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Typescript. / Thesis (Ph. D.)--University of Hawaii, 1969. / Bibliography: leaves [174]-183. / xiii, 183 l illus

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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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The sites of biosynthesis and incorporation of the abundant CNS myelin proteins 2' , 3' -cyclic nucleotide-3'-phosphodiesterase (CNPase) and P2 protein into the growing myelin membrane were investigated. Cell-free translation systems programmed with mRNA from rat brain, rabbit spinal cord, free and bound polysomes and purified myelin demonstrated conclusively that both CNPase and P2 are synthesized on free polysomes like the myelin basic proteins (MBPs) but unlike the proteolipid protein (PLP), the major intrinsic membrane protein of CNS myelin, which is known to be synthesized at the oligodendrocyte endoplasmic reticulum on bound polysomes (Colman et al., 1982) . These observations were supported by labelling studies on rats in vivo during the period of maximal myelin deposition. Newly synthesized CNPase associated with the myelin membrane very rapidly after labelling (~2 minutes) and this is consistent with the view that there is only a brief delay between synthesis and incorporation into their target membrane for extrinsic-type plasma membrane proteins. An RNA fraction isolated from purified CNS myelin was not enriched in mRNAs coding for CNPase and P2 but a considerable enrichment of mRNAs coding for MBPs was observed. This phenomenon has important implications for the cell biology of myelination since it suggests that although MBPs, CNPase and P2 are all basic extrinsic membrane proteins, and synthesized on free polysomes, different mechanisms for their transport to the myelin membrane exist. The addition of dog pancreatic microsomes (DPM) during translation showed no membrane association for CNPase however, at least 50% of MBPs were observed to non-specifically associate with these membranes. When newly synthesized MBP and P2 were incubated post-translationally with DPM or rabbit spinal cord myelin P2 only associated with myelin whereas MBP showed an equal affinity for both types of membranes. The segregation of MBP free polysomes at the myelin membrane during synthesis ensures that the nascent MBP polypeptides associate with the correct membrane. Recent evidence has shown that the free polysome-mRNA complex is bound to the cytoskeleton during protein synthesis. After extensive characterization of the purified rat brain oligodendrocyte and myelin-associated cytoskeletons it was shown that the synthesis of MBPs and CNPase only occurs from mRNA that is associated with the cytoskeleton and not when it is part of the cytoplasmic mRNA pool. Lipid analysis of the purified rat brain myelin-associated cytoskeleton revealed the presence of tightly bound lipid with a considerable enrichment of cerebroside and sphingomyelin (the latter at the expense of phosphatidylethanolamine). These studies on the cytoskeletal involvement in myelinogenesis suggest that extrinsic CNS myelin proteins are synthesized on the cytoskeleton and that post-translational cytoskeletal transport of these proteins to the growing myelin membrane may take place.

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