• 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.

• 미생물학회지
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• 제26권2호
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• pp.73-81
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• 1988

$\alpha$-Amylase (EC.3.2.1.1) of Neurospora crassa (ATCC9279) was cloned in E. coli HB101 using shotgun method, and the enzymes isolated from both N. crassa and E. coli were compared. Chromosomal DNA isolated from the spores of N. crassa was partially digested with PstI restriction endonuclease and rejoined to pBR322 which had been digested with the same enzyme. The resulting recombinant DNA were introduced into E. coli HB101 which had competancy by treating with $CaCl_{2}$. As the result, about 8000 colonies which showed tetracycline resistance were selected and two of the colonies which had 13.5Kb recombinant plasmid exhibit starch degrading activity on starch-containing plate when treated with D-cycloserine. $\alpha$-Amylases from both N.crassa and E. coli were isolated by using ammonium sulfate precipitation, DEAE-cellulose ion exchange column chromatography and Bio-Gel P150 gel foltration column. As the result, about 81.3 fold and 5.6 fold purifications in specific activities were obtained respectively, and specific activities of the gel filtrates were 6.1u/mg and 85u/mg respectively. The properties of both enzymes were compared and they showed quite the similar patterns in optimal temperature, optimal pH and had same molecular weight about 100,000 daltons on gel filtration method. Optimal temperatures for both enzymes were $70^{\circ}C$ and optimal pH were about 6 and 10.

• 한국어병학회지
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• 제12권2호
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• pp.115-121
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• 1999

양식넙치에서 분리된 Edwardsiella tarda가 갖는 약제내성을 전달하는 R plasmid의 특성을 알아보기 위하여, 16종의 화학요법제에 대한 E. tarda의 감수성 정도를 비교하였으며, 내성형태 및 내성전달을 확인하고 transferable R plasmid를 분리하였다. E. tarda는 ampicillin, amoxicillin, erythromycin, flumequine, doxycycline(DOXY), nalidixic acid, novobiocin, oxolinic acid, oxytetracycline(OTC), thiamphenicol(TP) 그리고 sulfonamide의 11약제에 대하여 다양한 조합으로 복합내성을 보였으며, DOXY, OTC, TP 내성이 Escherichia coli로 전달되었다. 복합내성의 두 균주에서 transconjugant가 형성되었으며 이들로부터 분리된 transferable R plasmid는 서로 상이한 DNA 구조였다. 이는 넙치에서 분리되는 E. tarda에는 적어도 두 종류 이상의 thansferable R plasmid가 분포한다는 것을 의미한다.

• BMB Reports
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• 제33권2호
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• pp.155-161
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• 2000

The saccharomyces cerevisiae Rad27, a structure-specific endonuclease for the okazaski fragment maturation has been known to interact genetically and biochemically with Dna2, an essential enzyme for DNA replication. In an attempt to define the significance of the interaction between the two enzymes, we expressed and purified both Dna2 and Rad27 proteins. In this report, Rad27 could not form a complex with Dna2 in the three different analyses. The analyses included glycerol gradient sedimentation, protein-column chromatography, and coinfection of baculoviruses followed by affinity purification. This is in striking contrast to the previous results that used crude extracts. These results suggest that the interaction between the two proteins is not sufficiently stable or indirect, and thus requires an additional protein(s) in order for Rad27 and Dna2 to form a stable physical complex. This result is consistent with our genetic findings that Schizosaccharomyces pombe Dna2 is capable of interacting with several proteins that include two subunits of polymerase $\delta$, DNA ligase I, as well as Fen-1. In addition, we found that the N-terminal modification of Rad27 abolished its enzymatic activity. Thus, as suspected, we found that on the basis of the structure determination, N-terminal methionine indeed plays an important role in the nucleolytic cleavage reaction.

• BMB Reports
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• 제33권2호
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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.

• 약학회지
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• 제31권2호
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• pp.116-125
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• 1987

It is still difficult and time consuming to obtain cDNA sequences that contain the entire nucleotide sequence of the corresponding mRNA. A rapid and high efficient cloning method to obtain full-length cDNA segments is thus developed. The cloning procedure described here consists of the construction of oligo(dT)-tailed vector primer using pWR34 plasmid, polyadenylation of mRNA-cDNA heteroduplex using terminal deoxytransferase, and replacement of MRNA strand with DNA by RNase H and DNA polymerase I. The restriction endonuclease analysis shows that the size of inserted-cDNA is in the range of 1.5~4.0 kb long suggesting that most of cloned cDNA are full-length or nearly full-length cDNA. The plasmid-DNA recombinants obtained were 4$\times$$10^5$~$10^{6}$ per $\mu\textrm{g}$ of rat liver poly (A$^+$)mRNA, which is 4 to 10 fold higher cloning efficiency in comparison to the presently used methods for full-length cDNA cloning. The results indicate that the described cloning system is much simpler, less time consuming, and very efficient cloning method to construct a cDNA library.

• 환경생물
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• 제18권1호
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• pp.53-61
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• 2000

서울시 소재 지하수 중 중금속으로 오염되어 음용수 이외의 생활 용수로 사용하고 있는 1개 정점과 음용수로 사용하고 있는 1개 정점, 대조군으로 강화도 천연 동굴의 1개 정점을 대상으로 실험을 실시하였다. 지하수 세균군집의 유전적 다양성의 변화를 보기 위해 지하수 세균 군집에서 16SrDNA를 증폭하는 primer로 PCR(polymerase chain reaction)을 실시한 후 ARDRA(amplified ribosomal DNA restriction analysis) 지문 분석으로 비교하였다. 16S rDNA를 증폭하여 제한효소 지문분석을 한 결과 in situ와 음용수에서 유전적 다양성이 상대적으로 크게 나타났다. 지하수 세균 군집의 ARDRA지문 분석은 상이한 환경과 서식지를 반영한 유전적 차이를 빠르게 비교 분석할 수 있었으며 지하수의 오염도에 따른 미생물의 다양성(천연 동굴>음용수>오염수)을 확인할 수 있었다.

• 한국환경성돌연변이발암원학회지
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• 제22권4호
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• pp.243-247
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• 2002

Prolonged exercise is known to increase steady-state serum high-density lipoprotein cholesterol (HDL-cholesterol) and apolipoprotein AI(apo AI) concentrations. We investigated the effect of adaptation to endurance exercise on the association of the genetic polymorphism in the apo AI gene with these biochemical parameters. 108 male subjects were randomly selected from a group of elite athletes, and 65 male samples used as sedentary control group from Korean general population. The genetic polymorphism in the apo AI gene locus was detected by polymerase chain reaction(PCR) and DNA digestion with Msp I restriction endonuclease. The genotype frequency for the Msp I RFLP was significantly different between the elite athletes and sedentary controls(P<0.05). There were, however, no significant associations between the Msp I RFLP of the apo AI gene and the biochemical parameters in elite athletic group. Therefore, our findings indicate that the Msp I RFLP of the apo AI gene was not associated with the serum apo AI and HDL-cholesterol concentrations in Korean male elite athletes.

• BMB Reports
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• 제51권7호
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• pp.315-316
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• 2018

Genome engineering with clustered regularly interspaced short palindromic repeats (CRISPR) system can be used as a tool to correct pathological mutations or modulate gene expression levels associated with pathogenesis of human diseases. Owing to well-established local administration methods including intravitreal and subretinal injection, it is relatively easy to administer therapeutic genome engineering machinery to ocular tissues for treating retinal diseases. In this context, we have investigated the potential of in vivo genome engineering as a therapeutic approach in the form of ribonucleoprotein or CRISPR packaged in viral vectors. Major issues in therapeutic application of genome engineering include specificity and efficacy according to types of CRISPR system. In addition to previous platforms based on ribonucleoprotein and CRISPR-associated protein 9 derived from Campylobacter jejuni, we evaluated the therapeutic effects of a CRISPR RNA-guided endonuclease derived from Lachnospiraceae bacterium ND2006 (LbCpf1) in regulating pathological angiogenesis in an animal model of wet-type age-related macular degeneration. LbCpf1 targeting Vegfa or Hif1a effectively disrupted the expression of genes in ocular tissues, resulting in suppression of choroidal neovascularization. It was also notable that there were no significant off-target effects in vivo.

• 한국식물병리학회지
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• 제10권3호
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• pp.235-239
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• 1994

Viral RNA was extracted from purified Chinese cabbage strain of turnip mosaic virus (TuMV-Ca) from infected leaves of turnip. Polyadenylated genomic viral RNA was recovered by oligo (dT) cellulose column chromatography and used as a template for the synthesis of complementary DNA (cDNA). Recombinant plasmids contained cDNA ranged from about 900 bp to 2, 450 bp were synthesized. Among the selected 41 transformants, pTUCA31 and pTUCA35 had over 2 Kbp cDNA insert. Restriction endonuclease patterns of the clones examined were very similar among them. Clones pTUCA23 and pTUCA31 were overlapped with pTUA35. The longest clone pTUCA35, encoding 3'-end, showed that it contained two sites for EcoRI, and one site for BamHI, ClaI, HincII, SacI and XbaI, respectively. The restriction mapping indicated that the clone pTUCA35 contained partial nuclear inclusion body gene, complete coding region of the coat protein and 3' untranslated region of TuMV-Ca genomic RNA.