Purification and Characterization of Pst I Methylase from Providencia stuartii 164

김용석,노현모,Kim, Yong-Sok,Rho, Hyune-Mo 생화학분자생물학회 1984 한국생화학회지 Vol.17 No.2

본 논문에서는 Providencia stuartii 164 균주로부터 Pst I methylase를 분리하여 특성을 관찰하였다. 분리 방법으로는 $(NH_4)_2SO_4$ 분획법, DEAE-Cellulose 크로마토그라피법, Phosphocellulose 크로마토그라피법, 그리고 Heparin agarose 크로마토그라피법을 순서적으로 사용했다. 분리된 효소는 site-specific한 methylase였는데, 이것은 Pst I 제한효소로 자른 pBR322 DNA는 분리한 methylase에 의해 methylation이 일어나지 않은 사실과 분리한 methylase로 methylation 시킨 pBR322 DNA는 Pst I 제한효소로 잘라지지 않는다는 사실로 확인되었다. 또한 Pst I restriction-modification 유전자를 가지는 plasmid인 pRL829 DNA가 이 효소에 의해 더 이상 methylation이 일어나지 않은 사실은 분리해 낸 효소가 Pst I methylase임을 확신시켜 주었다. 이 효소는 pH 7.5에 서 가장 높은 활성도를 보여 주었고 50 mM 농도의 NaCl이 있으면 활성도가 증가했다. 또한 Pst I methylase는 외가닥으로 된 ${\Phi}X$ 174 DNA를 잘 methylation 시키는 것으로 나타났다. In this report, the purification and characterization of Pst I methylase from Providencia stuartii 164 strain was described. Pst I methylase was purified by the following procedures; ammonium sulfate fractionation, DEAE-cellulose chromatography, Phosphocellulose chromatography and Heparin agarose chromatography. Pst I methylase was a site-specific methylase which has been proven by the facts that pBR322 DNA cleaved by Pst I restriction endonuclease was not methylated by Pst I methylase, and pBR322 DNA methylated by Pst I methylase was not cleaved by Pst I restriction endonuclease. The plasmid pRL829, which contains Pst I restriction-modification genes and thus was fully methylated in vivo, was not methylated by Pst I methylase. These results strongly suggest that the methylase has a site specificity on Pst I recognition site. Pst I methylase activity has optima at pH 7.5 and in the presence of 50 mM NaCl. In addition, Pst I methylase has also been found to have methylation activity on single-strand ${\Phi}X174$ DNA.

Providencia stuartii 164로부터 Pst ⅠMethylase 의 분리 및 특성에 관한 연구

김용석,노현모 ( Yong Sok Kim,Hyune Mo Rho ) 생화학분자생물학회 1984 BMB Reports Vol.17 No.2

In this report, the purification and characterization of Pst I methylase from Providencia stuartii 164 strain was described. Pst I methylase was purified by the following procedures; ammonium sulfate fractionation, DEAF-cellulose chromatography, Phosphocellulose chromatography and Heparin agarose chromatography. Pst I methylase was a site-specific methylase which has been proven by the facts that pBR322 DNA cleaved by Pst I restriction endonuclease was not methylated by Pst I methylase, and pBR322 DNA methylated by Pst I methylase was not cleaved by Pst I restriction endonuclease. The plasmid pRL829, which contains Pst I restriction-modification genes and thus was fully methylated in vivo, was not methylated by Pst I methylase. These results strongly suggest that the methylase has a site specificity on Pst I recognition site. Pst I methylase activity has optima at pH 7.5 and in the presence of 50 mM NaCl. In addition, Pst I methylase has also been found to have methylation activity on single-strand ΦX174 DNA.

한국형 B형 간염바이러스 ( Subtype - adr - k ) 의 내면항원 유전자의 염기서열

김용석,현상원,노현모 ( Yong Sok Kim,Sang Won Hyun,Hyune Mo Rho ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.3

The nucleotide sequence of core antigen-coding region of hepatitis B virus (subtype adr-k) cloned in E. coli has been determined by the dideoxy chain termination method using M13 phage template. This sequence we determined is about 1.3 kb long and also includes the open reading frame for gene B (designated gene X previously). Our results showed 96% homology to adr, 92% to adw and 91% to ayw subtype compared with corresponding region of reported sequence. The nucleotide sequence we determined showed differences from reported adr subtype DNA by 27 nucleotides addition at the position of 1791-1817, that was also reported in adw and ayw subtype HBV DNA, and differed by one nulceotide (T) addition at the position 1826. Signal Sequences such as RNA polymerase binding sequences, direct repeat sequences and poly(A) addition site were also detected. Possible roles of these sequences were discussed.

Nucleotide Sequence of the Core Antigen-Coding Region of Hepatitis B Virus (Subtype adr-k)

김용석,현상원,노현모,Kim, Yong-Sok,Hyun, Sang-Won,Rho, Hyune-Mo 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.3

대장균내에 크론잉된 한국형 B형 간염바이러스(adr-k)의 내면항원 유전자(HBcAg)의 염기서열을 M13파아지/dideoxy chain termination 방법을 이용하여 결정하였다. 결정한 염기서열은 약 1.3 kb로서 내면 유전자의 전부분과 후부분을 포함하여 intron이 없는 것으로 생각된다. 보고된 다른 subtype의 염기서열과 비교해 볼 때 adr와는 96%, adw와는 92%, 그리고 ayw와는 91%의 염기서열이 같음을 알 수 있었다. 이미 보고된 adr에 없던 27개의 염기서열이 adr-k에는 1791-1817위치에 나타났는데, 이는 adw나 ayw에서는 나타났었다. 또한 1826 위치에 하나의 염기(T)가 더 끼어 있음이 특징이다. HBcAg 유전자의 앞부분에 조절 및 기능부위로 보이는 RNA polymerase binding sequence, direct repeat sequences 및 poly(A) addition site가 발견되었으며, 이들의 signal siquences에 대한 가능한 역할에 대해서 논의하였다. The nucleotide sequence of core antigen-coding region of hepatitis B virus (subtype adr-k) cloned in E. coli has been determined by the dideoxy chain termination method using M13 phage template. This sequence we determined is about 1.3 kb long and also includes the open reading frame for gene B (designated gene X previously). Our results showed 96% homology to adr, 92% to adw and 91 % to ayw subtype compared with corresponding region of reported sequence. The nucleotide sequence we determined showed differences from reported adr subtype DNA by 27 nucleotides addition at the position of 1791-1817, that was also reported in adw and ayw subtype HBV DNA, and differed by one nulceotide (T) addition at the position 1826. Signal Sequences such as RNA polymerase binding sequences, direct repeat sequences and poly(A) addition site were also detected. Possible roles of these sequences were discussed.

Blunt End Ligation 의 최적반응에 관한 연구

송옥규,김용석,노현모 ( Ok Kyu Song,Yong Sok Kim,Hyune Mo Rho ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.3

To determine the optimal condition of blunt-ended DNA ligation for the purpose of the efficient cloning, blunt-ended vector and insert DNA fragments were ligated under the various conditions of buffer, temperature, reaction time, and the molar ratio of vector to insert DNA. The efficiency of ligation was monitored by both agarose gel electrophoresis and the transforming frequency of genetic markers in plasmid pRSK 116 in vivo. The results showed that the optimal incubation temperature and the concentration of ATP were 20-25℃ and 0.5 mM, respectively. Spermidine (1 mM) and polyethylene glycol (3%) stimulated blunt-end ligation, but more than 10 mM of spermidine and 2 mM of ATP completely inhibited the reaction. Higher concentration of salt also inhibited blunt-end ligation. According to the transforming frequency assay, the optimal molar ratio of vector to insert DNA was 1, which was different from that of sticky-end ligation. However, the variation of pH (6.6-8.2) and bovine serum albumin concentration (0-500 ㎍/㎖) showed little influence on the ligation.

Studies on the Optimal Condition for Ligation of Blunt Ended DNA Fragments

송옥규,김용석,노현모,Song, Ok-Kyu,Kim, Yong-Sok,Rho, Hyune-Mo 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.3

Blunt end DNA ligation의 최적반응조건을 구하기 위하여 반응온도, 완충용액상태, vector와 insert DNA의 농도비율, 반응시간등의 최적조건을 연구하였다. 실험결과 반응최적온도는 $20-25^{\circ}C$였고, ATP의 최적농도는 0.5 mM이었다. Spermidine의 농도가 1 mM 인 경우와 4%의 PEG 4,000을 ligation buffer에 넣어준 경 우 반응이 잘 되는 것으로 나타났으나 spermidine이 10 mM이거나 ATP의 농도가 2 mM 이상에서 반응은 저해되었다. 높은 농도의 NaCl 또한 반응을 저해시키는 것으로 나타났다. Vector와 insert DNA의 농도비를 달리하여 ligation시켜 이것을 직접 E. coli를 형질 전환시켜 본 결과 농도비가 1일때 insert가 끼어 들어간 확율이 가장 높았다. Buffer의 pH변화(6.6-8.2)나 BSA의 농도변화 ($0-500\;{\mu}g/ml$)는 반응에 별 영향이 미치지 않는 것으로 나타났다. To determine the optimal condition of blunt-ended DNA ligation for the purpose of the efficient cloning, blunt-ended vector and insert DNA fragments were ligated under the various conditions of buffer, temperature, reaction time, and the molar ratio of vector to insert DNA. The efficiency of ligation was monitored by both agarose gel electrophoresis and the transforming frequency of genetic markers in plasmid pRSK 116 in vivo. The results showed that the optimal incubation temperature and the concentration of ATP were $20-25^{\circ}C$ and 0.5 mM, respectively. Spermidine (1 mM) and polyethylene glycol (3%) stimulated blunt-end ligation, but more than 10 mM of spermidine and 2 mM of ATP completely inhibited the reaction. Higher concentration of salt also inhibited blunt-end ligation. According to the transforming frequency assay, the optimal molar ratio of vector to insert DNA was 1, which was different from that of sticky-end ligation. However, the variation of pH (6.6-8.2) and bovine serum albumin concentration ($0-500\;{\mu}g/ml$) showed little influence on the ligation.

Complete Nucleotide Sequence of Hepatitis B Virus (subtype adr)

김기태,현상원,김용석,노현모,Kim, Kee-Tae,Hyun, Sang-Won,Kim, Yong-Sok,Rho, Hyune-Mo 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4

대장균내에 클로닝된 한국형 B형 간염 바이러스(subtype adr)의 전체염기서열을 M13 파아지/dideoxy chain termination 방법으로 결정하였다. 이 바이러스의 전체 genome은 3213 bp였으며 4개의 open reading frames(S, C, P and B)를 가지고 있었다. 우리의 전체염기서열을 보고된 다른 subtype과 비교할 때 adr과는 97%, adw와는 91%, ayw와는 90%의 염기서열이 동일한 것을 알 수 있었다. 여러 subtype간에 염기서열과 아미노산서열을 비교 연구하였다. 특히 하나의 염기(T)가 더 끼어 있어 B 유전자의 reading frame이 변경되었다. Direct repeat sequence와 poly(A) addition site 그리고 enhancer element가 확인되었다. The complete nucleotide sequence of hepatitis B virus DNA(subtype adr) from a Korean patient has been determined by dideoxy chain termination method. The viral genome is 3213 base pairs long and includes four open reading frames for surface antigen, core antigen, putative DNA polymerase, and B(or X) protein of unknown function in the L strand. It is unique that one nucleotide addition altered B open reading frame. Signal sequences such as direct repeat sequence and poly(A) addition site, and enhancer element were well conserved.

Cloing and Expression of Bdi Ⅰ Methylase Gene in E . coli

노현모,김용석,최경래 한국유전학회 1984 Genes & Genomics Vol.6 No.3

The gene for the Bdi I modification enzyme, which is a counterpart of Bdi I restriction endonuclease, from Brevibacterium divaricatum FERM 5948 was cloned and expressed in E. coli. First, we have isolated Bdi I restriction endonuclease by cleavage assay in vitro with three column steps and have proved that Bdi I restriction enzyme is an isoschizomer of Cla I whose recognition sequence is 5' ATCGAT 3'. For cloning of Bdi I methylase gene, we have initially used three cloning sites (Sal I, Bam HI and Eco RI) of pBR 322 and adopted the retransformation method after Bdi I restriction endonuclease cleavage. Selection of transformants carrying the gene was based on the resistance of the modified plasmid encoding the enzyme to cleavage by Bdi I restriction enzyme. Among colonies after retransformation, the recombinant plasmid, pBDIM 116 containing 5.2 Kb EcoRI insert was proved to carry the gene. Using the restriction map constructed, we inserted λ-Sal I fragment containing another Bdi I site into pBDIM 116 and subcloned various DNA fragment for Bdi I methylase gene mapping. The expression of Bdi I methylase gene in E. coli and the relationship between Bdi I restriction-modification genes will be also discussed.

Blunt - end 를 가진 DNA 의 재조합을 위한 유전자 운반체의 개발

노현모,김용석,송옥규 한국유전학회 1984 Genes & Genomics Vol.6 No.2

A blunt-end DNA cloning vector, pRSK 116, was constructed. This vector was derived from pBR 322 by opening with Sal I restriction endonuclease, filled with only TTP by Klenow fragment, trimmed with S1 unclease, and ligated with T4 DNA ligase. It appeared that the removal of three bases in the middle region of Tc^r gene did not change the genetic property of the marker gene of the pBR 322 and the unique Hinc II site in the Ap^r gene of the modified plasmid pRSK 116 en. dowed improved property for the selection of recombinants. The Hinc II site has been tested for the cloning of blunt-ended human DNA. The recombinant colonies selected were Ap sensitive and Tc resistant and inserted DNAs were easily recovered by Hinc II treatment.

Construction of pRSK116 and Modified Method for Blunt - end Ligation of HincⅡ Site of the Plasmid

노현모,김용석,송옥규 한국유전학회 1983 Genes & Genomics Vol.5 No.3

We have constructed plasmid pRSK116 which has only one site of HincII restriction endonuclease within the β-lactamase gene which is resistant to ampicillin. The pRSK116 was derived from pBR322 by the following procedures. Plasmid pBR322 was opened by treatment with SalI endonuclease, filled with only TTP by Klenow fragment, trimmed with S1 nuclease, and ligated with T4 ligase. We have used the HincII site of the plasmid, pRSK116, for the study of blunt-end ligation since the pBR322 does not have blunt-end restriction site within marker genes. We attempted to ligate blunt-end DNA fragments with T4 DNA ligase under various conditions (e.g. temperature, DNA concentration, and incubation time), and measured the ligation efficiency by agarose-gel electrophoresis and transformation in E.coli HB101 in the presence of proper antibiotics.