Fragmentation and Integrative Modification of YACs

The very large cloning capacity of yeast artificial chromosomes (YACs) has facilitated the analysis of complex genomes by bridging physical and genetic maps. The large size of YAC inserts also creates some unique problems, including identification of novel genes on large stretches of uncharacterized DNA, creating physical maps of large genomic inserts in YACs, and localizing defined sequences within the YAC. The well-characterized and highly efficient system of homologous recombination in Saccharomyces cerevisiae (1 ) can be used to introduce additional markers (e.g., neo ^R cassette, polylinkers, bacterial markers) and modifications to YACs that allow these problems to be addressed. This chapter covers fragmentation and integration, two recombination-based techniques for modifying the insert DNA and vector arm sequences of YACs. Specialized vectors containing cloned repetitive elements or sequences specific to YAC vector arms can recombine with homologous sequences in the YAC (2 ). Repetitive sequences are ideal for recombination-based modification because they are present at a high copy number and sufficiently widespread in the genome that most YACs will contain multiple representatives of these families.