Date of Award

1998

Degree Type

Thesis

Degree Name

M.Sc.

Department

Physics

First Advisor

Lee, Lana,

Keywords

Biology, Molecular.

Rights

CC BY-NC-ND 4.0

Abstract

Bovine platelet plasmin inhibitor (BPPI) is a novel Kunitz inhibitor, recently discovered in platelets (Li, 1992) for which an expression system is sought. Because the protein's origin of synthesis was unknown, a search for a genomic clone was undertaken. PCR was used in an attempt to amplify the BPPI gene from lymphocyte, bovine genomic DNA. The first set of primers used was designed according to the N and C termini of the amino acid sequence of BPPI (P1/P2). These were based on a degenerate nucleotide sequence derived from its amino acid sequence. Amplification with this set of primers was unsuccessful. The second approach in amplifying the gene was to increase the specificity of the primers in the 3' end (P3/P4) using a table of codon usage of the bovine genome. Two successive amplifications led to the formation of a 230 bp fragment. The sequence of this product was not comparable to the degenerate sequence of BPPI. A third set of primers (P6/P7) was designed according to the gene sequence of SI (II). An inferred amino acid sequence from the SI (II) gene, outside of the region encoding the mature protein shared almost identical homology with the amino acid sequence of BPPI, hence its use as a template in the design of primers. The use of P6/P7 for amplification, resulted in the formation of two products. One was sized at 450 bp and the other at 172 bp. Sequencing of these genes indicated no similarities with the BPPI or SI (II) genes. Amplification of the BPPI gene from genomic DNA was unsuccessful using these particular primers. Future work could involve the construction of a cDNA library from a suitable source such as megakaryocytes. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1997 .L32. Source: Masters Abstracts International, Volume: 39-02, page: 0452. Adviser: Lana Lee. Thesis (M.Sc.)--University of Windsor (Canada), 1998.

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