Student Theses and Dissertations

Anindya Dutta

Date of Award

1989

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Abstract

Transcription from the Rous sarcoma virus (RSV) Long terminal repeat (LTR) in untransformed rat 3Yl fibroblasts is dependent on the presence of serum. Within an hour of addition of serum to a serum-deprived culture there is a 5 fold stimulation in the level of transcripts initiated at the LTR. This stimulation does not require synthesis of new proteins. Mutations in the RSV LTR revealed that serum-stimulated transcription was mostly dependent on two CCAAT boxes in the LTR, though other upstream sequences may play a secondary role. Serum caused the rapid appearance of a nuclear protein that binds to the two CCAAT boxes. This serum-induced CCAAT factor was also bound by CCAAT sequences from other promoters, e.g. those of human heat shock protein 70, human c-Ha-ras, human histone 1 etc, but not by the adenovirus origin of replication, or the SV40 enhancer core sequence. This data suggests that the serum induced CCAAT factor is related to CPl or CP2 rather than the NFl or CjEBP types of CCAAT binding factors. The abundance of the factor in the nucleus is increased by serum even in the presence of inhibitors of new protein synthesis. The serum dependence of transcription from the RSV L TR is lost in v-src transformed 3Yl, and cross-feeding experiments showed that the mechanism did not involve the production of extra-cellular growth factors. Temperature-sensitive (ts) v-src transformed 3Yl was used to demonstrate that the tyrosine kinase activity of v-src can (a) substitute for the serum-requirement of the RSV LTR, (b) increase the level of transcripts initiated in the LTR even in the presence of serum, (c) exert its serum-sparing effect on the RSV L TR in the absence of new protein synthesis, and (d) increase the amount of CCAAT binding factor in the nucleus. Orthovanadate, an inhibitor of tyrosine-phosphatases, which non-specifically elevates the level of phosphotyrosine in the cells, can mimic the effects of serum and v-src on transcription from the RSV L TR. The RSV LTR directed accurate initiation of transcripts in Saccharomyces cerevisiae, about 60 bases downstream from the TATA box that is used in animal cells. Mutations in the LTR revealed that the authentic TATA box was absolutely necessary for the transcription, and the CCAAT boxes that are responsive to serum in animal cells, were acting as "upstream activating sequences". The absence of the TATA box or the CCAAT boxes gave a phenotype to the yeast carrying the mutant derivatives of RSVCAT, making it possible to establish a genetic system for cloning genes for yeast and mammalian transcription factors, and do structure-function analysis on them. The activity of the RSV L TR in yeast is not stimulated by la.ctate, and is not decreased by mutations in the HAP2 or HAP3 genes, suggesting that the "UAS" activity of the CCAAT boxes is mediated by yeast CCAAT binding factors other than HAP2/HAP3. While a high level of V-STC is toxic to the yeast, expression of very low levels of the oncogene may be stimulating the CAT activity from the RSV L TR about two fold. If this can be seen at the phenotypic level, it may be possible to establish a genetic system to study how v-src influences gene expression in yeast.

Comments

A thesis submitted to the faculty of the Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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