N4SSB 단백질의 C-말단기의 7개의 아미노산이 N4SSB 단백질의 in vivo 활성에 미치는 영향

최미영,Choi, Mieyoung 한국미생물학회 1998 미생물학회지 Vol.34 No.4

Bacteriophage N4, a lytic phage specific for Esherichia coli K12 strain encodes single-stranded DNA-binding protein, N4SSB (bacteriophage N4-coded single-stranded DNA-binding protein). N4SSB protein is originally identified as a protein required for N4 DNA replication. N4SSB protein is also required for N4 late transcription, which is catalyzed by E. coli ${\sigma}^{70}$ RNA polymerase. N4 late transcription does not occur until N4SSB protein is synthesized. Recently it is reported that N4SSB protein is essential for N4 DNA recombination. Therefore N 4SSB protein is a multifunctional protein required for N4 DNA replication, late transcription, and N4 DNA recombination. In this study, a variety of mutant N4SSB proteins containing internal deletions or substitutions were constructed to define and characterize domains important for N4 DNA replication, late transcription, and N4 DNA recombination. Test for the ill vivo activity of these mutant N4SSBs for N4 DNA replication, late transcription, and N4 DNA recombination was examined. The results suggest that C-terminal 7 amino acid residues are important for the activity of N4SSB. Three lysine residues, which are contained in this region play important roles on N4SSB activity. Esherichia coli(E. coli) K12 균주를 숙주세포로 삼는 박테리오파아지인 N4는 single-stranded DNA에 결합하는 단백질인 N4SSB(bacteriophage N4-coded single-stranded DNA-binding protein) 단백질을 만든다. N4SSB 단백질은 N4 DNA replication 뿐만 아니라 late transcription과 N4 DNA recombination에도 필요한 여러 가지 기능을 가진 단백질이다. N4 late transcription은 숙주세포인 E. coli의 $E{\sigma}^{70}$ RNA polymerase에 의해서 수행이 되나 N4SSB 단백질을 반드시 필요로 하기 때문에 N4SSB 단백질이 생성될 때까지는 N4 late promoter로부터 RNA 합성이 일어나지 않는다. 본 연구에서는 N4SSB의 N4 DNA replication과 late transcription, 그리고 N4 DNA recombination에 필요한 영역(domain)을 알아내기 위해서 여러 가지 돌연변이형 N4SSB 단백질을 만들어 N4 DNA replication과 late transcription, 그리고 N4 DNA recombination의 3가지 작용에 대한 in vivo 활성을 조사 분석하였다. 그 결과 N4SSB 단백질의 C-말단기에 있는 7개의 아미노산이 N4SSB 단백질의 활성에 중요하다는 것을 알 수 있었다. 특히 C-말단기의 7개의 아미노산에는 세 개의 lysine이 포함되어 있는데 이 lysine이 N4SSB 단백질의 활성에 중요한 역할을 한다는 것이 제시되었다.

과학 고등학교 학생을 대상으로 한 DNA 재조합 기술 적용의 탐색

유준희,김학현 韓國生物敎育學會 2004 생물교육 Vol.32 No.4

The rapid developments of a biotechnology in 20 century have resulted in enormous changes of our society as well as science. We have been living in the science-based society, and a genetic engineering among the biotechnology especially has affected various areas of our life. However, science high school students, anything but general high school ones, have not had chances of performing experiments for genetic engineering. Therefore, experiments related with recombinant DNA technology, a core of genetic engineering were applied to science high school students for 5 days in their school by their teacher not a professional researcher. All experiment were generally well done and students showed a significant increase of achievements (p<0.05). After doing all experimental procedures, they showed a high interest in a genetic engineering and an usual understanding in procedures of experiments. A little understanding for the experiments may be due to absence of pre-concepts about recombinant DNA and a little experimental experience of teacher. I suggest a proper time for performing the recombinant DNA experiments is after high school students fully obtaining basic concepts of recombinant DNA. Especially, plasmid isolation, DNA electrophoresis and transformation experiments among all procedures were more effective in science motivation and they are thought to be very important and useful within experimental courses for recombinant DNA technology. Therefore, I recommend the three experiments as core courses of recombinant DNA technology.

Rad52/Rad59-dependent Recombination as a Means to Rectify Faulty Okazaki Fragment Processing

Lee, Miju,Lee, Chul-Hwan,Demin, Annie Albert,Munashingha, Palinda Ruvan,Amangyeld, Tamir,Kwon, Buki,Formosa, Tim,Seo, Yeon-Soo American Society for Biochemistry and Molecular Bi 2014 The Journal of biological chemistry Vol.289 No.21

<P>The correct removal of 5′-flap structures by Rad27 and Dna2 during Okazaki fragment maturation is crucial for the stable maintenance of genetic materials and cell viability. In this study, we identified <I>RAD52</I>, a key recombination protein, as a multicopy suppressor of <I>dna2-K1080E</I>, a lethal helicase-negative mutant allele of <I>DNA2</I> in yeasts. In contrast, the overexpression of Rad51, which works conjointly with Rad52 in canonical homologous recombination, failed to suppress the growth defect of the <I>dna2-K1080E</I> mutation, indicating that Rad52 plays a unique and distinct role in Okazaki fragment metabolism. We found that the recombination-defective Rad52-QDDD/AAAA mutant did not rescue <I>dna2-K1080E</I>, suggesting that Rad52-mediated recombination is important for suppression. The Rad52-mediated enzymatic stimulation of Dna2 or Rad27 is not a direct cause of suppression observed <I>in vivo</I>, as both Rad52 and Rad52-QDDD/AAAA proteins stimulated the endonuclease activities of both Dna2 and Rad27 to a similar extent. The recombination mediator activity of Rad52 was dispensable for the suppression, whereas both the DNA annealing activity and its ability to interact with Rad59 were essential. In addition, we found that several cohesion establishment factors, including Rsc2 and Elg1, were required for the Rad52-dependent suppression of <I>dna2-K1080E</I>. Our findings suggest a novel Rad52/Rad59-dependent, but Rad51-independent recombination pathway that could ultimately lead to the removal of faulty flaps in conjunction with cohesion establishment factors.</P>

효모의 재조합 변이주를 이용한 인간 Centromeric Alphoid DNA Repeat의 안정성에 관한 연구

김광섭,신영선,이상엽,안은경,도은주,박인호,임선희,선우양일,Kim, Kwang-Sup,Shin, Young-Sun,Lee, Sang-Yeop,Ahn, Eun-Kyung,Do, Eun-Ju,Park, In-Ho,Leem, Sun-Hee,SunWoo, Yang-Il 한국미생물학회 2007 미생물학회지 Vol.43 No.4

Centromere는 채세포분열과 생식세포분열 등 맡은 주요 기능을 담당하는 고도로 분화된 구조이다. Alphoid DNA (${\alpha}$-satellite)는 인간뿐 아니라 모든 영장류의 염색체 내 centromere에서 발견되는 반복서열의 대부분을 차지한다. 인간 인공염색체(Human Artificial Chromosome, HAC)의 개발에서 가장 핵심적인 부분은 centromere의 분리 및 안정적인 유지에 있다. 이 영역은 출아효모에서 alphoid DNA 반복서열을 hook으로 이용하여 Transformation-associated recombination (TAR) cloning법을 사용하여 선택적으로 분리할 수 있다. 이러한 실험방법으로 먼저 repeat array를 rolling-circle amplication (RCA)를 통하여 약 5 kb까지 길이를 연장시킨 후, 효모내에서 상동성재 조합을 이용한 TAR cloning법을 사용하여 분리할 수 있다. 이렇게 분리된 35 kb-50 kb 길이의 4종류의 centromeric DNA repeat arrays (2,4,5,6 mer)를 사용하여, 반복서열의 안정성 유지를 조사하기 위해 상동성재조 합 변이주인 rad51, rad52, rad54를 사용하여 비교 분석하였다. 야생주, rad51과 rad54 변이주를 이용하여 형질전환을 수행한 결과, 반복서열의 크기에 있어서 많은 변화를 나타내었다. 반면, rad52 변이주는 야생주와 다르게 형질전환빈도가 매우 낮은 비율로 나타났으나, centromeric DNA repeat array의 안정성은 3배 이상으로 높게 나타냈다. 이러한 결과들을 미루어, rad52 변이주를 사용하여 centromeric DNA repeat arrays의 형질전환실험에서 발생하는 맡은 변이를 줄일 수 있을 것으로 보인다. 이러한 유전적 방법은 HAC 제작에서 반복서열의 유지에 훨씬 효율적으로 사용할 수 있을 것으로 사료된다. The centromere is a highly differentiated structure of the chromosome that fulfills a multitude of essential mitotic and meiotic functions. Alphoid DNA (${\alpha}$-satellite) is the most abundant family of repeated DNA found at the centromere of all human chromosomes, and chromosomes of primates in general. The most important parts in the development of Human Artificial Chromosomes (HACs), are the isolation and maintenance of stability of centromeric region. For isolation of this region, we could use the targeting hook with alphoid DNA repeat and cloned by Transformation-Associated Recombination (TAR) cloning technique in yeast Saccharomyces cerevisiae. The method includes rolling-circle amplification (RCA) of repeats in vitro to 5 kb-length and elongation of the RCA products by homologous recombination in yeast. Four types of $35\;kb{\sim}50\;kb$ of centromeric DNA repeat arrays (2, 4, 5, 6 mer) are used to examine the stability of repeats in homologous recombination mutant strains (rad51, rad52, and rad54). Following the transformation into wild type, rad51 and rad54 mutant strains, there were frequent changes in inserted size. A rad52 mutant strain showed extremely low transformation frequency, but increased stability of centromeric DNA repeat arrays at least 3 times higher than other strains. Based on these results, the incidence of large mutations could be reduced using a rad52 mutant strain in maintenance of centromeric DNA repeat arrays. This genetic method may use more general application in the maintenance of tandem repeats in construction of HAC.

만성 B 형 및 C 형 간염바이러스 재조합 DNA 백신 제조

김영호 수원대학교 기능성생명소재연구소 2002 자연과학연구논문집 Vol.1 No.-

Small-HBsAg DAN and HCV-E1 envelope DNA were fused into pcDNA3.1. HCV E1 envelope DNA was selected from highly conserved region. Middle-HBsAg DNA and HCV-E1 envelope DNA were also fused into pcDNA3.1. All these fused DNA were cloned with SOD sequence. COS cell line expressed the fusion proteins. Expressed protein were confirmed with Western Blot and ELISA test. Balb/C mice were primarily immunized with these recombinant Plasmid DAN and will be boostly immunized in the next month. One epitope of the HCV E1 envelope protein(amino acid 315-327) region were synthesized as a single stranded DNA and re-annealed by renaturation and also each strand contained with a cohesive ends was inserted into expression vector. Small-HBsAg DNA and middle-HBsAg DNA was fused with HCV-E1 epitope DNA. For efficient expression of fused proteins, SOD sequences were inserted into the front of the HCV-E1/HBsAg DNA sequences. This chimeric DNA were ready for the insertion into yeast vector or Pichia vector system for the highly expression of fusion proteins. Chimeric proteins expressing HCV-E1/HBsAg were confirmed by western blot and ELISA method. This proteins will be confirmed by neutralization test against hepatitis patient's serum due to HCV infection.

DNA-dependent Protein Kinase Mediates V(D)J Recombination via RAG2 Phosphorylation

Hah, Young-Sool,Lee, Jung-Hwa,Kim, Deok-Ryong Korean Society for Biochemistry and Molecular Biol 2007 Journal of biochemistry and molecular biology Vol.40 No.3

V(D)J recombination, a site-specific gene rearrangement process occurring during the lymphocyte development, begins with DNA double strand breaks by two recombination activating gene products (RAG1/2) and finishes with the repair process by several proteins including DNA-dependent protein kinase (DNA-PK). In this report, we found that RAG2 was specifically phosphorylated by DNA-PK at the $365^{th}$ serine residue, and this phosphorylated RAG2 affected the V(D)J recombination activity in cells in the GFP expression-based assay. While the V(D)J recombination activity between wild-type RAG2 and mutant S365A RAG2 in the assay using a signal joint substrate was undistinguishable in DNA-PK deficient cells (M059J), the activity with wild-type RAG2 was largely increased in DNA-PK proficient cells (M059K) in comparison with mutant RAG2, suggesting that RAG2 phosphorylation by DNA-PK plays a crucial role in the signal joint formation during V(D)J recombination.

Foldback Intercoil DNA and the Mechanism of DNA Transposition

Kim, Byung-Dong Korea Genome Organization 2014 Genomics & informatics Vol.12 No.3

Foldback intercoil (FBI) DNA is formed by the folding back at one point of a non-helical parallel track of double-stranded DNA at as sharp as $180^{\circ}$ and the intertwining of two double helixes within each other's major groove to form an intercoil with a diameter of 2.2 nm. FBI DNA has been suggested to mediate intra-molecular homologous recombination of a deletion and inversion. Inter-molecular homologous recombination, known as site-specific insertion, on the other hand, is mediated by the direct perpendicular approach of the FBI DNA tip, as the attP site, onto the target DNA, as the attB site. Transposition of DNA transposons involves the pairing of terminal inverted repeats and 5-7-bp tandem target duplication. FBI DNA configuration effectively explains simple as well as replicative transposition, along with the involvement of an enhancer element. The majority of diverse retrotransposable elements that employ a target site duplication mechanism is also suggested to follow the FBI DNA-mediated perpendicular insertion of the paired intercoil ends by non-homologous end-joining, together with gap filling. A genome-wide perspective of transposable elements in light of FBI DNA is discussed.

Foldback Intercoil DNA and the Mechanism of DNA Transposition

김병동 한국유전체학회 2014 Genomics & informatics Vol.12 No.3

Foldback intercoil (FBI) DNA is formed by the folding back at one point of a non-helical parallel track of double-stranded DNAat as sharp as 180° and the intertwining of two double helixes within each other’s major groove to form an intercoil with adiameter of 2.2 nm. FBI DNA has been suggested to mediate intra-molecular homologous recombination of a deletion andinversion. Inter-molecular homologous recombination, known as site-specific insertion, on the other hand, is mediated bythe direct perpendicular approach of the FBI DNA tip, as the attP site, onto the target DNA, as the attB site. Transposition ofDNA transposons involves the pairing of terminal inverted repeats and 5–7-bp tandem target duplication. FBI DNAconfiguration effectively explains simple as well as replicative transposition, along with the involvement of an enhancerelement. The majority of diverse retrotransposable elements that employ a target site duplication mechanism is alsosuggested to follow the FBI DNA-mediated perpendicular insertion of the paired intercoil ends by non-homologousend-joining, together with gap filling. A genome-wide perspective of transposable elements in light of FBI DNA is discussed.

Recombinant DNA and Protein Vaccines for Foot-and-mouth Disease Induce Humoral and Cellular Immune Responses in Mice

배지영,문선화,최정아,박종석,한범수,김기영,김병한,송재영,권대혁,이석찬,김종범,양주성 대한면역학회 2009 Immune Network Vol.9 No.6

Foot-and-mouth disease virus (FMDV) is a small single- stranded RNA virus which belongs to the family Picornaviridae, genus Apthovirus. It is a principal cause of FMD which is highly contagious in livestock. In a wild type virus infection, infected animals usually elicit antibodies against structural and non-structural protein of FMDV. A structural protein, VP1, is involved in neutralization of virus particle, and has both B and T cell epitopes. A RNA-dependent RNA polymerase, 3D, is highly conserved among other serotypes and strongly immunogenic, therefore, we selected VP1 and 3D as vaccine targets. VP1 and 3D genes were codon-optimized to enhance protein expression level and cloned into mammalian expression vector. To produce recombinant protein, VP1 and 3D genes were also cloned into pET vector. The VP1 and 3D DNA or proteins were co-immunized into 5 weeks old BALB/C mice. Antigen-specific serum antibody (Ab) responses were detected by Ab ELISA. Cellular immune response against VP1 and 3D was confirmed by ELISpot assay. The results showed that all DNA- and protein-immunized groups induced cellular immune responses, suggesting that both DNA and recombinant protein vaccine administration efficiently induced Ag-specific humoral and cellular immune responses. Foot-and-mouth disease virus (FMDV) is a small single- stranded RNA virus which belongs to the family Picornaviridae, genus Apthovirus. It is a principal cause of FMD which is highly contagious in livestock. In a wild type virus infection, infected animals usually elicit antibodies against structural and non-structural protein of FMDV. A structural protein, VP1, is involved in neutralization of virus particle, and has both B and T cell epitopes. A RNA-dependent RNA polymerase, 3D, is highly conserved among other serotypes and strongly immunogenic, therefore, we selected VP1 and 3D as vaccine targets. VP1 and 3D genes were codon-optimized to enhance protein expression level and cloned into mammalian expression vector. To produce recombinant protein, VP1 and 3D genes were also cloned into pET vector. The VP1 and 3D DNA or proteins were co-immunized into 5 weeks old BALB/C mice. Antigen-specific serum antibody (Ab) responses were detected by Ab ELISA. Cellular immune response against VP1 and 3D was confirmed by ELISpot assay. The results showed that all DNA- and protein-immunized groups induced cellular immune responses, suggesting that both DNA and recombinant protein vaccine administration efficiently induced Ag-specific humoral and cellular immune responses.

재조합 DNA probe에 의한 Fusarium oxysporum 분화형간의 분류 및 유전적 변이 분석

김영태,김홍기 충남대학교 생물공학연구소 1998 생물공학연구지 Vol.6 No.-

재조합 DNA probe를 이용하여 국내 Fusarium oxysporum 분화형간의 분류 가능성을 탐색하고 그들의 유전적 변이를 분석하였다. F. oxysporum f. sp. lycopersici, cucumerinum, fragariae, garlic, sesami 등 5종의 분화형을 공시하여 RELP분석을 실시하였다. Repetitive copy clone인 세종의 재조합 클론 pFC46, pFC52, pFC57을 이용하여 HindⅢ로 처리한 F. oxysporum의 genomic DNA에 대해 southern hybridization한 결과 나타난 밴드의 양상은 분화형에 따라 차이가 명확히 밝혀져 이들을 이용한 분류가 가능하였다. 또한 RFLP 분석 결과 f. sp. sesami는 다른 분화형에 비해 다소 변이가 심했으나 다른 분화형들은 변이가 거의 없어 f. sp. sesami를 제외한 f. sp. lycopersici 등 4종의 Fusarium oxysporum 분화형의 균주들은 채집 지역에 관계없이 대체로 유전적으로 안정되어 있는 것으로 판단되었다. Hybridization 밴드의 양상에 기초하여 유전적 유연관계를 집괴 분석한 결과 각 분화형별로 유사도가 매우 높게 별도의 유사군을 형성하였다. Five formae speciales of Fusarium oxysporum in Korea were examined using RFLP analysis to find the possibility for classification and analyze genetic variations. DNAs from F. oxysporum f. sp. lycopersici, cucumerinum, fragariae, garlic and sesami were used with three recombinant probes such as pFC46, pFC52 and pFC57. Distinct differences among five formae speciales of this fungus were detected in RFLP band patterns based on southern hybridization of genomic DNA using each recombinant clone, which was a repetitive copy probe. Strains belong to four formae speciales could be very stable in genetic variation except f. sp. sesami which has more variation than the others based on the RFLP analysis. They formed their own cluster which has high similarity within the same formae specialis resulted from the UPGMA analysis for genetic relationship analysis and each cluster represented its own formae specialis. The method using three recombinant DNA probes could be a good tool for classification of formae speciales in F. oxysporum.