• The Plant Pathology Journal
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• 제25권1호
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• pp.47-53
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• 2009

Sequence analysis of the Chlorella virus SS-2 revealed one putative nuclease gene that is 807 bp long and encodes a 31kDa protein. Multiple sequence alignment analysis reveals the presence of highly conserved PD-(D/E)XK residues in the encoded protein. The gene cloned into an expression vector was expressed as a His-tagged fusion protein in chaperone containing pKJE7 cells. The recombinant protein was purified using a His-Trap chelating HP column and used for functional analysis. Exonuclease activity of the SS-2 nuclease was detected when the DNA substrates, such as linear ssDNA, PCR amplicon, linear dsDNA with 5'-overhang ends, 3'-overhang ends, or blunt ends were used. Covalently closed circular DNA was also degraded by the SS-2 recombinant protein, suggesting that the SS-2 nuclease has an endonuclease activity. Stable activity of SS-2 nuclease was observed between $10^{\circ}C$ and $50^{\circ}C$. The optimum pH concentrations for the SS-2 nuclease were pH 6.0-8.5. Divalent ions inhibited the SS-2 nuclease activity.

• BMB Reports
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• 제36권5호
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• pp.520-523
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• 2003

The proliferation and differentiation signals of myelogeneous U937 cells are provided by extracellular stimuli, such as lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA). In a DNA-native-polyacrylamide gel assay system, we demonstrated that a particular nuclease activity is expressed in PMA-stimulated U937 cells and secreted into the culture medium. The nuclease activity was induced in U937 cells by LPS treatment, while the secretion of the enzyme was undetected in the culture medium. Therefore, it is likely that the expression and secretion of the particular nuclease in U937 cells are controlled by extracellular stimulations, such as PMA and LPS treatment.

• BMB Reports
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• 제31권4호
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• pp.405-408
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• 1998

Endonuclease-sensitive sites detected by T4 endonuclease V or UvrABC nuclease treatments were compared in the dihydrofolate reductase gene of UV-irradiated Chinese hamster ovary B-11 cells. The number of endonuclease-sensitive sites detected by T4 endonuclease V treatment followed by NaOH denaturation was twice that of formamide denaturation. Repeated treatment of damaged genomic DNA with T4 endonuclease V resulted in no further increase in the number of endonuclease-sensitive sites detected. The numbers of endonuclease-sensitive sites detected by UvrABC nuclease using each denaturation condition were similar. Sequential treatment with the two endonucleases using formamide denaturation resulted in twice the number of endonuclease-sensitive sites detected by treatment of each nuclease alone. Due to a lack of AP endonuclease activity these results suggest the presence of T4 endonuclease V-sensitive sites which could be complemented by alkaline gel separation or by UvrABC nuclease treatment.

• 대한수의학회지
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• 제21권2호
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• pp.99-104
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• 1981

A total of 251 clinical isolates (human origin, 43 strains and bovine udder origin 208 strains) of the Staphylococcus that fermented mannitol aerobically were tested for their ability to produce coagulase, DNase, and thermostable nuclease. Of these, 158 isolates coagulated human or bovine plasma, produced DNase, and thermostable, nuclease and were identified as St. aureous, 146 of which produced a 1+ to 3+ clot. The remaining 12 isolated produced a -clot in citrate treated plasma but produced 1+ to 3+ clot in ethylenedi-aminetetraacetic acid (EDTA) treated plasma. It was found that 7 coagulase positive isolates failed to produced thermostable nuclease. In these organisms, we found out of the clot formation is not by coagulase activity but utilization of citrate, because EDTA treated plasma is not coagulated. Among 93 isolates which did not coagulate citrate-or EDTA treated plasma and thermostable nuclease negative, 28 strains produced DNase were identified as St. epidermidis, and other strains were not identification further. It was found that thermostable nuclese production appears to be a consistent property of St. aureus and the test is easy to perform, is rapid became quite distinct within 2 to 4 hour, and is not influenced by as many factors and variations as the coagulase test.

• Journal of Microbiology and Biotechnology
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• 제14권1호
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• pp.178-181
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• 2004

A gene encoding an extracellular nuclease was cloned from Aeromonas hydrophila strain ATCC14715. The gene was overexpressed and the enzyme was purified by fusing to maltose binding protein. It was shown that the protein possessed DNase activity on both single-stranded and double-stranded DNAs. It exhibited both endo- and exonuclease activities. It was also shown that the protein had an RNase activity. Possible roles of this extracellular enzyme in the A. hydrophila life cycle are discussed.

• Asian-Australasian Journal of Animal Sciences
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• 제3권2호
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• pp.115-120
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• 1990

Extracellular nuclease(s) in buffalo rumen fluid were purified from strained rumen fluid by a procedure involving Seitz filtration, acetone fractionation and gel filtration on Sephadex G-100. The enzyme resolved into two peaks exhibiting both DNase and RNase activities. The molecular weight of enzyme corresponding to peaks I and II were approximately 30,000 and 12,000 respectively. The properties of enzymes from the two peaks, however, were same. Optimum temperature for both DNase and RNase activities was at $50^{\circ}C$. Whereas DNase activity was stable upto $60^{\circ}C$, RNase activity was stable only up to $50^{\circ}C$. DNase activity recorded two pH optima, one at pH 5.5 and the other at pH 7.0. RNase activity recorded a broad pH optimum between pH 6.0-8.0. pH stability of the enzyme coincided with pH optima for both the activities. DNase activity was stimulated by $Mg^{2+}$ and $Mn^{2+}$ and inhibited by $Fe^{2+}$, $Zn^{2+}$, $Hg^{2+}$ and $Ag^+$. RNase activity was also stimulated by $Mg^{2+}$ and $Mn^{2+}$ and inhibited by $Cu^{2+}$, $Fe^{2+}$, $Zn^{2+}$, $Hg^{2+}$ and $Ag^+$. Reducing agents stimulated both the activities.

• Bulletin of the Korean Chemical Society
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• 제35권10호
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• pp.3005-3008
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• 2014

Human ChlR1 protein (hChlR1), a member of the cohesion establishment factor family, plays an important role in the segregation of sister chromatids for maintenance of genome integrity. We previously reported that hChlR1 interacts with hFen1 and stimulates its nuclease activity on the flap-structured DNA substrate covered with RPA. To elucidate the relationship between hChlR1 and Okazaki fragment processing, the effect of hChlR1 on in vitro nuclease activities of hFen1 and hDna2 was examined. Independent of ATPase activity, hChlR1 stimulated endonuclease activity of hFen1 but not that of hDna2. Our findings suggest that the acceleration of Okazaki fragment processing near cohesions may aid in reducing the size of the replication machinery, thereby facilitating its entry through the cohesin ring.

• BMB Reports
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• 제42권11호
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• pp.713-718
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• 2009

Apoptotic DNA fragmentation, the hallmark of apoptosis, is mediated primarily by caspase-activated DFF40 (CAD) nuclease. DFF40 exists as a heterodimer with DFF45 (ICAD), which is a specific chaperone and inhibitor of DFF40 under normal conditions. To understand the mechanism through which the DFF40/DFF45 system is regulated, we analyzed the structural and biochemical properties of apoptotic DNA fragmentation mediated by DFF40/DFF45. Using limited proteolysis, we show that residues 1-281 of DFF45 form a rigid, crystallized domain, whereas the loop formed by residues 277-281 is accessible by trypsin. These results show that the C-terminal helix formed by residues 281-300 is dynamic and necessary for the chaperone activity of DFF45, but not for inhibition of DFF40.

• 미생물학회지
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• 제32권2호
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• pp.147-154
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• 1994

Serratia marcescens가 세포외로 분비하는 nuclease의 유전자가 발현된 Escherichia coli JM107을 배양하여 다량의 효소를 정제하였다. Matrex green gel과 heparin agarose gel column chromatography법으로 약 50배 정제한 효소는 분자량이 29KDa였으며, 전기영동 상에서 단일 띠를 보였다. 이 단백질을 이용하여 polyclonal antibody를 만들고, 면역조직화학법으로 세포내의 분포를 조사하였다. Nuclease는 주로 세포막에 존재하였고, 이를 토대로 효소가 세포질에서 합성된 후 세포막으로 빠르게 이동함을 알 수 있었다. 이 결과는 세포의 막분획에서 효소의 활성의 대부분이 회수되며, 면역블럿 방법으로 효소의 대부분이 세포막에서 검출된다는 결과와 일치하였다.

• Molecules and Cells
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• 제38권9호
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• pp.773-780
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• 2015

3D8 single chain variable fragment (scFv) is a recombinant monoclonal antibody with nuclease activity that was originally isolated from autoimmune-prone MRL mice. In a previous study, we analyzed the nuclease activity of 3D8 scFv and determined that a HeLa cell line expressing 3D8 scFv conferred resistance to herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). In this study, we demonstrate that 3D8 scFv could be delivered to target tissues and cells where it exerted a therapeutic effect against PRV. PRV was inoculated via intramuscular injection, and 3D8 scFv was injected intraperitoneally. The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays. Intraperitoneal injection of 5 and $10{\mu}g$ 3D8 scFv resulted in no detectable toxicity. The survival rate in C57BL/6 mice was 9% after intramuscular injection of 10 $LD_{50}$ PRV. In contrast, the 3D8 scFv-injected C57BL/6 mice showed survival rates of 57% ($5{\mu}g$) and 47% ($10{\mu}g$). The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models.