• BMB Reports
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• 제37권4호
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• pp.466-473
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• 2004

A new acid deoxyribonuclease (DNase) was purified from the cultured mycelia of Cordyceps sinensis, and designated CSDNase. CSDNase was purified by $(NH_4)_2SO_4$ precipitation, Sephacryl S-100 HR gel filtration, weak anion-exchange HPLC, and gel filtration HPLC. The protein was single-chained, with an apparent molecular mass of ca. 34 kDa, as revealed by SDS-PAGE, and an isoelectric point of 7.05, as estimated by isoelectric focusing. CSDNase acted on both double-stranded (ds) and single- stranded (ss) DNA, but preferentially on dsDNA. The optimum pH of CSDNase was pH 5.5 and its optimum temperature 55. The activity of CSDNase was not dependent on divalent cations, but its enzymic activity was inhibited by high concentration of the cation: $MgCl_2$ above 150 mM, $MnCl_2$ above 200 mM, $ZnCl_2$ above 150 mM, $CaCl_2$ above 200 mM, NaCl above 300 mM, and KCl above 300 mM. CSDNase was found to hydrolyze DNA, and to generate 3-phosphate and 5-OH termini. These results indicate that the nucleolytic properties of CSDNase are essentially the same as those of other well-characterized acid DNases, and that CSDNase is a member of the acid DNase family. To our knowledge, this is the first report of an acid DNase in a fungus.

• 한국식물병리학회지
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• 제11권1호
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• pp.73-79
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• 1995

Complementary DNAs (cDNAs) to the coat protein gene of an ordinary Korean strain of potato virus Y (PVY-OK) isolated from potato (cv. Superior) were synthesized and cloned into a plasmid pUC119 and sequenced. The RNA of the virus propagated in tobacco (Nicotinaa sylvestris) was extracted by the method of phenol extraction. The first strand of cDNAs to the coat protein penomic RNA of the virus was made by Moloney murine leukemia virus reverse transcriptase. The cDNA were synthesized and amplified by the method of polymerase chain reaction (PCR) using a pair of oligonucleotide primers. PVYCP3P and PVYCP3M. The size of cDNAs inserted in pUC119 plasmid was estimated as about 840 bp upon agarose gel electrophoresis. Double stranded cDNAs were transformed into the competent cell of E. coli JM109. Sequence analysis of cDNAs was conducted by the dideoxynucleotide chain termination method. Homology of cDNAs of the PVY-OK coat protein genomic RNA with those of PVY-O (Japan), PVY-T (Japan), PVY-TH (Japan), PVYN (The Netherlands),and PVYY (France) was represented as 97.3%, 88.9%, 89.3%, 89.6% and 98.5%, respectively. Homology at the amino acid level turned out to the be 97.4%, 92.5%, 92.9%, 92.9% and 98.5%, respectively.

• BMB Reports
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• 제36권2호
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• pp.201-206
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• 2003

Two lymphoid-specific proteins, RAG1 and RAG2, are required for the initiation of the V(D)J recombination in vitro. The V(D)J cleavage that is mediated by RAG proteins at the border between the coding and signal sequences results in the production of a hairpin at the coding end and a double-stranded break at the signal end. Two hairpin coding ends are re-opened, modified, and sealed; whereas, the signal ends are directly ligated. Here I report that only RAG1 can carry out a distinct endonucleolytic activity in vitro using an oligonucleotide substrate that is tethered by a short single-stranded DNA. The purified RAG1 protein alone formed a nick at the near position to the recombination signal sequence. This endonucleolytic activity was eliminated by immunoprecipitation using the RAG1-specific antibody, and required the 3'-hydroxy group. All of the RAG1 mutants that were incapable of the nick and hairpin formation in the V(D)J cleavage analysis also showed this new endonucleolytic activity. This suggests that the nicking activity that was observed might be functionally different from the nick formation in the V(D)J cleavage.

• 한국동물생명공학회지
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• 제34권3호
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• pp.197-204
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• 2019

This study was investigated to test whether the zygote recognized the topoisomerase II beta (TOP2B) mediated DNA fragmentation in epididymal spermatozoa or the nuclease degradation in vas deferens spermatozoa by testing for the presence of gammaH2AX (γH2AX). The γH2AX is phosphorylation of histone protein H2AX on serine 139 occurs at sites flanking DNA double-stranded breaks (DSBs). The presence of γH2AX in the pronuclei of mouse zygotes which were injected with DNA broke epididymal spermatozoa was tested by immunohistochemistry at 5 and 9 h post fertilization, respectively. Paternal pronuclei that arose from epididymal spermatozoa treated with divalent cations did not stain for γH2AX at 5 h. On the other hand, in embryos injected with vas deferences spermatozoa that had been treated with divalent cations, γH2AX was only present in paternal pronuclei, and not the maternal pronuclei at 5 h. Interestingly, both pronuclei stained positively for γH2AX for all treatments and controls at 9 h after sperm injection. In conclusion, the embryos recognize DNA that is damaged by nuclease, but not by TOP2B because H2AX in phosphorylated in paternal pronuclei resulting from spermatozoa treated with fragmented DNA from vas deferens spermatozoa treated with divalent cations, but not from epididymal spermatozoa treated the same way.

• 핵의학기술
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• 제18권1호
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• pp.153-157
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• 2014

Anti double-stranded DNA (Anti-ds DNA)항체의 검출은 SLE의 진단에 중요하며 American College of Rheumatologists의 SLE 진단기준에 포함되어 있다. 또한 SLE 질병의 활성도와 Anti-ds DNA 항체 수준의 상관성이 보고되어 있으며 Anti-ds DNA 항체 정량검사는 SLE의 치료 전, 후 추적에 매우 유용하다. Anti-ds DNA 항체의 검출을 위한 검사 방법으로 방사면역측정법(radioimmunoassay, RIA)을 이용한 Farr assay, Crithidia luciliae를 이용한 면역형광 측정법(immunofluorescence Test, CLIFT), 효소면역측정법(enzyme-linked immunosorbent assay, ELISA), 화학발광면역 측정법(chemiluminescence immunoassay, CLIA)이 있다. 본원에서 방사면역측정법(radioimmunoassay, RIA)으로 Anti-ds DNA 항체 검사 과정에서 lipemic한 검체의 경우 침전물의 형성이 원활하지 않거나 침전물도 같이 흡입되는 상황이 발생 하였고, 이러한 문제점을 해결하기 위해 lipemic 검체가 분석 결과에 미치는 영향 정도를 평가하였다. 2012년 9월부터 2013년 2월까지 Anti-ds DNA 항체 검사가 의뢰된 검체 중 lipemic한 검체(n=81)를 선택하여 마이크로 원심분리기로 전처리(고속 원심분리: 14,000 rpm 5 mins)한 후 동시에 Anti-ds DNA 항체 검사(Anti-ds DNA kit, Trinity Biotech, Ireland)를 시행하였다. 실험군 1 (lipemic 검체의 Anti-ds DNA 항체 농도 ${\leq}7IU/mL$)에서 y=0.3636x+4.7322, $R^2=0.0238$, Pearson 상관계수는 0.154, paired t-test (P=0.003), Difference (%) mean 65.7의 결과를 얻었으며 통계적으로 유의한 차이를 보였다. 그러나 실험군 2 (lipemic 검체의 Anti-ds DNA 항체 농도 ${\geq}8IU/mL$)에서 y=0.9837x+0.2982, $R^2=0.994$, Pearson 상관계수는 0.997, paired t-test (P=0.181), Difference (%) mean -5.53을 보였고 통계적으로 유의한 차이가 없음을 확인할 수 있었다. 임상에서 SLE (systemic lupus erythematosus)의 진단에 중요한 역할을 하는 Anti-ds DNA 항체 검사는 lipemic 검체의 영향을 배제하기 위해서 반드시 전처리(고속 원심분리: 14,000 rpm 5 mins) 과정을 통해 혈청 내 지질 성분을 제거한 후 검사를 시행하여야 한다.

• Journal of Microbiology and Biotechnology
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• 제26권9호
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• pp.1629-1635
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• 2016

A novel bacteriophage, PBES 02, infecting Cronobacter sakazakii was isolated and characterized. It has a spherical head of 90 nm in diameter and a tail of 130 nm in length, and belongs to Myoviridae as observed under a transmission electron microscope. The major virion protein appears to be 38 kilodaltons (kDa) in size. The latent period of PBES 02 is 30 min and the burst size is 250. Infectivity of the phage remained intact after exposure to temperatures ranging from 4℃ to 55℃ for 1 h. It was also stable after exposure to pHs ranging from 6 to 10 for 1 h. The phage effectively removed contaminating Cronobacter sakazakii from broth infant formula. PBES 02 has a double-stranded DNA genome of 149,732 bases. Its GC ratio is 50.7%. Sequence analysis revealed that PBES 02 has 299 open reading frames (ORFs) and 14 tRNA genes. Thirty-nine ORFs were annotated, including 24 related to replication and regulation functions, 10 related to structural proteins, and 5 related to DNA packaging. The genome of PBES 02 is closely related to that of two other C. sakazakii phages, CR3 and CR8. Comparison of DNA sequences of genes encoding the major capsid protein revealed a wide geographical distribution of related phages over Asia, Europe, and America.

• The Plant Pathology Journal
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• 제28권4호
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• pp.433-438
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• 2012

Dinoflagellates are considered one of the most abundant and diverse groups of marine microplankton and viruses are recognized as one of the significant factors affecting the plankton dynamics. Here, we report basic characteristics of a new dinoflagellate-infecting virus, Heterocapsa pygmaea DNA virus (HpygDNAV) which infects a toxic dinoflagellate, H. pygmaea. HpygDNAV is a polyhedral large virus (ca. 160-170 nm in diameter) propagating in its host's cytoplasm. Because of the virion size, appearance in thin sections, and propagation characteristics, HpygDNAV is assumed to harbor a large double-stranded DNA genome; i.e., HpygDNAV is most likely a nucleocytoplasmic large DNA virus (NCLDV) belonging to the family Phycodnaviridae. Its infectivity is strain-specific, rather than species-specific, as is the case for other algal viruses. The burst size and latent period are estimated to be roughly 100-250 infectious units $cell^{-1}$ and < 96 h, respectively.

• Journal of Microbiology
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• 제37권4호
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• pp.229-233
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• 1999

The mitochondrial plasmid pMLP1 from a white-rot fungus, Pleurotus ostreatus, is a double-stranded DNA containing 381 bp terminal inverted repeat (TIR) whose 5'-ends are covalently bound by terminal proteins. The plasmid contains two major open reading frames (ORFs), encoding putative DNA and RNA polymerases, and a minor ORF encoding a small, highly basic protein. To identify the DNA binding activity that recognizes the TIR region of pMLP1, gel retardation assays were performed with mitochondrial extracts. A specific protein binding to a region between 123 and 248 nt within TIR was observed. We examined whether the gene product of mORF1 bindes to this region specifically. E. coli cell extract which contains an overproduced mORF1 protein formed a complex specific to the region between 123 and 248 nt. Inclusion of mORF1 protein in the specific complex formed between P. ostreatus mitochondrial extract and TIR was confirmed by a supershift assay using polyclonal antibodies against the mORF1 protein. Our result suggest that the product of mORF1 may function as a terminal region recognition factor (TRF), recognizing an internal region in TIR.

• BMB Reports
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• 제56권2호
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• pp.102-107
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• 2023

Genome editing using CRISPR-associated technology is widely used to modify the genomes rapidly and efficiently on specific DNA double-strand breaks (DSBs) induced by Cas9 endonuclease. However, despite swift advance in Cas9 engineering, structural basis of Cas9-recognition and cleavage complex remains unclear. Proper assembly of this complex correlates to effective Cas9 activity, leading to high efficacy of genome editing events. Here, we develop a CRISPR/Cas9-RAD51 plasmid constitutively expressing RAD51, which can bind to single-stranded DNA for DSB repair. We show that the efficiency of CRISPR-mediated genome editing can be significantly improved by expressing RAD51, responsible for DSB repair via homologous recombination (HR), in both gene knock-out and knock-in processes. In cells with CRISPR/Cas9-RAD51 plasmid, expression of the target genes (cohesin SMC3 and GAPDH) was reduced by more than 1.9-fold compared to the CRISPR/Cas9 plasmid for knock-out of genes. Furthermore, CRISPR/Cas9-RAD51 enhanced the knock-in efficiency of DsRed donor DNA. Thus, the CRISPR/Cas9-RAD51 system is useful for applications requiring precise and efficient genome edits not accessible to HR-deficient cell genome editing and for developing CRISPR/Cas9-mediated knockout technology.

• BMB Reports
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• 제56권5호
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• pp.302-307
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• 2023

Lyn, a tyrosine kinase that is activated by double-stranded DNA-damaging agents, is involved in various signaling pathways, such as proliferation, apoptosis, and DNA repair. Ribosomal protein S3 (RpS3) is involved in protein biosynthesis as a component of the ribosome complex and possesses endonuclease activity to repair damaged DNA. Herein, we demonstrated that rpS3 and Lyn interact with each other, and the phosphorylation of rpS3 by Lyn, causing ribosome heterogeneity, upregulates the translation of p-glycoprotein, which is a gene product of multidrug resistance gene 1. In addition, we found that two different regions of the rpS3 protein are associated with the SH1 and SH3 domains of Lyn. An in vitro immunocomplex kinase assay indicated that the rpS3 protein acts as a substrate for Lyn, which phosphorylates the Y167 residue of rpS3. Furthermore, by adding various kinase inhibitors, we confirmed that the phosphorylation status of rpS3 was regulated by both Lyn and doxorubicin, and the phosphorylation of rpS3 by Lyn increased drug resistance in cells by upregulating p-glycoprotein translation.