Gene sequence of ovine cathepsin B : a study of the gene sequence encoding the lysosomal protease cathepsin B

Abstract

Cathepsin B is a lysosomal protease whose activity has been implicated in the processes of meat tenderization (Koohmaraie 1988; Ouali 1990; Etherington 1991; Dransfield 1991; Geesink 1993) and tumour metastasis (Sloane et al. 1986; Qian et al. 1989; Rozhin et al. 1990; Murnane et al. 1991; Dalet-Fumeron et al. 1992; Buck et al. 1992) both of which are characterized by phenotypic variation. It is not known which genetic factors are responsible for this variation. Gene sequences for the protein coding region of cathepsin B have been described in cattle, human and mouse (Mordier et al. 1993) but only one case of sequence polymorphism in the gene has been described (Hseih et al. 1991). Due to the putative involvement of cathepsin B in both meat tenderization and tumor metastasis, characterization of the cathepsin B gene sequence may be of commercial importance. The sheep is a suitable animal model for study of the cathepsin B gene sequence due to the it's importance in the meat in industry, and the reported incidence of tumor development in this animal (Ross 1984). Mordier et al. (1993) were able to amplifiy a 560 bp fragment of bovine cathepsin B DNA using PCR primers designed from the bovine genomic DNA sequence. The experiments described in this thesis were developed in order to (1) amplifiy a region of the ovine cathepsin B gene using the primers designed by Mordier et al. (1993), and failing that, to (2) amplify a region of the ovine gene using alternative primers. A PCR reaction was initially optimised using bovine primers and ovine genomic DNA, and a 450 bp fragment was amplified. Sequencing of this fragment revealed that it was not homologous to the cathepsin B gene sequence from other species, and it was concluded that this DNA fragment was not derived from ovine the cathepsin B gene. However, the fragment amplified was found to show a limited degree of homology the the human retinoblastoma susceptability gene, a tumor supressor gene. A second peR reaction was optimised using ovine genomic DNA and a new primer, derived from a region of the cathepsin B gene sequence showing 100 % homology between humand and bovine cDNA. This primer was used in combination with the upstream primer designed by Mordier et al. 1993). A DNA fragment of 400 bp was found to be amplified in the second PCR reaction, and a 363 bp nucleotide sequence of this DNA fragment was found to show a high degree of homology to exon 7 of the bovine cathepsin B gene. It was concluded that the second PCR fragment was derived from the ovine cathepsin B gene. Five nucleotides from the ovine sequence were unable to be resolved and restriction enzymes, cleaving DNA sequences at the sites where these nucleotides occurred, were identified. Further restriction analysis of the ovine cathepsin B gene fragment will reveal whether these unresolved nucleotides are situated in polymorphic regions of the gene.