CONSTRUCTION OF HUMAN-CHROMOSOME-16-SPECIFIC AND HUMAN-CHROMOSOME-5-SPECIFIC CIRCULAR YAC BAC LIBRARIES BY IN-VIVO RECOMBINATION IN YEAST (TAR CLONING)/

Transformation-associated recombination (TAR) in yeast was exploited f or the selective isolation of human DNAs as large circular yeast artif icial chromosomes (YACs) from two rodent/human hybrid cell lines conta ining human chromosomes 5 and 16. TAR cloning vectors containing the F -factor origin of replication were constructed for use in these experi ments. Presence of the F-factor origin in TAR vectors provides the cap ability of transferring the YACs generated by in vivo recombination in yeast into Escherichia coil cells and propagating them as bacterial a rtificial chromosomes (BACs). A high enrichment of human versus rodent YACs was observed during isolation of human DNA from the rodent/human hybrid cell lines. Although <3% of the DNA content in the hybrid cell s was human, as many as 75% of the transformants contained human YACs. In contrast to the standard YAC cloning method based on in vitro liga tion, no human/mouse chimeras were observed during TAR cloning. The co nstructed human chromosome 16 YAC library had approximately 2.6x cover age, represented by 4320 YAC clones with an average insert size of 80 kb. YAC clones generated from chromosome 16 were successfully converte d into BACs by electroporation of DNA isolated from yeast transformant s into E. coli. The BAC clones represent similar to 0.6x chromosomal c overage. Pilot YAC and BAC libraries of chromosome 5 have been also co nstructed. The chromosomal distribution of YAC/BACs from chromosome 5 and chromosome 16 was evaluated by fluorescence in situ hybridization (FISH). The distribution of FISH signals appeared random along the len gth of each chromosome. We conclude that TAR cloning provides an effic ient means for generating representative chromosome-specific YAC/BAC l ibraries. (C) 1998 Academic Press.