Masters Theses

Date of Award

12-1991

Degree Type

Thesis

Degree Name

Master of Science

Major

Life Sciences

Major Professor

David A. Brian

Committee Members

W. Stuart Riggsby, Leon Potgieter

Abstract

The 5' end terminus of the porcine transmissible gastroenteritis virus (TGEV) leader sequence was determined on the messenger RNAs (mRNAs) for the hydrophobia protein and the nucleocapsid protein. A new method developed in our laboratory for the copy DNA cloning and 5' end analysis of mRNAs was employed in this study, and it is comprised of the following steps: 1) A radiolabeled oligodeoxynucleotide primer, having a G+C content ≥ 50%, was annealed downstream of the start codon on the mRNA species. 2) Reverse transcriptase was used to extend the primer to the end of the template, and the single-stranded first-strand copy DNA products were isolated on a denaturing polyacrylamide sequencing gel. 3) The isolated, first-strand copy DNA molecules were ligated head-to-tail with T4 RNA ligase, then amplified by the polymerase chain reaction. 4) The double-stranded products from the polymerase chain reaction were ligated into the plasmid vector pGEM-3Z and the recombinant plasmid was used to transform E. coli strain JM109. 5) Positive clones were detected by colony hybridization with a leader-detecting oligodeoxynucleotide probe, and positive clones were sequenced using the dideoxynucleotide chain-termination method. The 5' end of the TGEV leader was determined to be 5' GACTTTTAAA... This contrasts with the previously established leader sequence (as determined by Maxam and Gilbert sequencing), which was believed to start with a 5-base poly-T tract. Additional evidence was sought for the presence of an intraleader open reading frame in swine testicle cells persistently infected with TGEV. Earlier studies with the bovine coronavirus have shown that an intraleader open reading frame exists on a majority of viral mRNAs isolated from human rectal tumor cells persistently infected but not acutely infected with the bovine coronavirus. This open reading frame, which results from a G-->A mutation at the +5 position of the leader, potentially encodes an 11 amino acide peptide that is neutral and hydrophilic. It has been hypothesized that this intraleader open reading frame attenuates the translation of downstream open reading frames, thus possibly attenuating coronavirus replication. To test the hypothesis that this is a general phenomenon among coronaviruses, a TGEV intraleader open reading frame was sought. In the current study, total cytoplasmic RNA was isolated from 1) swine testicle cells that were infected with TGEV and carried for over 1 year and 2) persistently infected swine testicle cells after 72 and 81 days postinfection. Northern hybridization analysis did not show any viral specific RNA being made in any of the persistently infected cells. Using primer extension analysis an attempt was made to copy the 5' end terminus of the mRNA for the nucieocapsid protein in cells after 81 days postinfection, but an extended product could not be visualized by autoradiography. These results suggest that the mRNA levels for TGEV in persistently infected cells are too low to be used for this study. The sequence of the 5' end of the TGEV leader will be important for the construction of reporter RNA molecules which can be transfected into cells so that the phenomenon of mRNA replication can be observed in vitro. It will also prove useful for designing antisense RNA experiments for blocking virus replication.

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