Date of Award

1988

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Ding S. Shih

Abstract

To understand the initiation of translation of picornavirus RNA, cell-free translation of the RNA of encephalomyocarditis virus (EMCV) was examined after hybridization of chemically synthesized cDNA fragments to different sites of the 5$\sp\prime$ untranslated region (5$\sp\prime$ UTR) of the viral RNA and after deletion of specific segments of the 5$\sp\prime$ UTR by RNase H digestion. In addition, "translation competition" experiments, using RNA fragments derived from different parts of the 5$\sp\prime$ UTR, were performed. The results of these experiments are summarized as follows: (i) Binding of cDNA fragments to sequences between the 5$\sp\prime$ terminus and nucleotide 338 caused no effect on translation of the viral RNA; (ii) binding of cDNA fragments to the sequence between nucleotides 420 and 449 caused a slight inhibition; (iii) binding of fragments to sites between nucleotides 450 and the initiator AUG codon (nucleotide 834) caused strong inhibition; (iv) deletion from the 5$\sp\prime$ terminus to nucleotide 338 caused no effect on translation of the coding region of the RNA, whereas deletion from the 5$\sp\prime$ terminus to nucleotide 450 or beyond totally abolished translation of the coding region; and (v) presence of a 5$\sp\prime$ fragment (from 5$\sp\prime$ terminus to approximate nucleotide 338) caused no effect on translation of EMCV RNA, but presence of either of two other fragments, one from approximately nucleotides 338 to 589, the second from approximately nucleotides 338 to 813, caused inhibition of translation. These results together indicate that the first part of the 5$\sp\prime$ UTR, at least to nucleotide 338, is not required for EMCV RNA translation but the sequence near, and possibly also sequences beyond nucleotide 450 are critical for translation of the viral RNA. For the purpose of future more detailed analysis of the sequence elements that are important for translational initiation, cDNAs corresponding to the 5$\sp\prime$ UTR and a beginning portion of the viral protein coding region of the viral RNA were synthesized and cloned, and two sets of sequential deletion mutants were constructed from the cloned cDNA. Results from an initial in vitro translation study of the synthetic mRNAs from some of these deletion clones are presented.

Pages

115

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