• Title/Summary/Keyword: Replication origin

Search Result 50, Processing Time 0.028 seconds

NMR Structural Study of the 3'-T.G Mismatched DNA Decamer Duplex Containing the T-T (6-4) Adduct

  • Lee, Joon-Hwa;Park, Yun-Jeong;Park, Byong-Seok
    • Journal of the Korean Magnetic Resonance Society
    • /
    • v.3 no.1
    • /
    • pp.60-70
    • /
    • 1999
  • The pyrimidine(6-4) pyrimidone photoproduct [(6-4) adduct] is one of the major photoproducts induced by UV irradiation of DNA and occurs at TpT sites. The (6-4) adduct is highly mutagenic and specific during translesion replication. The marked preference for insertion of A opposite the 5'-T and G opposite the 3--T of the (6-4) adducts leads to a predominantly 3'-T\longrightarrowC transition with 85% replicating error rate. In order to obtain insight into the origin of 3'-T\longrightarrowC transition induced by the (6-4) adduct, we have performed one - and two-dimensional NMR experiment. The 3'-Tof the (6-4) lesion forms the stable hydrogen bonding to the imino proton of an opposed G, which stabilizes the overall helix and diminishes the highly distorted conformation caused by the (6-4) lesion in the (6-4)/AA duplex. We proposed that the greater insertion of a G over an A opposite the 3'-T of the (6-4) lesion These results may account for the greater preference for the insertion of a G over a A opposite the 3'-T of the (6-4) lesion. Thus this insertion leads to the highly specific 3'-T\longrightarrowC multation at the (6-4) lesion site.

  • PDF

Structure of a DNA Duplex Containing a Site-Specific Dewar Isomer: Structural Influence of the 3'-T.G base pair of the Dewar product.

  • Lee, Joon-Hwa;Choi, Byong-Seok
    • BMB Reports
    • /
    • v.33 no.3
    • /
    • pp.268-275
    • /
    • 2000
  • In contrast to the pyrimidine (6-4)pyrimidone photoproduct [(6-4) adduct], its Dewar valence isomer (Dewar product) is low mutagenic and produces a broad range of mutations with a 42 % replicating error frequency. In order to determine the origin of the mutagenic property of the Dewar product, we used experimental NMR restraints and molecular dynamics to determine the solution structure of a Dewar·lesion DNA decamer duplex, which contains a mismatched base pair between the 3'-T residue and an opposed G residue. The 3'-T of the Dewar lesion forms stable hydrogen bonds with the opposite G residue. The helical bending and unwinding angles of the DW/GA duplex, however, are much higher than those of the DW/AA duplex. The stable hydrogen bonding of the G 15 residue does not increase the thermal stability of the overall helix. It also does not restore the distorted backbone conformation of the DNA helix that is caused by the forming of a Dewar lesion. These structural features implicate that no thermal stability, or conformational benefits of G over A opposite the 3'-T of the Dewar lesion, facilitate the preferential incorporation of an A. This is in accordance with the A rule during translesion replication and leads to the low frequent $3'-T{\rightarrow}C$ mutation at this site.

  • PDF

Identification of the Capsid Protein-binding Region of the SL1(+) RNA Located at the 5' Region of the Potato virus X Genome

  • Cho, Sang-Yun;Kim, Kook-Hyung
    • The Plant Pathology Journal
    • /
    • v.28 no.1
    • /
    • pp.75-80
    • /
    • 2012
  • $Potato$ $virus$ $X$ (PVX) contains $cis$-acting elements including stem-loop 1 (SL1) RNA at the 5' region; SL1 is conserved among all potexviruses. The SL1 at the positive-sense RNA, SL1(+), is required for PVX RNA replication, cell-to-cell movement, and translation. Previous research demonstrated that SL1(+) RNA also serves as the origin of assembly for encapsidation of PVX RNA. To identify the essential sequences and/or regions for capsid protein (CP) subunit recognition within SL1(+) RNA, we used electrophoretic mobility shift assays (EMSA), UV cross-linking, and yeast three-hybrid analyses. The EMSA and UV cross-linking analyses with PVX CP subunits and RNA transcripts corresponding to the SL1(+) RNA showed that the SL1(+) RNA formed complexes with CP subunits. We also conducted EMSA and yeast three-hybrid analyses with RNAs containing various mutations of SL1(+) RNA elements. These analyses indicated that SL1(+) RNA is required for the interaction with PVX CP and that the RNA sequences located at the loop C and tetra loop of the SL1(+) are crucial for CP binding. These results indicate that, in addition to being important for RNA accumulation, the SL1(+) RNA from the 5' region of the PVX genome is also required for specific binding of PVX CP.

Expression of recombinant plasmids harboring glucoamylase gene STA in saccharomyces cerevisiae (Glucoamylase 유전자 STA를 포함한 재조합 플라스미드들의 saccharomyces cerevisiae에서의 발현)

  • 박장서;박용준;이영호;강현삼;백운화
    • Korean Journal of Microbiology
    • /
    • v.28 no.3
    • /
    • pp.181-187
    • /
    • 1990
  • STA gene coding glucoamylase was introduced into haploid Saccharomyces cerevisiae SHY3 and polyploid Saccharomyces cerevisiae 54. We constructed the recombinant plasmid by substituting the promoter region of alcohol dehydrogenase isoenzyme I gene for that of STA gene to increase the expression of STA gene and found that the activity of glucoamylase was increased in transformants. The plasmid stability was improved remarkably when we got the STA gene into the plasmid which had centromere. The activity of glucoamylase and transformation frequency of it, however, was decreased because of low copy number. Industrial polyploid strain was transformed with the recombinant plasmid having the $2\mu$ origin of replication and STA gene. It produced more alcohol than host when fermented in liquefied starch media. The industrial strain, however, was not transformed with the autonomously replicating plasmid containing centromere.

  • PDF

Cloning of the Hepatitis B Surface Antigen Containing Pre-surface Antigen Region and Poly(A) Addition Site (Pre-surface antigen 지역과 poly(A) addition site가 포함된 B형 간염 표면항원 유전자의 재조합)

  • Kim, Sang-Hae;Kim, Yong-Sok;Park, Mee-Young;Park, Hyune-Mo
    • The Korean Journal of Zoology
    • /
    • v.28 no.3
    • /
    • pp.166-178
    • /
    • 1985
  • In order to express hepatitis B surface antigen $(HB_sAg)$ containing pre-surface antigen region in mammalian calls, 2.7 kb DNA fragment containing pre-surface region-$HB_sAg$ gene poly(A) addition site of HBV genome was cloned into simian virus 40(SV 40) based chimeric vector pSVOB. 2.7 kb DNA fragment was derived from pHBVD 107 containing tandem copies of the HBV genome in a head-to-tail arrangement by Bgl II digestion. Construction of the vector pSVOE involved the incorporation of SV40 sequences spanning the viral origin of replication and 72 bp repeats (enhancer) into a pBR 322 derivative lacking sequences which inhibit replication in mammalian cells. Bam HI linker was inserted at the Pvu II site in the proximity of SV40 late promoter of pSVOE and named as pSVOB. To construct the recombinant plasmid pSVBS, pHBVD 107 was digested with Bgl II to isolate 2.7kb DNA fragment and the fragment was ligated into the Bam HI site of pSVOB by ligation. Preliminary result showed that the recombinant plasmid pSVBS produced $HB_sAg$ in the monkey cell producing large T antigen (COS cell).

  • PDF

Host Construction by Curing the Octopine Type Ti and Cryptic Plasmids in Agrobacterium tumefaciens KU12 (Agrobacterium tumefaciens KU12로부터 Octopine형 Ti 및 잠재 플라스미드의 제거에 의한 숙주 개발)

  • Ha, Un-Hwan;Lee, Yong-Woog;Moon, Hye-Yeon;Sim, Woong-Seop
    • Korean Journal of Microbiology
    • /
    • v.32 no.1
    • /
    • pp.53-59
    • /
    • 1994
  • Agrobacterium tumefaciens KU12 contains pTiKU12 (240kb) of the octopine type Ti plamsid and pTi12 (45 kb) of the cryptic plasmid. To make the avirulent A. tumefaciens, the octopine type Ti plasmid, pTiKU12, was cured with elevated temperature (37${\circ}C$) and ethidium bromide (EtBr), respectively. Also the cryptic plasmid, pTi12, was cured by the introduction of recombinant plasmid, pYWXP, made by pTi12 replication origin and pUC19. pYWXP was cured by elevated temperature (37${\circ}C$) and EtBr simultaneously.

  • PDF

Combination of Epstein-Barr Virus-Based Plasmid and Nonviral Polymeric Vectors for Enhanced and Prolonged Gene Expression

  • Choi, Hye;Park, Key Sun;Bae, Seon Joo;Song, Su Jeong;Kim, Kyoon Eon;Park, Jong-Sang;Choi, Joon Sig
    • Bulletin of the Korean Chemical Society
    • /
    • v.33 no.11
    • /
    • pp.3676-3680
    • /
    • 2012
  • An Epstein-Barr virus (EBV)-based plasmid contains the EBV nuclear antigen 1 (EBNA1) gene and EBV replication origin (oriP) sequence. Since EBNA1 (the only EBV-encoded protein) is combined with oriP, it is replicated simultaneously with chromosomal DNA in human, primate, and canine cells and is faithfully segregated at a stable copy number upon cell division. Consequently, it can be used to stably express gene inserts over a prolonged time in target cells. We have previously shown that the polyamidoamine (PAMAM) dendrimer can be surface-modified with L-arginine. Arginine is present at a high frequency in the transactivator of transcription (Tat) sequences of human immunodeficiency virus (HIV). It presents high membrane permeability and permits effective transfer of DNA inside the cells. In this study, we constructed two kinds of recombinant DNA by inserting the luciferase gene and enhanced green fluorescence protein (eGFP) gene as reporter genes into the pCEP4 plasmid vector. We measured dynamic light scattering (DLS) and zeta potential after preparing PAMAM-based cationic polymer/EBV-based plasmid complexes. We performed transfection of HEK 293 cell lines with the polyplexes, and monitored luciferase activity and green fluorescence protein (GFP) expression. Our results show that PAMAM-based cationic polymer/EBV plasmid complexes provide enhanced and sustained gene expression.

Use of the Cellulase Gene as a Selection Marker of Food-grade Integration System in Lactic Acid Bacteria

  • Lee, Jung-Min;Jeong, Do-Won;Lee, Jong-Hoon;Chung, Dae-Kyun;Lee, Hyong-Joo
    • Food Science and Biotechnology
    • /
    • v.17 no.6
    • /
    • pp.1221-1227
    • /
    • 2008
  • The application of the cellulase gene (celA) as a selection marker of food-grade integration system was investigated in Lactobacillus (Lb.) casei, Lactococcus lactis, and Leuconostoc (Leu.) mesenteroides. The 6.0-kb vector pOC13 containing celA from Clostridium thermocellum with an integrase gene and a phage attachment site originating from bacteriophage A2 was used for site-specific recombination into chromosomal DNA of lactic acid bacteria (LAB). pOC13 was also equipped with a broad host range plus replication origin from the lactococcal plasmid pWV01, and a controllable promoter of nisA ($P_{nisA}$) for the production of foreign proteins. pOC13 was integrated successfully into Lb. casei EM116, and pOC13 integrants were easily detectable by the formation of halo zone on plates containing cellulose. Recombinant Lb. casei EM 116::pOC13 maintained these traits in the absence of selection pressure during 100 generations. pOC13 was integrated into the chromosome of L. lactis and Leu. mesenteroides, and celA acted as an efficient selection marker. These results show that celA can be used as a food-grade selection marker, and that the new integrative vector could be used for the production of foreign proteins in LAB.

Stage specific transcriptome analysis of liver tissue from a crossbred Korean Native Pig (KNP × Yorkshire)

  • Kumar, Himansu;Srikanth, Krishnamoorthy;Park, Woncheol;Lee, Kyung-Tai;Choi, Bong-Hwan;Kim, Jun-Mo;Lim, Dajeong;Park, Jong-Eun
    • Journal of Biomedical and Translational Research
    • /
    • v.19 no.4
    • /
    • pp.116-124
    • /
    • 2018
  • Korean Native Pig (KNP) has a uniform black coat color, excellent meat quality, white colored fat, solid fat structure and good marbling. However, its growth performance is low, while the western origin Yorkshire pig has high growth performance. To take advantage of the unique performance of the two pig breeds, we raised crossbreeds (KNP ${\times}$ Yorkshire to make use of the heterotic effect. We then analyzed the liver transcriptome as it plays an important role in fat metabolism. We sampled at two stages: 10 weeks and at 26 weeks. The stages were chosen to correspond to the change in feeding system. A total of 16 pigs (8 from each stage) were sampled and RNA sequencing was performed. The reads were mapped to the reference genome and differential expression analysis was performed with edgeR package. A total of 324 genes were found to be significantly differentially expressed (${\left|log2FC\right|}$ > 1 & q < 0.01), out of which 180 genes were up-regulated and 144 genes were down-regulated. Principal Component Analysis (PCA) showed that the samples clustered according to stages. Functional annotation of significant DEGs (differentially expressed genes) showed that GO terms such as DNA replication, cell division, protein phosphorylation, regulation of signal transduction by p53 class mediator, ribosome, focal adhesion, DNA helicase activity, protein kinase activity etc. were enriched. KEGG pathway analysis showed that the DEGs functioned in cell cycle, Ras signaling pathway, p53 signaling pathway, MAPK signaling pathway etc. Twenty-nine transcripts were also part of the DEGs, these were predominantly Cys2His2-like fold group (C2H2) family of zinc fingers. A protein-protein interaction (PPI) network analysis showed that there were three highly interconnected clusters, suggesting an enrichment of genes with similar biological function. This study presents the first report of liver tissue specific gene regulation in a cross-bred Korean pig.

The Abuse and Invention of Tradition from Maintenance Process of Historic Site No.135 Buyeo Gungnamji Pond (사적 제135호 부여 궁남지의 정비과정으로 살펴본 전통의 남용과 발명)

  • Jung, Woo-Jin
    • Journal of the Korean Institute of Traditional Landscape Architecture
    • /
    • v.35 no.2
    • /
    • pp.26-44
    • /
    • 2017
  • Regarded as Korea's traditional pond, Gungnamj Pond was surmised to be "Gungnamji" due to its geological positioning in the south of Hwajisan (花枝山) and relics of the Gwanbuk-ri (官北里) suspected of being components to the historical records of Muwang (武王)'s pond of The Chronicles of the Three States [三國史記] and Sabi Palace, respectively, yet was subjected to a restoration following a designation to national historic site. This study is focused on the distortion of authenticity identified in the course of the "Gungnamji Pond" restoration and the invention of tradition, whose summarized conclusions are as follows. 1. Once called Maraebangjuk (마래방죽), or Macheonji (馬川池) Pond, Gungnamji Pond was existent in the form of a low-level swamp of vast area encompassing 30,000 pyeong during the Japanese colonial period. Hong, Sa-jun, who played a leading role in the restoration of "Gungnamji Pond," said that even during the 1940s, the remains of the island and stone facilities suspected of being the relics of Gungnamji Pond of the Baekje period were found, and that the traces of forming a royal palace and garden were discovered on top of them. Hong, Sa-jun also expressed an opinion of establishing a parallel between "Gungnamji Pond" and "Maraebangjuk" in connection with a 'tale of Seodong [薯童說話]' in the aftermath of the detached palace of Hwajisan, which ultimately operated as a theoretical ground for the restoration of Gungnamj Pond. Assessing through Hong, Sa-jun's sketch, the form and scale of Maraebangjuk were visible, of which the form was in close proximity to that photographed during the Japanese colonial period. 2. The minimized restoration of Gungnamji Pond faced deterrence for the land redevelopment project implemented in the 1960s, and the remainder of the land size is an attestment. The fundamental problem manifest in the restoration of Gungnamji Pond numerously attempted from 1964 through 1967 was the failure of basing the restorative work in the archaeological facts yet in the perspective of the latest generations, ultimately yielding a replication of Hyangwonji Pond of Gyeongbok Palace. More specifically, the methodologies employed in setting an island and a pavilion within a pond, or bridging an island with a land evidenced as to how Gungnamji Pond was modeled after Hyangwonji Pond of Gyeongbok Palace. Furthermore, Chihyanggyo (醉香橋) Bridge referenced in the designing of the bridge was hardly conceived as a form indigenous to the Joseon Dynasty, whose motivation and idea of the misguided restoration design at the time all the more devaluated Gungnamji Pond. Such an utterly pure replication of the design widely known as an ingredient for the traditional landscape was purposive towards the aesthetic symbolism and preference retained by Gyeongbok Palace, which was intended to entitle Gungnamji Pond to a physical status of the value in par with that of Gyeongbok Palace. 3. For its detachment to the authenticity as a historical site since its origin, Gungnamji Pond represented distortions of the landscape beauty and tradition even through the restorative process. The restorative process for such a historical monument, devoid of constructive use and certain of distortion, maintains extreme intimacy with the nationalistic cultural policy promoted by the Park, Jeong-hee regime through the 1960s and 1970s. In the context of the "manipulated discussions of tradition," the Park's cultural policy transformed the citizens' recollection into an idealized form of the past, further magnifying it at best. Consequently, many of the historical sites emerged as fancy and grand as they possibly could beyond their status quo across the nation, and "Gungnamji Pond" was a victim to this monopolistic government-led cultural policy incrementally sweeping away with new buildings and structures instituted regardless of their original space, and hence, their value.