| To help clarify the biological role(s) of poly (ADP-ribose) polymerase (PADPRP), the expression in mammalian cell lines of a number of mutant PADPRP enzymes was studied. Three PADPRP mutant constructs were prepared to correlate changes in the functional domains of PADPRP with its physiological roles in cells. Establishment of clonal cell lines expressing various forms of PADPRP analogs has allowed modulation of the cellular PADPRP levels and should allow initiation of future studies on poly(ADP-ribosyl)ation of nuclear proteins.;In order to properly modulate PADPRP levels in cells by the above approaches and study the consequential effects on cell functions, it was important initially to understand various features of the normal expression of the PADPRP gene in NIH 3T3 cells. As a continuation of earlier work (Kang, M. S. Thesis), the expression of the endogenous PADPRP mRNA was examined in NIH 3T3 cells.;The first PADPRP analog designed in an attempt to modulate the endogenous activity of PADPRP in various mammalian cell lines was a cDNA (pSVNO3;As an alternative way to modulate PADPRP activity in 3T3 cells, and to examine the possible roles of PADPRP in proliferation, DNA repair, DNA strand breaks, and differentiation, antisense PADPRP cDNA, under the control of MMTV promoter (pMAM-As) was stably transfected into 3T3-L1 preadipocytes. Transfected cells that expressed antisense PADPRP RNA, when induced with dexamethasone, showed a decreased level of PADPRP at both the mRNA and protein levels. The PADPRP activity was reduced up to 75% in these clones. Studies on the effect of expression of antisense RNA on the differentiation of transfected 3T3-L1 cells into adipocytes indicated that the differentiation of the cells was inhibited, as measured by cytoplasmic triglyceride accumulation. The data suggested that PADPRP may play a role in critical mitosis events prior to terminal differentiation. |
