P-445 A suicide gene therapy of metastasized liver tumors

Abstract

The cell-division-cycle gene, CDC7, of Saccharomyces cerevisiae has been cloned in a large plasmid (pCM6) containing an insert of about 45 kb of yeast chromosomal DNA in the yeast-Escherichia coli shuttle vector, YRp7. A subclone (pCM39), having a 7-kb insert with a unique BamHI site, was capable of conferring the ts + phenotype on a cdc7ts− strain of S. cerevisiae. When this insert was placed in the vector Ylp28, and then cut with BamHI and integrated into the ts − strain, genetic analysis showed the CDC7-complementing activity and an associated vector marker to be linked to the TRP1 locus i.e., to chromosome IV. Analysis of subclones covering the pCM39 insert showed that pCM52, in which a 3.3-kb EcoRI-HpaI fragment with the unique BamHI site has been cloned, possessed the full complementing activity. Although the CDC7 gene lies across the BamHI site, the main part of the gene appears to be in the 2.2-kb BamHI-HpaI segment of the pCM52 insert. A Northern blot analysis showed this region to give rise to a 1.7-kb mRNA which we conclude is the transcript of CDC7.The large insert of pCM6 also contains a 1.5-kb XhoI fragment which several observations suggest is that containing the centromere (CEN4) of chromosome IV to which the CDC7 gene is closely linked. The 45-kb DNA fragment in pCM6 is believed to be an uninterrupted piece of chromosome IV.