Student Theses and Dissertations

Date of Award

1983

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Abstract

This thesis has focused on the avian Fujinami sarcoma virus (FSV) and its role in transformation. Specifically, it included the biochemical characterization of temperature-sensitive (ts) mutants of FSV, the identification of the product of a cellular gene homologous to the transforming gene of FSV, and the isolation and characterization of phenotypic revertants of rat cells transformed by FSV.

Analysis of the defect of the two ts mutants of FSV revealed that Pl30 is the transforming protein of FSV and that its kinase activity plays a crucial role in cell transformation by this virus.

v-fps, the transforming gene of FSV is homologous to a cellular gene, c-fps, present in uninfected cells. We obtained a specific antiserum directed against the v-fps domain of P130. This antiserum specifically crossreacts with a cellular protein of MW 98,000 daltons, present in uninfected chicken bone marrow cells. Like P130, this normal cellular protein (NCP98) is a phosphoprotein which is associated in vitro with a protein kinase activity specific for tyrosyl residues. In addition, tryptic peptides analysis showed that NCP98 is structurally related to P130. The expression of NCP98 is tissue-specific and highest in bone marrow cells of the granulocytic lineage.

The isolation of phenotypic revertants of rat cells transformed by FSV suggested a relationship between the expression of P130, its enzymatic activity, and cellular transformation. Three types of revertants have been characterized, all of them susceptible to retransformation after superinfection with FSV. Type I revertants (3 clones) have lost the complete FSV provirus. Type II revertant (1 clone) has a mutated FSV provirus which encodes a transformation defective protein of MW 130,000. This protein has no kinase activity in vitro, is underphosphorylated, and lacks phosphotyrosine in vivo. In superinfected clones, it appears to be a substrate of P140, the transforming protein of another strain of FSV used in the superinfection. The mutated protein is associated in vivo with two cellular proteins (90K and sOK) which have been implicated in the binding of the transforming proteins coded by conditional mutants of avian sarcoma viruses. Type III revertants (11 clones) have the following properties: (1) The FSV provirus and integration site are identical to those of the parental clones. (2) No or very little v-􁪽 mRNA is synthesized likewise, P130 is not detected, or is present in a very small amount. (3) Each revertant segregates transformants with a frequency ranging from 10-6 to 10-'. (4) There is an inverse correlation between the level of methylation of FSV proviral DNA and the level of expression of FSV mRNA. (5) DNase I hypersensitivity of the FSV provirus in revertants is abolished.

Finally, in clones of rat fibroblasts showing different degrees of transformation after FSV infection, we observed a good correlation between the amount of v-􁪽 mRNA in a cell and its phenotype. Therefore, we propose that transformation by FSV is a function of the dosage of the v-fps gene, and not an all or none phenomenon.

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