Strategies for Expressing Analogs of PADPRP in Eukaryotic Cells

Abstract

Towards our goal of obtaining cell lines expressing altered levels of poly(ADP-ribose) polymerase (PADPRP), we have constructed expressing plasmids containing various mutagenized forms of PADPRP and have stably introduced these into NIH3T3 cells. We reasoned that it might be feasible to compete out or enhance the activity of the endogenous PADPRP in intact cells with a homologous or partial PADPRP protein. This might molecularly perturb the various functions of PADPRP in vivo. One recombinant plasmid was constructed with the heavy-metal inducible mouse metallothionein (MT) promoter upstream of the partial cDNA coding the PADPRP DNA-binding and part of the automodification domains. Another mutagenized PADPRP with a deletion of 132bp in the NAD-binding domain and shown in in vitro assay to be inactive, was subcloned downstream of SV40 early promoter. Each plasmid was stably co-transfected with pNeomycin vector into NIH3T3 cells.