Molecular cloning and sequence analyses of cDNA coding for canine gastrin, and studies on the DNA protection by type Ⅱ methylases

Abstract

In this thesis, the content will be classified into two main parts ; (1) molecular cloning and sequence analyses of cDNA coding for canine gastrin, (2) studies on the DNA protection by type II restriction and modification enzymes. For the first part, cDNA encoding the precursor of canine gastrin was cloned. The full length cDNA sequence has been determined by overlapping the partial gastrin sequences of the two cDNA clones, pDG 1917 and pDG 1647. The nucleotide sequences of the two cDNA clones, pDG 1917 and pDG 1647. The nucleotide sequence of the combined cDNA insert (446 nucleotides) revealed 315 nucleotides in the entire mRNA coding region, 49 nucleotides in the 5``-untranslated region and 82 nucleotides in the 3``untranslated region. Comparison of this sequence with those of porcine, human and rat gastrin reveals extensive (83\%) homology in the gastrin coding region as well as short regions of conserved nucleotides in the noncoding regions. The canine sequence encodes a preprogastrin of 104 amino acids which consists of a signal peptide, a 37 amino acid prosegment, and the gastrin 34 sequence, followed by a glycine (the amide donor), and flanked by pairs of arginine residues. Cleavage at an internal pair of lysine residues yields gastrin 17. A considerable homology between the deduced amino acid sequence of canine precursor and that from other mammalian species has been found; the canine sequence shows 85\% homology with porcine gastrin, 79\% homology with human gastrin and 75\% homology with rat gastrin. For the second part, the DNAs methylated by central tetranucleotide specific methylases (Alu I, Hpa II, Hha I) were resistant against cleavages of related hexameric endonucleases indicating that the methyl group of the C5 position of the inmost cytosine nucleotides interferes with the interaction between the enzymes and the hexameric recognition sequences. The results imply that one can predict the specificities of the related hexameric methylases which h...