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Title:A Study of Cell Surface Change After Viral Transformation by Iodination and Cell-Cycle Dependent Phosphorylation of Sv-40 Large Tumor Antigen
Author(s):Jone, Cyrenius Mun-Hong
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Biochemistry
Abstract:This thesis consists of two parts. Part I deals with iodination of the cell surface protein before and after viral transformation and Part II deals with cell-cycle dependent phosphorylation of the large tumor antigen induced by SV-40 in human cells.
Part I: Iodination of Zeta Protein by Lactoperoxidase, Chloroperoxidase and Chloramine T. Lactoperoxidase has been used widely to specifically label cell surface structures with iodine atoms because under certain conditions, the active iodinating species remains enzyme-bound and hence cannot penetrate into the cell. Using this enzymic labeling reaction, a cell surface protein, termed Zeta protein, has been found in abundance in normal chicken embryo fibroblasts but has been found in greatly diminished quantity in Rous sarcoma virus transformed cells. Zeta protein is also susceptible to trysin digestion and can be stripped from normal cells by brief treatment with trysin.
It has been suggested that the specific labeling of Zeta protein by lactoperoxidase might be the result of a special interaction between lactoperoxidase and Zeta protein. Thus, it has been argued that other surface glycoproteins might also appear or disappear as the result of transformation but would not be detected by the lactoperoxidase label. In this investigation, two enzymic methods and one chemical method were employed to label a normal and Rous sarcoma virus transformed chicken embryo fibroblasts with ('125)I. The enzymic systems were chloroperoxidase and lactoperoxidase in conjunction with hydrogen peroxide generated by glucose oxidase and glucose. The chemical method used chloramine T as the oxidant for iodide ion. It has been found that no specific interaction is required for the iodination of Zeta protein by lactoperoxidase. Chloroperoxidase and chloramine T also serve to iodinate Zeta protein. Zeta protein is the only protein identified by iodination in normal cells. Disappearance of Zeta protein is the only surface change detected by these three methods after viral transformation.
Part II: Cell-Cycle Dependent Phosphorylation of SV-40 Large Tumor Antigen. Early after infection of permissive and nonpermissive cells, large T-antigen can be detected in the nuclei of the cells by immunofluorescent staining with antiserum from hamsters bearing SV-40 induced tumors. T-antigen is a phosphoprotein. The amino acid residue which is phosphorylated has been determined to be a serine residue.
Since it has been established that phosphorylation and dephosphorylation of proteins serves to regulate their biological activities, it follows that phosphorylation of T-antigen may also play a regulatory role. If the degree of phosphorylation of T-antigen varies in a predictable manner through the cell cycle, this fact could be a good indication that phosphorylation serves a regulatory role. Since T-antigen binds to DNA, possibly the DNA binding ability may be affected by phosphorylation.
SV80, a SV-40 transformed human skin fibroblast cell line has been synchronized by a 5 mM double thymidine block. The scheme consists of two blocks of 13 hours with a 10-hour growth period in between. Phosphorylation of T-antigen has been found to be a cell-cycle dependent phenomenon in the synchronized cells. A higher degree of phosphorylation of T-antigen takes place in the S phase with a lower degree of phosphorylation in G(,1). The difference in degree of phosphorylation is possible due to additional sites available in the S phase. When the cell finishes its DNA synthesis phase, these additional sites become dephosphorylated. Finally, the degree of phosphorylation of T-antigen does not affect the ability to bind to calf thymus DNA at pH 8.
Issue Date:1980
Description:172 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1980.
Other Identifier(s):(UMI)AAI8017957
Date Available in IDEALS:2014-12-14
Date Deposited:1980

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