Restriction digests of the clone give the following sizes (kb): EcoRI--7.4; BamHI--7.4; EcoRI/BamHI--5.7, 1.7. The insert contains the following restriction sites (approximate kb from the 5' end): PstI--0.09; ClaI--0.67, 1.28; AccI--0.89. Murine fibroblast cell lines such as NIH 3T3 and PA317, transformed with pCD2, express cytosine deaminase. If transformed cells are grown in the presence of non-toxic 5-flurocytosine (5FC), the cytosine deaminase will convert the 5FC into the lethal metabolite 5-fluoruracil which inhibits DNA and RNA synthesis. This cytosine deaminase negative selection system (CDNSS) could prove useful in selectively controlling gene therapy experiments. PA317/pCD2 transformed cell lines expressed 16.3U of cytosine deaminase while NIH 3T3/pCD2 transformed cell lines expressed 3.6U (1U = 1pmol of uracil converted from cytosine/10+E08 cells/minute). Recombinant cytosine deaminase is expressed poorly in eukaryotic cells if 88 bp 5' to naturally occurring GTG start site is present. pCD2 was engineered to remove these 88 bp and convert the GTG start to ATG. |
