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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA025078-09A1
Application #
3166676
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1981-08-01
Project End
1991-12-31
Budget Start
1987-04-01
Budget End
1987-12-31
Support Year
9
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Type
Schools of Medicine
DUNS #
129348186
City
Hershey
State
PA
Country
United States
Zip Code
17033
Ghoshal, K; Jacob, S T (1991) Ara-ATP impairs 3'-end processing of pre-mRNAs by inhibiting both cleavage and polyadenylation. Nucleic Acids Res 19:5871-5
Chrislip, K M; Hengst-Zhang, J A; Jacob, S T (1991) Polyadenylation of SV40 late pre-mRNA is dependent on phosphorylation of an essential component associated with the 3' end processing machinery. Gene Expr 1:197-206
Jacob, S T; Zhang, J; Garg, L C et al. (1991) Multiple functional enhancer motifs of rat ribosomal gene. Mol Cell Biochem 104:155-62
Raju, V S; Jacob, S T (1991) Anti-poly(A) polymerase antibodies in the sera of tumor-bearing rats can inhibit specific cleavage and polyadenylation reactions. Carcinogenesis 12:917-20
Jacob, S T; Terns, M P; Hengst-Zhang, J A et al. (1990) Polyadenylation of mRNA and its control. Crit Rev Eukaryot Gene Expr 1:49-59
Hengst-Zhang, J A; Jacob, S T (1990) Tissue and species distribution of liver type and tumor type nuclear poly(A) polymerases. Biochem Biophys Res Commun 169:325-31
Garg, L C; Dixit, A; Jacob, S T (1989) A 37-base pair element in the far upstream spacer region can enhance transcription of rat rDNA in vitro and can bind to the core promoter-binding factor(s). J Biol Chem 264:220-4
Garg, L C; Dixit, A; Webb, M L et al. (1989) Interaction of a positive regulatory factor(s) with a 106-base pair upstream region controls transcription of metallothionein-I gene in the liver. J Biol Chem 264:2134-8
Terns, M P; Jacob, S T (1989) Role of poly(A) polymerase in the cleavage and polyadenylation of mRNA precursor. Mol Cell Biol 9:1435-44
Jacob, S T; Terns, M P; Maguire, K A (1989) Polyadenylate polymerases from normal and cancer cells and their potential role in messenger RNA processing: a review. Cancer Res 49:2827-33

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