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Bibliography: leaves 133-141.

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The use of ribonucleoside phosphoramidites protected at the 2$ sp prime$-hydroxyl with alkylsilyl ethers was developed for the solid phase synthesis of oligoribonucleotides. A series of pentadecameric oligomers of various base composition was synthesized using this methodology and a complete protocol for the deprotection of synthetic oligoribonucleotides was developed. The use of $ beta$-cyanoethyl phosphate protection was compared to that of methyl protection in the synthesis of pentadeca-uridylic acid sequences and no significant difference between the use of the two protecting groups was found. Concurrently a procedure for the derivatization of long chain alkylamine controlled pore glass beads with ribonucleosides was used to provide an improved solid phase support for the efficient automated synthesis of long oligomers. A side reaction on this support was identified and a method for its prevention developed. / A reaction occurring at the O$ sp6$-position of guanosine residues, which leads to chain cleavage, was identified. The basis of this reaction was characterized by both $ sp{31}$P nuclear magnetic resonance and the synthesis of oligoriboguanylate sequences. Three methods involving: protection of the O$ sp6$-position, the use of an alternate coupling cycle, and the use of a different phosphoramidite activator, were successfully applied to the resolution of this problem. The aforementioned procedures were utilized in the synthesis of a half E. Coli N- f - met tRNA molecule 43 units in length. / Finally the cumulative knowledge gained from the above studies was applied to the synthesis of an entire E. Coli N- f - met analogue 77 units in length along with a number of other very long sequences. Methods for the deprotection of these oligomers were investigated culminating in the isolation of oligoribonucleotides which were successfully characterized by polyacrylamide gel electrophoresis, enzyme degradation, enzymatic and chemical sequencing, terminal nucleotide analysis, and, in the case of the E. Coli f - met tRNA analogue, an aminoacylation assay.

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Transcription in mammalian and yeast mitochondria proceeds from a few well defined promoters, with processing of polycistronic transcripts producing the mature RNAs. The levels of different sequences in the steady-state RNA populations depend on differential promoter strengths, transcription attenuation and/or selective termination, and differential RNA stabilities. To gain insights into the processes governing transcription and RNA levels in plant mitochondria, a system using isolated maize mitochondria, which synthesize bona fide mitochondrial RNAs, was developed and partially characterized with respect to exogenous requirements and sensitivity to inhibitors of DNA-dependent RNA synthesis. / Although initiation and processing probably occur at reduced levels in isolated maize mitochondria, endogenous DNA templates are extensively transcribed at the same relative rates as in vivo. Isolated maize mitochondria were used to demonstrate that differential rates of both synthesis and turnover help determine the steady-state abundances of various mitochondrial RNA sequences and that mitochondria from certain lines possess an autonomously-replicating, RNA-based genetic system.

The in vitro translation products directed by cucumber necrosis virus (CNV) RNA were analyzed in both rabbit reticulocyte lysate and wheat germ extract cell-free translation systems. In rabbit reticulocyte lysates, one major protein of ca. 33 Mr was produced. In wheat germ extracts, four proteins of ca. 41, 33, 21 and 20 Mr were produced. Hybrid-arrested translation (HART) studies using synthetic CNV antisense RNA corresponding to the entire CNV genome demonstrated that the four major proteins synthesized from CNV virion RNA in wheat germ extracts are virus-specific translation products. The genomic locations of the CNV in vitro translation products were determined using a number of experimental approaches including: (1) HART using antisense RNA corresponding to selected regions of the CNV genome; (2) in vitro translation of synthetic messenger-sense CNV transcripts; (3) immunoprecipitation of in vitro translation products with CNV polyclonal antisera and (4) in vitro translation of size-fractionated CNV virion RNA. Together, these experiments demonstrated that the ca. 33 Mr protein is derived from the 5' proximal coding region, the ca. 41 Mr protein is derived from an internal coding region, and that at least one but probably both of the ca. 20 and 21 Mr proteins are derived from the 3' terminal coding region(s) of the CNV genome. In addition, immunoprecipitation experiments provided further evidence that the ca. 41 Mr protein is the viral coat protein. The size, number, and genomic locations of the CNV in vitro translation products reported here are in agreement with those predicted from nucleotide sequence data (Rochon & Tremaine, 1989). The natural template for the expression of downstream cistrons in the CNV genome was investigated by in vitro translation of sucrose fractionated CNV virion RNA as well as in vitro translation of messenger-sense synthetic transcripts. These studies indicate that in vitro, both subgenomic and genomic-length CNV RNA molecules may act as templates for the synthesis of the ca. 41,21 and 20 Mr proteins as well as the ca. 33 Mr protein. / Land and Food Systems, Faculty of / Graduate

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