Studies in our laboratory have shown that nuclear poly(A) polymerase (PAP) from normal rat liver is structurally and immunologically distinct from the corresponding enzyme in either a Morris rat hepatoma or primary hepatoma induced by feeding an azo dye. This enzyme is an oncofetal antigen and is not expressed in regenerating liver. We will now test the direct role of PAP in mRNA polyadenylation and explore the possibility that the liver-type and tumor-type enzymes might be polyadneylating different classes of mRNAs. This will be done by (a) red cell mediated microinjection of anti- PAP antibodies followed by transfection of a specific gene and by measurement of polyadenylation of mRNA and its translation and (b) by coupled transcription-polyadenylation of a specific gene in cell-free systems. We will complete cDNA cloning of tumor PAP and use it as a probe (a) in Northern blot analysis to determine the level of mRNA for the tumor enzyme (b) to determine the distribution of mRNA for the tumor enzyme in different tissues and (c) to investigate whether the gene for the tumor enzyme is related to known oncogenes. We will also clone cDNA for the liver enzyme, compare the cDNA sequences of the liver and tumor enzymes and use the liver cDNA for investigating alterations in the expression of the liver PAP gene in response to physiological and pathological stimuli. We will study interaction of PAP with specific regions of mRNA and determine the nature of the trans factor(s) essential for accurate polyadenylation. We will also study the potential role of 5' capping on mRNA precursor in the cleavage/polyadenylation reaction and the possible existence of a """"""""polyadenylating complex"""""""" similar to that associated with splicing. Finally, we will investigate synthesis of the tumor enzyme in vivo and in vitro. It is hoped these studies will reveal the role of PAP in mRNA polyadenylation and the mechanism(s) by which two structurally distinct nuclear poly(A) polymerases direct polyadenylation of mRNA and elucidate the factors controlling this reaction in normal and cancer cells.
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