글루타치온의 효소적 생합성에 관계되는 E . coli γ-Glutamylcysteine Synthetase의 특성 연구

남용석,곽준혁,이세영 ( Yong Suk Nam,Joon Hyeok Kwak,Se Yong Lee ) 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.40 No.6

γ-Glutamylcysteine synthetase was purified from E. coli K-12 strain and its properties related to the in vitro synthesis of glutathione by enzymatic method were investigated. The activity of purified γ-glutamylcysteine synthetase was increased with increasing concentration of L-glutamate up to 60 mM, while it was decreased by about 50% and 40% under 60 mM of L-cysteine and 45 mM of glycine, respectively. The enzyme activity was reduced not only by ADP, one of the reaction products, but also by the reduced form of glutathione. Therefore, because the reduced glutathione as well as glycine which is the substrate for glutathione synthetase inhibit the activity of γ-glutamylcysteine synthetase, it is recommended to design a bioreactor system with two separate reactions for glutathione synthesis : one with γ-glutamylcysteine synthetase reaction and the other glutathione synthetase reaction. In addition since ADP, resulted from these reactions, reduces the activity of γ-glutamylcysteine synthetase, it is necessary to introduce an ATP regeneration system for glutathione synthesis.

Studies on the Properties of E. coli ${\gamma}-Glutamylcysteine$ Synthetase in Relation to the Enzymatic Synthesis of Glutathione

남용석,곽준혁,이세영,Nam, Yong-Suk,Kwak, Joon-Hyeok,Lee, Se-Yong 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.40 No.6

E. coli K-12 균주에서 ${\gamma}-Glutamylcysteine$ synthetase를 정제하고 효소적 방법에 의한 글루타치온 합성에 관련된 특성을 검사하였다. 정제한 효소의 활성은 L-glutamate의 농도가 60 mM 까지 증가와 더불어 증가하였으나, 60 mM L-cysteine 에서는 50% 그리고 45 mM glycine 에서는 40%의 효소활성이 감소되었다. 효소의 활성은 반응산물 중의 하나인 ADP 뿐만 아니라 환원형 글루타치온에 의해서 감소되었다. 그러므로 환원형 글루타치온 뿐만 아니라 glutathione synthetase의 기질인 glycine은 ${\gamma}-glutamylcysteine$ synthetase 활성을 저해하므로 글루타치온 생산을 위해서는 ${\gamma}-glutamylcysteine$ synthetase 반응과 glutathione synthetase의 두 분리된 반응으로 이루어진 생반응계를 고안하는 것이 바람직하다. 또한 글루타치온 합성반응으로 부터 생성되는 ADP는 ${\gamma}-glutamylcysteine$ synthetase의 활성을 감소시키므로 글루타치온 합성을 위해서 ATP 재생계의 도입이 필요하다. ${\gamma}-Glutamylcysteine$ synthetase was purified from E. coli K-12 strain and its properties related to the in vitro synthesis of glutathione by enzymatic method were investigated. The activity of purified ${\gamma}-glutamylcysteine$ synthetase was increased with increasing concentration of L-glutamate up to 60 mM, while it was decreased by about 50% and 40% under 60 mM of L-cysteine and 45 mM of glycine, respectively. The enzyme activity was reduced not only by ADP, one of the reaction products, but also by the reduced form of glutathione. Therefore, because the reduced glutathione as well as glycine which is the substrate for glutathione synthetase inhibit the activity of ${\gamma}-glutamylcysteine$ synthetase, it is recommended to design a bioreactor system with two separate reactions for glutathione synthesis : one with ${\gamma}-glutamylcysteine$ synthetase reaction and the other glutathione synthetase reaction. In addition since ADP, resulted from these reactions, reduces the activity of ${\gamma}-glutamylcysteine$ synthetase, it is necessary to introduce an ATP regeneration system for glutathione synthesis.

Molecular Breeding of Glutathione Producing Bacterial Strains

남용석(Yong Suk Nam),(Se Yong Lee) 한국미생물생명공학회(구 한국산업미생물학회) 1991 한국미생물생명공학회 학술발표초록집 Vol.1991 No.1

시중 음식점에서 판매되는 쇠고기의 유전자 분석을 이용한 한우육 감별

김진만,남용석,최지훈,이미애,정종연,김천제,Kim Jin-Man,Nam Yong-Suk,Choi Ji-Hun,Lee Mi-Ae,Jeong Jong-Yon,Kim Cheon-Jei 한국축산식품학회 2005 한국축산식품학회지 Vol.25 No.2

유전자수준의 염기서열에 근거를 둔 유전자 감식기법을 활용하여 개체간의 유전적 차이로 한우육과 젖소육 혹은 수입육의 구별방법을 개발하기 위한 연구가 많이 수행되었다. 그 중 real-time PCR 판별방법은 기존 PCR의 단점인 허위양성을 배제시켜 그 결과의 신뢰성이 높으며, PCR 산물들의 제한효소에 의한 절단 및 겔에서의 확인 등의 과정을 생략시킬 수 있어 짧은 시간 내에 다량의 시료를 분석할 수 있고 민감도가 높아 쇠고기가 포함된 가공식품의 분석도 가능하다는 장점이 있다. 본 연구는 시중에서 한우로 유통되는 쇠고기(생육 또는 양념육)의 DNA를 추출하여 real-time PCR을 통해서 모색유전자의 유전자 형(C-type, C/T-type or T-type)으로 한우육과 젖소육 및 수입육을 구분하였다. 한우육만을 판매한다는 시중 음식점 41개소에서 쇠고기(생육 또는 양념육)를 수거하여 분석한 결과, 분석된 41개의 시료 중 29개가 한우 유전자형으로, 12개의 시료가 젖소 또는 수입육 유전자형으로 판별되었다. 따라서 분석된 시료들의 한우육(T-type) 비율은 $70.1\%$, 그리고 젖소육 또는 수입육 비율은 $29.3\%$(C/T-type; $12.2\%$, C-type; $17.1\%$)이었다. Real time-polymerase chain reaction (RT-PCR) is currently considered as the most sensitive method to detect low abundant DNAs in samples. Compared to conventional PCR, real-time PCR has a high reliability because of excluding false-positive results and can allow a simultaneous faster detection and quantification of target DNAs. This study was carried out to identify the Hanwoo (Korean native cattle) beef by genotyping after DNA extraction of commercial beef in 41 restaurants. Since Hanwoo, Holstein and imported cattle meat have different patterns in the MC1R gene associated with the coat colors of cattles (C-type, C/T-type or T-type), we could identify the genotype using real-time PCR The result of real-time PCR assay for beef samples in 41 restaurants which are asserted to sell Hanwoo beef only, showed that 29 of 41 samples were Hanwoo beef gene type (T-type) and 12 of 41 samples were Holstein or imported cattle gene type (C-type or C/T-type). Therefore, the proportion of Han-woo beef was $70.7\%$ and the proportion of Holstein or imported cattle meat was $29.3\%(C/T-type; 12.2\%,\;C-type; 17.1\%)$.

삼풍백화점 붕괴사고 희생자들의 신원확인을 위한 유전자검사

남용석,이혜린,김경훈,김희선,이희석,황적준 大韓法醫學會 1996 대한법의학회지 Vol.20 No.1

A DNA typing was performed to identify decomposed body remains from Sampoong Department mass disaster in June 1995. These body parts include bone fragment, skin tissue, hairs, from which the extracted DNAs were highly degraded. Two VNTR loci, 4STR loci, and amelogenin gene were chosen for AMP-FLP, and mtDNA sequence analysis for the confirmation of maternal relationship. The results of AMP-FLP of the selected polymorphic loci showed different sucess rate for PCR. DIS80 and D17S5 loci were amplified successfully form 64.5%, and 67% of the samples, respectively. HUMTHOI, HUMCSF1PO, and HUMTPOX loci were amplified successfully from 90.3% of the samples each. HUMACTBP2 and amelogenin was amplified in 87% of the cases submitted. THE DNA types of 33 remains were compared with those of 81 bereaved families consisting of 173 member. Thirty three samples were reduced to 28 in numbers according to results of the same DNA types. Among them, the DNA types of 15 remains matched with those of bereaved families and the identified remains were reconfirmed by amelogenin sex typing and mitochondrial DNA sequence analysis. The others were not identified a family by failures of PCR amplification or non-matching of DNA types. Also it is confirmed that one hair sample should be artificial by non-digestion of protease and another be animal bone by result of dot blotting with human Alu probe. Our results indicate that multiplex PCR system consisting of several STR loci like HUMCSF1PO, HUMTPOX, and HUMTHO1 is more effective for the identification of highly decomposed human remains from mass disaster.

韓國人의 HLA DQA1 遺傳座位에 대한 集團 遺傳學的 特性

남용석,김희선,이희석,이혜린,황적준 大韓法醫學會 1995 대한법의학회지 Vol.19 No.1

Using reverse dot blotting technique, genotype of HLA DQAl locus have been determined from 142 unrelated Korean individuals. Twenty genotypes were found from possible twenty one genotypes - the missing one was A2/A2 that had lowest expected frequency. All of known 6 alleles were found with each of its frequency being 15.1% for A1. 1, 16.6%for A1.2, 12.7% for A1.3, 11.6% for A2, 25.7% for A3 and 18.3% for A4. After X?-test(p>0.1), G-test(p>0.05), and by comparision of expected (0.82) and observed heterozygosity(0.81), the population was confirmed to be on the state of Hardy-Weinberg equilibrium. The gene diversity(0.82) of Korean population, which generally thought to be a group of single unity, actually was higher than that of most other populations. The pattern of alelic distribution was different from that of other populations, especially allele A1.3 which displayed heterogeneity between other goups with significance(p<0.01), as it turned out to have anthropological significance. After all, this HLA DQA1 system, even though its small number of alleles, having high degree of heterozygosity, was proven to be effective in individual identification, and paternity testing in Koean population.

E. coli K-12 균주로부터 글루타치온 합성 유전자의 클로닝

남용석,박영인,이세영 한국산업미생물학회 1991 한국미생물·생명공학회지 Vol.19 No.6

글루타치온 생산증대를 도모하기 위하여 글루타치온 합성에 관여하는 효소들인 GSH-I 및 GSH-II 유전자들을 pBR322 벡터에 클로닝하였다. gshI 유전자를 클로닝하기 위하여 GSH-I 효소활성이 결여된 GS903 균주를 분리하였다. E. coli K-12 염색체 DNA로부터 분리된 3.6Kb PstI DNA 절편내에 gshI 유전자가 존재하였으며 이 절편을 pBR322 벡터에 클로닝하였다. gshII 유전자는 2.2Kb PstI-BamHI DNA 절편내에 존재하며 이 절편을 pUC13 벡터에 클로닝하였다. 플라스미드의 copy number와 gsh 유전자들을 포함하고 있는 삽입 DNA의 크기의 차이에 의한 gsh 유전자들의 발현 정도를 조사하기 위하여 pGH100, pGH101, pGH200, pGH201, pLF4, pLF6 그리고 pGH300같은 여러 플라스미드를 제조하였다. pBR322 플라스미드 대신 pUC계통의 플라스미드를 사용함으로써 gshI 유전자의 발현이 의해 2배 이상 증가하였다. 그러나 벡터 플라스미드내에 존재하는 gsh 유전자들을 포함하는 삽입 DNA의 크기의 차이에 의해서는 gsh 유전자들의 발현이 영향을 받지 않았다. To increase the production of glutathione by the expression of recombinant gsh plasmids, two genes responsible for the biosynthesis of glutathione were isolated and cloned. To clone a gshI gene, the GS903 mutant strain, which is deficient in γ-glutamylcysteine synthetase activity, has been raised. A gshI gene was cloned using pBR322 plasmid as a 3.6Kb PstI DNA fragment isolated from E. coli K-12 chromosomal DNA. Also a gshII gene was cloned using pUC13 plasmid as a 2.2Kb PstI-BamHI DNA fragment. To study the effects of plasmid copy number and passenger DNA size on the expression levels of the gsh genes, various recombinant plasmids containing different sets of genes were constructed. The expression levels of the gsh genes were increased approximately twice higher in pUC series plasmids than that in pBR322 plasmid. But the sizes of the passenger DNA containing the gsh genes in the vector plasmid did not affect on the expression levels of the gsh genes.

미토콘드리아 DNA 염기서열 분석법에 의한 가족관계의 규명

남용석,이희석,김희선,이혜린,황적준 大韓法醫學會 1995 대한법의학회지 Vol.19 No.1

The human mitochondrial DNA has two characteristics that make it possible to identify individuals and establish family relationships. First, it is haploid, being exhibited only maternal inheritance. Second, it is highly variable on the hypervariable control region of mitochondrial DNA. Taking advantage of two characteristics of mitochondrial DNA, individual relationships in dispute were identified by combining PCR amplification with direct mitochondrial DNA sequencing. Two persons who alleged the same maternal lineage were identical on the mitochondrial DNA sequences from 15,960 to 16,569 and from 1 to 533. Other two persons were identical on the mitochondrial DNA sequences from 16221 to 16390 each other. However, seven bases are different on the sequences from 16221 to 16390 between two groups,. Even though four persons are kinship, these results suggest that they should come from two different maternal lineage.

pV47-2 다좌위탐식자를 이용한 인체 게놈에서 다형성 유전좌위의 분리

남용석,이혜린,한길로,황적준 大韓法醫學會 1997 대한법의학회지 Vol.21 No.2

Two polymorphic loci, so- called FS106 and FS185, have been isolated from the human genome, using a multilocus probe pV47-2, which is extensively used in Korea for forensic investigation such as resolving paternity disputes. Among the several plaques selected from λ Fix-II genomic libraries, fourteen clones have been characterized. Restriction maps of 14 clones were constructed to define the flanking as well as repeat parts. The repeat-free flanking DNA fragments were tested for single locus specific polymorphism, and repeat containing DNA fragments were sequenced for the design of PCR primers. None of the repeat-free flanking DNA fragments was not shown any polymorphisms by RFLP analysis. The (GGT)??-rich sequences in most of repeat containing DNA fragments were identified by sequencing analysis. Most of repetitive sequences consists of major units of (GGT)??, but a regular repetition pattern can not be found in all clones. Two sets of primers designed from flanking sequences of repeat containing DNA fragments were shown length polymorphisms by PCR analysis, when tested in 50 unrelated individuals. Three and four alleles were detected at FS106 and 0.58 for FS185. In addition, two loci, FS106 and FS185, have been mapped on chromosome 5 and 3, respectively, by somatic cell hybrid analysis.

創傷의 經過時間

黃迪駿,李羲碩,南容碩 大韓法醫學會 1994 대한법의학회지 Vol.18 No.2

When examining a victim of viloence, no forensic pathologist is fulfilling his role when one confines the report to merely a numbered lists of wounds found on the corpse. Of course, the nature, exact position, and dimensios of every injury should be described, and it should e photographed. However, the distinction between antemortem and postmortem injuries and their proper timing is one of the cardinal problems of froensic medicine. It help not only to convict guilty but also to acquit persons who are suspect but in factnot guilty. The cause of death is sometimes of less importance than the recsotruction of events, which may become possible with careful examination of the wounds and their proper timing. As with time of death, it can be a very dificult matter in forensic medical investigations to determine whether a wound found at autopsy was inflicted before or after death, and if ante-mortem, how long before death was it sustained? Unfortunately, as with so many problems, biological variability introduces a wide range of uncertainty about the time of wound, so that a range of probabilities can be offered, but never a definite time interval. Therefore, the purpose of this paper is to discuss pros and cons about various dating methods of ante-mortem wound that have been published.