Doctoral Dissertations

Date of Award

12-1985

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

David A. Brian

Committee Members

W. Stuart Riggsby, Beth Mullin, Karl Sirotkin

Abstract

Two approaches were taken to examine the genome structure of the porcine transmissible gastroenteritis coronavirus (TGEV). The first approach was designed to compare the degree of nucleotide sequence homology between the genomes of TGEV and the bovine enteric coronavirus (BCV), a member of an antigenically distinct coronavirus subgroup. For this study, the RNA genomes of TGEV and BCV were separately metabolically labeled with 32P-orthophosphate, and analyzed by two-dimensional Tl oligonucleotide fingerprinting. From an analysis of comigrating oligonucleotides in a mixing experiment, it was determined that TGEV and BCV share a sequence homology of approximately 91%. This degree of homology supports the notion that the two viruses, while antigenically dissimilar, arose from a common ancestor. It further raises the question of whether conserved sequences might be confined to restricted regions of the genome allowing identification of conserved functional domains. The second approach was designed to systematically examine the primary structure of the TGEV genome thereby allowing rigorous genetic analyses. For this approach, the 3' end of the 20 Kb TGEV genome was cloned and sequenced, and the properties of two genes were deduced and analyzed. TGEV genomic RNA was copied into cDNA after priming with oligo(dT) and the double stranded product was cloned into the Pst 1 site of the pUC9 vector. One clone of 2.0 Kb contained part of the poly(A) tail and was sequenced in its entirety using the chemical method of Maxam and Gilbert. Another clone of 0.7 Kb also contained part of the poly(A) tail and was sequenced in part to confirm the primary structure of the most 3' end of the genome. Two potential nonoverlapping genes were identified within the 3' terminal 1663 bases from an examination of open reading frames. The first gene encodes a 382 amino acid protein of 43,426 molecular weight, that is the apparent nucleocapsid protein on the basis of size, chemical properties, and amino acid homology with other coronavirus nucleocapsid proteins. It is flanked on its 5' side by at least part of the matrix protein gene. The second gene encodes a hypothetical 78 amino acid protein of 9101 molecular weight that is hydrophobic at both ends. A 3' non-coding sequence of 273 bases was also determined and a sequence of 9 nucleotides near the poly A tail was found to be conserved for TGEV, the mouse hepatitis coronavirus, and the avian infectious bronchitis coronavirus.

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