Student Theses and Dissertations
Date of Award
1976
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Norton Zinder
Keywords
RNA, viroids, gene expression, iodination, fingerprinting, regulatory RNA
Abstract
This dissertation concerns studies of plant viroid RNA and other RNA species of unusual function. By "RNA species of unusual function" we mean RNA species which do not function as transfer, ribosomal or messenger RNA. In the studies to be reported here we have laid the experimental and theoretical foundation for investigating the role of RNA in the control of gene expression. In Chapter I the various standard techniques of RNA fingerprinting and sequencing have been applied to RNA molecules labeled in vitro with 125I. Fingerprints of human 5S RNA and bacteriophage f2 RNA resemble those of their noniodinated counterparts both in complexity and in specific pattern. Iodination as used here is thus a general labeling procedure, and appears principally to label cytidine residues. This iodination method shows little sensitivity to potential structure in single-stranded RNA molecules, yields stable oligonucleotide products in a reproducible manner, and does not change the specificity of RNases T1, T2, and U2, nor does it affect the specificity of digestion by pancreatic RNase or spleen phosphodiesterase. Some difficulties arising in the handling of iodinated RNA are also discussed. In Chapter II we have employed these techniques to study several properties of plant viroid RNA which was previously unobtainable as a radioactively labeled species of high specific activity. Highly purified isolates of potato spindle tuber viroid (PSTV) from tomatoes and citrus exocortis viroid (CEV) from Gynura aurantiaca have been compared. Two-dimensional fingerprinting analysis of PSTV and CEV RNA labeled in vitro with 125I has demonstrated that (i) each of these RNA species has a complexity compatible with the size estimate of 250-350 nucleotides and (ii) these RNA molecules do not have the same sequence. In the course of these studies we have developed a technique for the extraction of very small amounts (less than one microgram) of low molecular weight RNA from polyacrylamide gels of nucleic acid preparations from healthy and viroid-infected plant tissues. The purity of such samples is high enough to allow subsequent iodination (to a specific activity of 1-5 x 106 cpm/μg) and characterization by two-dimensional fingerprinting analysis. Our original interest in the study of viroids arose through a consideration of potential roles for RNA in the control of gene expression. Because viroids are composed entirely of RNA, it is possible that they could interact directly with the regulatory machinery of the host cells and could constitute aberrant forms of regulatory substances. In Chapter III we have considered the features of RNA which make it especially suitable for a regulatory role, then suggest a possible source for such proposed regulatory RNA, and finally describe possible modes of action. We have considered possible roles for extra RNA regions which are removed from RNA precursor molecules during their maturation, and have suggested that specific cleavage by RNA processing enzymes can give rise to reproducible fragments from these regions which may function after their removal. Nucleotide sequence analysis of an RNA processing site from bacteriophage T7 early messenger RNA precursor, as well as of potential cleavage sites from HeLa cell heterogeneous nuclear RNA, has been carried out.
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Recommended Citation
Dickson, Elizabeth, "Studies of Plant Viroid RNA and Other RNA Species of Unusual Function" (1976). Student Theses and Dissertations. 491.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/491
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy