• Title/Summary/Keyword: DNA ligase

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'Restriction-PCR' - a Superior Replacement for Restriction Endonucleases in DNA Cloning Applications

  • Klimkait, Thomas
    • BMB Reports
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    • v.33 no.2
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    • pp.162-165
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    • 2000
  • Polymerase chain reaction (PCR) is well established as an indispensable tool of molecular biology; and yet a limitation for cloning applications continues to be that products often require subsequent restriction to be that products often require subsequent restriction digests, blunt-end ligation, or the use of special linear vectors. Here a rapid, PCR-based system is described for the simple, restriction enzyme-free generation of synthetic, 'restriction-like' DNA fragments with staggered ends. Any 3'- or 5'-protruding terminus, but also non-palindromic overhangs with an unrestricted single strand length are specifically created. With longer overhangs, "Restriction-PCR" does not even require a ligation step prior to transformation. Thereby the technique presents a powerful tool e.g. for a successive, authentic reconstitution of sub-fragments of long genes with no need to manipulate the sequence or to introduce restriction sites. Since restriction enzyme-free and thereby devoid the limitations of partial DNA digests, "Restriction-PCR" allows a straight one-step generation and cloning of difficult DNA fragments that internally carry additional sites for specific sequence insertions or deletions can be precisely engineered into genes of interest. With these properties "Restriction-PCR" has the potential to add significant speed and versatility to a wide variety of DNA cloning applications.

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Identification and Molecular Characterization of Parkin in Clonorchis sinensis

  • Bai, Xuelian;Kim, Tae Im;Lee, Ji-Yun;Dai, Fuhong;Hong, Sung-Jong
    • Parasites, Hosts and Diseases
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    • v.53 no.1
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    • pp.65-75
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    • 2015
  • Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to $Zn^{2+}$ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.

Detection of the Recovery Substance for Cell Divison in UV-Irradiated Escherichia coli B -Stabilization of the Active Substance by Magnesium- (자외선 조사한 대장균 B 주의 세포분열 회복활성물질 -Magnesium에 의한 활성물질의 안정화-)

  • Song, Bang-Ho
    • Microbiology and Biotechnology Letters
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    • v.7 no.3
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    • pp.165-173
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    • 1979
  • Recovery component for cell division in UV-irradiated E. coli B was detected with use of the cell extract of E. coli B/r which is a resistant mutant of E. coli B against UV-irradiation. The active substance was non-dialyzable and increased the activity by adding B-NAD remarkably. One more factor for increasing or promoting the restoration recognized was magnesium. Magnesium was effective to stabilze the substance in procedure of isolation. Two active substances were obtained from sucrose gradient centrifugation. One of them was recovred from the botton area and the other from top area just below below surface. the former was not stabilized by magnesium, while the latter stabilized the activity by it remarkably. The former which did not require magnesium was insensitive to protease and the latter which required magnesium was sensitive to it. Both were insensitive to RNase and DNase. Recovery ratio was doubled by using nitrogen gas than aeration in purification process. DNA-ligase less mutant was revealed same activity on it's recovery ratio with the parent strain of E. coli K-12. The active substance stimulating the filament cell may exist as a complex which is inactivated easily in the dissociated state ana requrie B-NAD or magnesium.

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Characterization of Agarose Product from Agar Using DMSO

  • Jeon, You-Jin;Athukorala, Yasantha;Lee, Je-Hee
    • ALGAE
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    • v.20 no.1
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    • pp.61-67
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    • 2005
  • Agar was extracted from Gelidium amansii, which was harvested at the shores of Jeju Island in South Korea. As a unique solvent, the ability of dimethyl sulfoxide (DMSO) was used to separate agarose from agar by removing agaropectine and quality of the resultant agarose was characterized for chromatography purposes. Agar sample was agitated by motor-driven stirrer with DMSO in a water bath (at 70$^{\circ}C$ for 2 h) and centrifuged (3,000 rpm for 20 min). Resultant upper agarose layer was gelled, washed, dried and milled. The quality of agarose was evaluated by the analysis of proximate chemical composition, sulfate content, gelling strength and DNA migration. In this study, the separated agarose showed low sulfate amount (0.28%) and showed high gel strength (1190 g ${\cdot}\;cm^{-2}$). The resolution power and the ligase activities gave clear picture about the suitability of the present agarose for practical purposes.

Construction of Complementary DNA Library and cDNA Cloning for Cy Strain of Odontoglossum Ringspot Virus Genomic RNA (오돈토글로썸 윤문 바이러스 Cy계통 게놈 RNA의 cDNA 구축 및 유전자 크로닝)

  • 류기현;박원목
    • Korean Journal Plant Pathology
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    • v.10 no.3
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    • pp.228-234
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    • 1994
  • Genomic RNA was extracted from Cy strain of odontoglossum ringspot tobamovirus (ORSV-Cy) isolated from infected leaves of tobacco cv. Samsun. Size of the genomic RNA was about 6.6 kb in length. The genomic RNA was fractionated using Sephadex G-50 column chromatography into 2 fractions. They were polyadenylated at their 3'-end using E. coli poly(A) polymerase. Polyadenylated viral RNA was recovered by oligo (dT) primer adapter containing NotI restriction site and Moloney murine leukemia virus SuperScript reverse transcriptase (RNase H-). Second-strand cDNA was synthesized by using E. coli DNA ligase, E. coli DNA polymerase I and E. coli RNase H. Recombinant plasmids containing cDNAs for ORSV-Cy RNA ranged from about 800 bp to 3,000 bp. Among the selected 238 recombinants, pORCY-124 clone was the largest one covering 3'-terminal half of the viral RNA. This clone contained two restriction sites for EcoRI and XbaI and one site for AccI, AvaI, BglII, BstXI, HindIII, PstI, and TthIII 1. respectively. The clone contained partial viral replicase, a full-length movement protein and a complete coat protein genes followed by a 3' untranslated region of 414 nucleotides based on restriction mapping and nucleotide sequencing analyses. Clones pORCY-028, -068, -072, -187 and -224 were overlapped with the pORCY-124. Clones pORCY-014 and -095 covered 5' half upstream from the middle region of the viral RNA, which was estimated based on restriction mapping and partial sequence analysis. Constructed cDNA library covered more than 90% of the viral genome.

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Fluorometric Detection of Low-Abundance EGFR Exon 19 Deletion Mutation Using Tandem Gene Amplification

  • Kim, Dong-Min;Zhang, Shichen;Kim, Minhee;Kim, Dong-Eun
    • Journal of Microbiology and Biotechnology
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    • v.30 no.5
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    • pp.662-667
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    • 2020
  • Epidermal growth factor receptor (EGFR) mutations are not only genetic markers for diagnosis but also biomarkers of clinical-response against tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). Among the EGFR mutations, the in-frame deletion mutation in EGFR exon 19 kinase domain (EGFR exon 19-del) is the most frequent mutation, accounting for about 45% of EGFR mutations in NSCLCs. Development of sensitive method for detecting the EGFR mutation is highly required to make a better screening for drug-response in the treatment of NSCLC patients. Here, we developed a fluorometric tandem gene amplification assay for sensitive detection of low-abundance EGFR exon 19-del mutant genomic DNA. The method consists of pre-amplification with PCR, thermal cycling of ligation by Taq ligase, and subsequent rolling circle amplification (RCA). PCR-amplified DNA from genomic DNA samples was used as splint DNA to conjugate both ends of linear padlock DNA, generating circular padlock DNA template for RCA. Long stretches of ssDNA harboring multiple copies of G-quadruplex structure was generated in RCA and detected by thioflavin T (ThT) fluorescence, which is specifically intercalated into the G-quadruplex, emitting strong fluorescence. Sensitivity of tandem gene amplification assay for detection of the EGFR exon 19-del from gDNA was as low as 3.6 pg, and mutant gDNA present in the pooled normal plasma was readily detected as low as 1% fraction. Hence, fluorometric detection of low-abundance EGFR exon 19 deletion mutation using tandem gene amplification may be applicable to clinical diagnosis of NSCLC patients with appropriate TKI treatment.

Identification and Characterization of Coronatine-Producing Pseudomonas syringae pv. actinidiae

  • Han, Hyo-Shim;Koh, Young-Jin;Hur, Jae-Seoun;Jung, Jae-Sung
    • Journal of Microbiology and Biotechnology
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    • v.13 no.1
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    • pp.110-118
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    • 2003
  • Pseudomonas syringae pv. actinidiae strains, which cause canker disease in kiwifruit, were collected from kiwifruit orchards in Korea and identified using biochemical and physiological tests. The nucleotide sequences of the 16s rDNA and 16s-23s internally transcribed spacer of the isolates were found to be Identical to those of' the pathotype strain, Kwl 1, of P syringae pv. actinidiae. Remarkably, no coding sequence for phaseolotoxin biosynthesis or phaseolotoxin- resistant ornithine carbamoyltransferase was found by PCR amplification in any of the new Korean isolates of pseudomonas syringae pv. actinidiae, although this was clearly identified in the control pathotype Kwl 1 reference strain. In contrast, three primer sets derived from the coronatine biosynthetic gene cluster and DNA from the Korean strains yielded amplified DNA fragments of the expected size. A sequence analysis of the PCR products revealed that P. syringae pv. actinidiae and the Korean strains of pv. actinidiae contain coronafncate ligase genes (cfl)with identical sequences, whereas their. corR genes exhibited 91% sequence similarity. The production of coronatine, instead of phaseolotoxin, by the Korean strains of P. syringae pv. actinidiae was confirmed by a bioassay using reference pathovars known to produce coronatine and phaseolotoxin. The genes for coronatine biosynthesis in the Korean strains of P. syringae pv. actinidiae were found to be present on plasmids.

Ubiquitin E3 ligases controlling p53 stability

  • Lee, Seong-Won;Seong, Min-Woo;Jeon, Young-Joo;Chung, Chin-Ha
    • Animal cells and systems
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    • v.16 no.3
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    • pp.173-182
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    • 2012
  • The p53 protein plays a pivotal role in tumor suppression. The cellular level of p53 is normally kept low by proteasome-mediated degradation, allowing cell cycle progression and cell proliferation. Under stress conditions, such as DNA damage, p53 is stabilized and activated through various post-translational modifications of itself as well as of its regulatory proteins for induction of the downstream genes responsible for cell cycle arrest, DNA repair, and apoptosis. Therefore, the level of p53 should be tightly regulated for normal cell growth and for prevention of the accumulation of mutations in DNA under stress conditions, which otherwise would lead to tumorigenesis. Since the discovery of Mdm2, a critical ubiquitin E3 ligase that destabilizes p53 in mammalian cells, nearly 20 different E3 ligases have been identified and shown to function in the control of stability, nuclear export, translocation to chromatin or nuclear foci, and oligomerization of p53. So far, a large number of excellent reviews have been published on the control of p53 function in various aspects. Therefore, this review will focus only on mammalian ubiquitin E3 ligases that mediate proteasome-dependent degradation of p53.

Tumor suppressor Parkin induces p53-mediated cell cycle arrest in human lung and colorectal cancer cells

  • Byung Chul Jung;Sung Hoon Kim;Yoonjung Cho;Yoon Suk Kim
    • BMB Reports
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    • v.56 no.10
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    • pp.557-562
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    • 2023
  • Dysregulation of the E3 ubiquitin ligase Parkin has been linked to various human cancers, indicating that Parkin is a tumor suppressor protein. However, the mechanisms of action of Parkin remain unclear to date. Thus, we aimed to elucidate the mechanisms of action of Parkin as a tumor suppressor in human lung and colorectal cancer cells. Results showed that Parkin overexpression reduced the viability of A549 human lung cancer cells by inducing G2/M cell cycle arrest. In addition, Parkin caused DNA damage and ATM (Ataxia telangiectasia mutated) activation, which subsequently led to p53 activation. It also induced the p53-mediated upregulation of p21 and downregulation of cyclin B1. Moreover, Parkin suppressed the proliferation of HCT-15 human colorectal cancer cells by a mechanism similar to that in A549 lung cancer cells. Taken together, our results suggest that the tumor-suppressive effects of Parkin on lung and colorectal cancer cells are mediated by DNA damage/p53 activation/cyclin B1 reduction/cell cycle arrest.