63. Preparation of New BAC Vectors for BAC Cloning and Transformation- Associated Recombination ("TAR") Cloning 

Changjiang Zeng¹, Yu Wang¹, Kazutoyo Osoegawa¹, Natasha Kouprina², Vladimir Larionov², and Pieter J. de Jong^1 
1Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY 14263 and ²Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC 27709 
zeng@dejong.med.buffalo.edu 

Recently, efficient procedures have been reported for the re-cloning of large genomic DNA fragments in yeast as circular YACs. This approach of "Transformation Associated Recombination" cloning utilizes homologous recombination between a linear YAC vector and homologous sequences in complex genomic DNA to generate circular yeast artificial chromosomes. For this purpose, a specific YAC vector equipped with short (unique) genomic sequences from the targeted region needs to be constructed. The sequences are positioned at the ends of the linear YAC fragment used for transformation into yeast spheroplasts. To make the process of TAR rescue of genomic DNA more universally applicable, we constructed several hybrid BAC/YAC vectors designated as pTARBAC-1, -2 and -4. These vectors differ from our earlier BAC vector (pBACe3.6) by the presence of a yeast centromere (CEN3) and a yeast-selectable marker (his3). The TARBAC vectors have been used to prepare BAC libraries for several species, including Trypanosoma brucei, Giardia and Cat. Clones lacking inserts do not generate viable yeast colonies after transformation of yeast spheroplasts and selection for his-function. However, most (25out of 30) of the Trypanosome BACs with 130 kb average inserts, transform yeast at high efficiency, indicating the presence of ARS elements in most of the genomic insert fragments. Most of the insert sequences in the Trypanosome BACs can be deleted by treating the BACs with a restriction enzyme (e.g. EcoRI) which lacks corresponding sites in the TARBAC vector. Such EcoRI-deleted BAC clones are functionally similar to the previous TAR-rescue vectors because they have (unique) genomic sequences at the ends of a hybrid BAC/YAC vector. Hence, most of the BAC clones in a TARBAC library can be used to generate TAR-rescue vectors to (re-)clone genomic segments from different haplotypes or from related species. We have confirmed that most of the deleted TARBACs have also lost the ARS elements as indicated by the loss of their capability to successfully transform yeast spheroplasts. We are currently exploring the re-isolation of the deleted sequences by co-transformation of deleted BAC DNA with genomic Trypanome DNA. The less-complex genomes of unicellular eukaryotes are used to model future work with mammalian TARBAC libraries. Information on our current libraries can be obtained from our Web page: http://bacpac.med.buffalo.edu .

* Supported in part by grants from the U.S. DOE , NHGRI and the German Forschungs Gemeinschaft (DFG).