships of genes can be studied by introducing a large YAC that contains a gene of interest into mammalian cells and eliciting regulated gene expression. If deletions of variable portions of DNA in and around the gene (interstitial deletions) can be obtained, YACs bearing such deletions could be introduced into cells to study the effects of the modifications. Recent successes in introducing YACs into mammalian cells (2-4) provide encouragement for the feasibility of these types of studies. The ability to make interstitial deletions would also be helpful in generating restriction maps of YACs and in assigning genetic markers to precise regions within a YAC.Homologous recombination (HR) in yeast has been used to generate terminal deletions in normal as well as artificial chromosomes in yeast (5, 6). Pavan et al. (6) constructed a vector that contains a yeast telomeric sequence, a yeast selectable marker, and a polylinker into which a human highly repetitive sequence (Alu repeat) was added. When a linearized version of this plasmid was introduced into yeast cells carrying human DNA in a YAC, the input plasmid was able to recombine with each of several homologous regions in the YAC, yielding different-size terminal deletions. The generation of these deletions requires a single crossover event between the target and the input DNA.We have used a modification of the terminal deletion strategy to generate interstitial deletions in a YAC carrying a 650-kb...
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