| Polyadenylation is an essential step in mRNA processing and in many aspects of gene expression. Nuclear poly(A) polymerase (nPAP) is the enzyme which catalyzes polyadenylation in the nucleus of eukaryotic cells. PAP purified from the adult rat liver nuclei differs from that purified from hepatoma nuclei structurally and immunologically.;In the second part of the thesis, I describe the development of an in vitro accurate polyadenylation system in which differences in function of the tumor-type and normal nPAP could be investigated. This system was capable of supporting accurate polyadenylation of an exogenously added mRNA precursor. Additionally, the system was characterized with respect pH, salt, and incubation temperature conditions and dependence on an intact polyadenylation system, amount of nPAP present, and dependence on magnesium for accurate polyadenylation. Finally, it was discovered that a polyadenylation system could be created using hepatoma and/or liver nuclear components. This system can be used in the future to further investigate differences in function of tumor-type and normal nPAP and their interaction with different polyadenylation system components.;In the first part of this thesis, I describe the results of efforts to investigate the significance of the occurrence of distinct forms of nPAP in tumors and normal adult tissue, we sought to determine at least a partial amino acid sequence of nPAP so that an oligonucleotide probe could be designed for use in screening a cDNA library. It was discovered that conventional nPAP preparations are usually contaminated with significant amounts of peptide elongation factor 1... |
