The Specificity and Catalytic Properties of Alu I Methylase

윤호섭,서향,김기태,한문희,유욱준,Yoon, Ho-Sup,Suh, Hyang,Kim, Ki-Tae,Han, Moon-H.,Yoo, O.-Joon 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.1

순수 정제된 Alu I methylase (Yoon et al., 1985)의 specificity와 그 효소적 특성을 연구하였다. 이 효소는 DNA의 염기 배열 5'-d($AG{\downarrow}CT$)-3'을 인식 하였으며 그 중 cytosine nucleotide에 methylation을 시키고 있음이 Maxam-Gilbert의 방업에 의한 DNA sequencing을 통하여 밝혀졌다. Alu I methylase는 Alu I site 당 두 개 의 methyl group을 붙여 주였으며 Eco RI에서와 같은 overmethylation현상은 보여주지 않았다. 효소반응은 pH 7.4에서 7.6사이, NaCl은 50 mM에서 최적 이었다. Alu I methylase는 Michaelis-Menten kinetics를 따랐으며 S-Adenosyl-methionine에 대한 $K_m$ 값은 $0.44\;{\mu}m$이었고 pBR 322 DNA를 사용 하였을 때의 DNA에 대한 $K_m$의 값은 $4.03\;{\mu}m$이었다. 이 $K_m$값들로부터 얻어진 turnover number는 각각 1.83/min과 1.61/min이었다. The specific methylation site for Alu I methylase was the cytosine nucleotide in Alu I sequence. The position of the methylated cytosine nucleotide was determined by the chemical cleavage reactions of the Maxam-Gilbert DNA sequencing procedure. As expected, the methylated cytosine nucleotide bands were disappeared on C+T and C lanes on 12% sequencing gels. Alu I methylase was maximally active at near pH 7.5 in the presence of 50 mM NaCl. The methylase did not require $Mg^{++}$ for activity, and obeyed Michaelis-Menten Kinetics with respect to both AdoMet and DNA. At $37^{\circ}C$, the $K_m$ for AdoMet was $0.44\;{\mu}m$, that for the Alu I site of pBR 322 DNA was 4.03 nM, and the corresponding turnover numbers were 1.83 methyl transfer per minute per monomer and 1.61 transfers per minute per monomer, respectively.

Purification and Characterization of Alu I Methylase

윤호섭,서향,한문희,유욱준,Yoon, Ho-Sup,Suh, Hyang,Han, Moon-H.,Yoo, O.-Joon 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.1

Alu I methylase를 순수 정제하였다. 300g (wet weight)의 Arthrobacter luteus에서 얻은 crude extract로부터 ammonium sulfate fractionation을 거친 후 phosphocellulose, DEAE-cellulose, Heparin-agarose, Hydroxylapatite등의 chromatography과정을 통하여 0.88mg의 Alu I methylase를 얻었다. Specific activity는 mg당 $1.32{\times}10^5\;unit$이었다. 이 methylase에 의하여 methylation된 DNA는 Alu I endonuclease에 의하여 절단되지 않았으며, 그 methylation site는 5'-d($AG^{\downarrow}CT$)-3'임이 확인되었다. 정제된 Alu I methylase는 10% SDS-polyacrylamide gel electrophoresis에서 한개의 major band로 나타났으며 그 분자량은 $56,000{\pm}1,000$ 이었다. Alu I methylase has been isolated from 300g (wet weight) cells of Arthrobacter luteus. After ammonium sulfate fractionation, the protein which has methylase activity was purified through phosphocellulose, DEAE-cellulose, Heparin agarose, and Hydroxy-lapatite column chromatography. The methylated DNA by the purified methylase was resistatnt against Alu I endonuclease. The purified Alu I methylase was essentially homogeneous as judged by 10% SDS-polyacrylamide gel electrophoresis, and the apparent subunit molecular weight was $56,000{\pm}1,000$. The specific activity of the enzyme was $1.32{\times}10^5$ units per mg protein.

Purification and Characterization of Hpa I endonuclease

윤호섭,강선철,유욱준,Yoon, Ho Sup,Kang, Sun Chul,Yoo, Ouk Joon 한국미생물 · 생명공학회 1985 한국미생물·생명공학회지 Vol.13 No.1

Hpa I endonuclease를 순수 정제하였다. 150g (wet weight)의 Haemophilus parainfluenzae로 부터 얻은 crude extract를 ammonium sulfate fractionation을 거친후 Heparin agarose, SP-sephadex, DEAE-sephadex, phosphocellulose 등 chromatography를 거쳐 최종적으로 0.2mg의 효소를 얻었다. Specific activity는 $2.2{\times}10^6units/mg$이었다. 얻어진 Hpa I endonuclease는 SDS-polyacrylamide gel에서 한개의 band를 보였고, 그 효소활성은 50mM NaCl과 pH 7.0과 7.5사이에서 가장 높았다. Hpa I endonuclease from Haemophilus parainfluenzae has been purified of homogeneity and its physical and ezymatic properties have been studied. For the purification of the enzyme, Heparin agarose, SP-sephadex C-25, DEAE-sephadex A-50 and phosphocellulose chromatography columns were used. The denatured and reduced form of the enzyme is a monomer of molecular weight of $30,000{\pm}1,000$ as judged by 10% polyacrylamide gel electrophoresis containing 0.1% sodium dodesyl sulfate. Hpa I endonuclease was maximally active at neutral pH (7.0 to 7.5) in the presence of 50 mM NaCl.

Hpa Ⅰ endonuclease 의 정제와 특성

윤호섭(Ho Sup Yoon),강선철(Sun Chul Kang),유욱준(Ouk Joon Yoo) 한국미생물생명공학회 1985 한국미생물·생명공학회지 Vol.13 No.1

제한효소 Hind III , Sst I , Pvu II , Sac I 의 DNA 절단 반응에 있어서 Alu I methylation 의 억제 효과

강선철,윤호섭,유욱준 ( Sun Chul Kang,Ho sup Yoon,Oog . Joon Yoo ) 생화학분자생물학회 1986 BMB Reports Vol.19 No.1

The cleavage reactions of Hind III, Sst I, Pvu II and Sac I endonucleases were inhibited by the methylation of Alu I methylase. To analyze the cleavage reactions quantitatively, tritium labeled pBR 322 and pPG 3282 plasmid DNAs (pBR 322 DNA contains Hind III and Pvu II sites, while pPG 3282 DNA which is a hog gastrin cDNA clone contains Sst I and Sac I sites) were used, and the degree of the inhibition against cleavages by the four kinds of endonucleases was measured. The results imply that we can predict the specificities of the related hexameric restriction methylases which have not been isolated yet. And the predictions are the followings: (1) The methylation sites of Sst I methylase is not identical with Alu I methylase since Sst I endonuclease slowly cut the sequences methylated by Alu I methylase. (2) The methylation sites of Pvu II and Sac I methylases could be identical with ALu I methylase since Pvu II and Sac I endonucleases could not cut the sequences methylated by Alu I methylase.

DNA Protection by Methylation with Alu I Methylase against Cleavages of Hind III, Sst I, Pvu II and Sac I Endonucleases

강선철,윤호섭,유욱준,Kang, Sun-Chul,Yoon, Ho-Sup,Yoo, O. Joon 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.1

Alu I methylase로 변형시킨 DNA는 Alu I 특정 염기 배열을 내부에 공통적으로 가지고 있는 제한효소들 즉, Hind III, Sst I, Pvu II, Sac I들에 의하여 정상적으로 절단되지 않았다. 이 효소들의 반응 결과를 정량적으로 분석하기 위하여 tritium으로 label된 pBR 322 DNA와 pPG 3282 DNA를 사용하였는데 pBR 322 DNA에는 Hind III와 Pvu II site가 각각 1 개씩, pPG 3282 DNA에는 Sst I과 Sac I site가 각각 1개씩 있는 점이 절단된 후의 분석하기에 용이하기 때문이다. 각각의 경우를 분석한 결과로부터 다음과 같은 결론을 얻을 수 있었다 : (1) Sst I endonuclease가 Alu I methylase에 의해 methylation된 DNA를 느리게라도 절단할 수 있는 점으로 보아 Sst I methylase가 발견된다면 그 specific methylation site는 Alu I methylase의 경우와 달리 internal cytosine이 아닐 것이다. (2) Pvu II와 Sac I endonuclease는 Alu I methylase에 의해 methylation 된 DNA를 전혀 절 단하지 못하는 점으로 보아 Pvu II와 Sac I methylase가 발견된다면 그 specific methylation site는 두 경우 모두 internal cytosine일 가능성이 높다. The cleavage reactions of Hind III, Sst I, Pvu II and Sac I endonucleases were inhibited by the methylation of Alu I methylase. To analyze the cleavage reactions quantitatively, tritium labeled pBR 322 and pPG 3282 plasmid DNAs (pBR 322 DNA contains Hind III and Pvu II sites, while pPG 3282 DNA which is a hog gastrin cDNA clone contains Sst I and Sac I sites) were used, and the degree of the inhibition against cleavages by the four kinds of endonucleases was measured. The results imply that we can predict the specificities of the related hexameric restriction methylases which have not been isolated yet. And the predictions are the followings: (1) The methylation sites of Sst I methylase is not identical with Alu I methylase since Sst I endonuclease slowly cut the sequences methylated by Alu I methylase. (2) The methylation sites of Pvu II and Sac I me thy lases could be identical with Alu I methylase since Pvu II and Sac I endonucleases could not cut the sequences methylated by Alu I methylase.

Implementation of a Spring Backboned Soft Arm Emulating Human Gestures

Hyun-Soo Yoon(윤현수),Jae Yeon Choi(최재연),Se Min Oh(오세민),Byung-Ju Yi(이병주),Ho Sup Yoon(윤호섭),Young-Jo Cho(조영조) 한국로봇학회(논문지) 2012 로봇학회 논문지 Vol.7 No.2

구조용강의 Waterjet Peening에 의한 압축잔류응력 유발조건

서용위(Yongwie Seo),신영삼(Young-Sam Shin),윤호섭(Ho-Sup Yoon) 대한기계학회 2008 대한기계학회 춘추학술대회 Vol.2008 No.10

Widely used structural steel SM45C was waterjet peened in air to investigate the optimum peening conditions for the induced compressive residual stress which is beneficial to improve the fatigue strength of the material. After peening the residual stresses generated by the waterjet peening on the surface were measured using X-ray diffraction technique and the results were analyzed to characterize the peening conditions for the maximum compressive stress. Also, the surface roughnesses of peened specimen were measured and the results showed the superior surface quality with minimal changes in surface topography before and after the waterjet peening process.

지능형 로봇을 위한 GCC-PHAT 기반 음원추적 기술의 성능분석

박범철(Park Beom-Chul),반규대(Ban Kyu-Dae),곽근창(Kwak Keun-Chang),윤호섭(Yoon Ho-Sup) 한국로봇학회(논문지) 2007 로봇학회 논문지 Vol.2 No.3

Expression of Cellular Oncogenes in Transitional Cell Carcinoma of Human Urinary Bladder

윤호섭,서정선,홍소희 한국유전학회 1986 Genes & Genomics Vol.8 No.1

DNA sequences homologous to the known viral oncogenes (v-onc) of certain retroviruses have been found in a variety of normal uninfected cells, including those of man. These genestermed cellular oncogenes (c-onc) appear to have been the evolutionary progenitors of the v-onc genes. It is suggested that cellular oncogenes may possess an oncogenic potential because of the structural similarity between c-onc genes and their viral homologues. One line of evidence supporting this idea is that transforming genes in human tumor cell lines and tumors have been identified as the c-onc genes known as c-ras^(Ha) and c-ras^(Ki). Activation of cellular oncogene found in human urinary bladder carcinoma seems to be activated by point mutation. Furthermore, considering the multistep carcinogenesis in human tumor in vitro, it seems to be very important to the expression level of multiple c-onc genes in freshly obtained human tumors. We, therefore, investigated the level of gene expression of five c-onc genes (c-ras^(Ha), c-ras^(Ki), c-myb, c-myc and c-fps) in 6 transitional cell carcinomas of human urinary bladder, using the presence of messenger RNA(mRNA) transcripts as a parameter of gene expression. DNA probes used in DNA-RNA hybridization technique were all cellular oncogenes except ras^(Ki) gene (v-ras^(Ki)). More than one cellular oncogenes are transcriptionally active in all of tumors examined and transcriptional activity of certain oncogenes was greater in the malignant than the normal tissue.