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Successful transfer of yeast artificial chromosomes (YACs) into human cells has been described in only a single study. We here report on the evaluation of YAC transfer strategies into a human renal cell carcinoma cell line by yeast spheroplast fusion and cationic lipids. While the latter approach proved inefficient, significant numbers of clones containing both vector arms were obtained by spheroplast fusion. FISH analyses on such clones revealed the presence of YAC integration and the co-localization of both vector arms with insert sequences. These data demonstrate that under certain experimental conditions efficient YAC transfer into human cells by spheroplast fusion is possible and may be useful for the cloning of human disease-related genes.

Chromosomal deletions and translocations of human chromosome region 3p14 are observed in various human malignancies and suggest the existence of a tumor suppressor gene locus within this region. Tumors most frequently affected by these aberrations are small-cell lung cancer and renal-cell carcinoma. In continuation of our previously published YAC contig of chromosome region 3p14.2-p14.3, we report here on the construction of a YAC contig of at least 11 Mb that consisted of 171 YACs and covers the entire subregion 3p14.1. This contig includes the t(3;8) breakpoint of a hereditary renal-cell carcinoma localized in 3p14.2 and extends into human chromosome region 3p12-p13. It defines the order of 34 DNA probes in relation to reference markers D3S6 and D3S30 as well as the human protein tyrosine phosphatase-gamma gene. For 31 DNA probes we identified nonchimeric YACs by fluorescence in situ hybridization. The minimal tilling pathway consists of 16 yeast artificial chromosomes. As a prerequisite for identification of a putative tumor suppressor gene within this region, this contig renders human chromosome region 3p14.1 accessible to gene isolation.