Acc Ⅰ 제한효소의 정제와 촉매적 성질에 관한 연구

학위논문(석사) - 한국과학기술원 : 생물공학과, 1985.2, [ 53 p. ]A large number of site-specific restriction endonucleases have been isolated from a wide variety of microorganisms because of their extensive usefulness in the structural analysis of DNA molecules, and for the construction of recombinant DNA in vitro. Through this thesis, I present a procedure of purification of restriction endonuclease AccI from Acinetobacter calcoaceticus ATCC 23055. I also describe the determination of physical and catalytic properties of this enzyme. For the purification of the enzyme, 300 g cells of wet weight were used and were broken by French press at 20,000 p.s.i.. After ammonium sulfate fractionation, the enzyme was further purified by heparin agarose column chromatography, DEAE-sephadex A-50 column chromatography, Affi - Gel Blue column chromatography, phosphocellulose column chromatography, and finally hydroxyl apatite column chromatography. The isolated enzyme has shown to have site-specific restriction endonuclease activity on the AccI recognition sequence which has a degeneracy i.e., : d (5``-G-T-A-T-A-C-3``) or d(5``-G-T-C-G-A-C-3``). AccI endonuclease has a single polypeptide species and its molecular weight of subunit was $45,000 \pm 1,000$ daltons, as judged by 10\% polyacrylamide gel electrophoresis in the presence of 0.1\% sodium dodecyl sulfate. In order to study the catalytic properties of AccI endonuclease, I was used (3H)-labeled DNA by HpaII methylase as a substrate. The enzyme showed maximum activity at pH values between 8.0 and 11.0, and in the presence of 5.0 to 20.0 mM magnesium chloride. But AccI endonuclease did not require salt (NaCl) and 2-mercaptoethanol for its activity. Furthermore the enzyme retained its maximum activity to the concentration of 100 mM sodium chloride and to the concentration of 300 mM 2-mercaptoethanol.한국과학기술원 : 생물공학과

개의 가스트린 유전자의 cDNA 합성과 염기서열에 관한 분자생물학적 연구 및 Type Ⅱ 메틸화 효소에 의한 DNA 보호효과에 관한 연구

학위논문(박사) - 한국과학기술원 : 생물공학과, 1988.2, [ xi, 148 p. ]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...한국과학기술원 : 생물공학과