• 미생물학회지
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• 제28권4호
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• pp.297-303
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• 1990

Secretion of Serratia marcescens nuclease by E. coli harboring pNUC4 was investigated. 29.2, 54.2 and 16.6% of total nuclease were observed in culture medium, periplasm, and cytoplasm of E. coli, respectively. To investigate the secretion mechanism of Serratia nuclease by E. coli, secretion kinetics of nuclease was examined in the presences of sodium azide, and energy metabolism inhibitor; procaine, an exoprotein processing inhibitor; and chloramphenicol, a protein synthesis inhibitor. In the presence of sodium azide, periplasmic unclease was gradually decreased and the extracellular nyclease was linearly increased according to the incubation time. Similar results were obtained in presences of procaine and chloramphenicol. From these results, we concluded that two transport processes are involved in nuclease secretion: secretion of nuclease through the inner membrane is occurred by an energy-dependent process and probably requiring precusor processing: secretion of nuclease through outer membrane does not require energy, de novo protein synthesis, and precursor processing.

• 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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• 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.

• Applied Biological Chemistry
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• 제37권5호
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• pp.334-338
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• 1994

Streptococcal nuclease가 Streptococcus sp. 발효액으로부터 stepwise CM-Sepharose 컬럼을 이용하여 완전히 분리 정제되었다. 역가 높은 효소 분획은 0.2 M 용리시 나왔고 분리 정제된 효소의 분자량은 35,000으로 전기영동상 밝혀졌으며 최적 pH와 온도는 각각 pH 9.0, $60^{\circ}C$로 밝혀졌다.

• 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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• 제4권4호
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• pp.364-366
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• 1994

Molecular properties of streptococcal nuclease purified from Streptococcus sp. were examined. The purified enzyme was stable in the range of pH 7 to 10 and easily inactivated above $60^{\circ}C$. Atomic spectroscopy analysis indicated that the enzyme contains Ca, Mg, Zn. Circular dichroism study showed 25% $\alpha$ -helix, 15% $\beta$-sheet and 30% $\beta$-tums.

• 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.

• Bulletin of the Korean Chemical Society
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• 제22권1호
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• pp.87-89
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• 2001

• 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.

• Journal of Microbiology and Biotechnology
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• 제16권12호
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• pp.2004-2007
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• 2006

An efficient method for the in vitro reassembly of homologous DNA sequences is presented. The proposed method involves obtaining single strands of homologous genes and hybridizing them to obtain partially hybridized heteroduplex DNA; cleaving the single-stranded regions of the heteroduplex DNA using S1 nuclease to generate double-strand DNA fragments; denaturing the double-strand DNA fragments to generate single-strand DNA fragments; conducting a series of polymerase chain reactions (PCR) using the single-strand DNA fragments as internal primers and a mixture of homologous DNA as templates to obtain elongated reassembled DNA; and finally, amplifying the reassembled DNA by a PCR using terminal primers. As a result, DNA reassembly could be achieved between homologous genes with a sequence similarity as low as 78%.