Date of Award

Summer 1986

Document Type

Thesis - Restricted

Degree Name

Master of Science (MS)

Abstract

It has been shown that as RNA is transcribed, it rapidly becomes associated with proteins. These proteins have been implicated in the processing of heterogeneous nuclear RNA (hnRNA) to mRNA. Complexes of protein with hnRNA and mRNA are termed hnRNP and m.RNP, respectively. Adenovirus 2 (Ad 2) was utilized as a tool for studying the function of these proteins. Ad 2 genome produces a very abundant, relatively well characterized class of RNA molecules and therefore serves as a useful system for studying gene expression in higher organisms. Hela cells were infected with Ad 2 for either 18 or 22 hours, followed by extraction of the RNA comp I exes from nuclei and cytoplasm. Covalent crosslinking of the protein-RNA complex with ultraviolet light irradiation (254nm) was done in nuclei and whole cells. Both the whole cells and nuclei were exposed to different levels of irradiation sufficient to cross link more than 50% of the RNP, complexes in either fraction. The proteins extracted from hnRNP from both irradiated and control fractions were separated on acrylamide gels; there was no obvious difference in protein content. The A, B and C hnRNP core proteins were present. To see if any viral proteins are associated with viral . poly A+ mRNA, cross linking of the RNP in infected cells was carried out followed by extraction of the polysomal fraction which was further purified by separation on an oligo dT affinity column which will bind only poly A+ RNA. The proteins isolated in this manner from viral RNP complexes were released from the RNA by ribonuclease treatment and electrophoresed on acrylamide gels. The proteins bound to the viral RNA late in the infection cycle appear to be cellular in origin, suggesting that viral proteins are not involved in an RNP complex.

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