• 제목/요약/키워드: Positive Clone

검색결과 117건 처리시간 0.03초

Quantitative Analysis of Human- and Cow-Specific 16S rRNA Gene Markers for Assessment of Fecal Pollution in River Waters by Real-Time PCR

  • Jeong, Ju-Yong;Park, Hee-Deung;Lee, Kyong-Hee;Hwang, Jae-Hong;Ka, Jong-Ok
    • Journal of Microbiology and Biotechnology
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    • 제20권2호
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    • pp.245-253
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    • 2010
  • The base sequences representing human- and cow-specific 168 rRNA gene markers identified in a T-RFLP analysis were recovered from clone libraries. The human- and cow-specific primers were designed from these sequences and their specificities were analyzed with fecal DNAs from human, cow, and pig. The AllBac primer set showed positive results for all human, cow, and pig samples, whereas the human-specific primer set showed positive result only for the human sample but not for the cow or pig samples. Likewise, the cow-specific primer set showed positive results only for the cow sample but not for the human or pig samples. Real-time PCR assay with these primers was developed for the identification and quantification of fecal pollution in the river water. The human- and cow-specific markers were detected in the order of 9 $\log_{10}$ copies per gram wet feces, which were two orders of magnitude lower than those of total Bacteroidales. For the river water samples, the human-specific marker was detected in $1.7-6.2\;\log_{10}$ copies/100 ml water, which was 2.4-4.9 orders of magnitude lower than those of total Bacteroidales. There was no significant correlation between total Bacteroidales and conventional fecal indicators, but there was a high correlation between Bacteroidales and the human-specific marker. This assay could reliably identify and quantify the fecal pollution sources, enabling effective measures in the watersheds and facilitating water quality management.

인체 급성백혈병 Jurkat T 세포에 있어서 L-canavanine에 의해 유도되는 세포자살기전에 미치는 단백질 티로신 키나아제 p56lck의 저해 효과 (A Natural L-Arginine Analog, L-Canavanine-Induced Apoptosis is Suppressed by Protein Tyrosine Kinase p56lck in Human Acute Leukemia Jurkat T Cells)

  • 박해선;전도연;우현주;류석우;김경민;김상국;박완;문병조;김영호
    • 생명과학회지
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    • 제19권11호
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    • pp.1529-1537
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    • 2009
  • L-arginine 구조유사체인 L-canavanine의 인체 급성백혈병 Jurkat T 세포에 대한 apoptosis 유도활성이 단백질 티로신키나아제 $p56^{lck}$에 어떻게 조절되는지를 규명하기 위해 $p56^{lck}$를 발현하는 Jurkat T 세포주 E6.1과 $p56^{lck}$-결손 Jurkat T 세포주 JCaM1.6에 있어서 L-canavanine의 세포독성, L-canavanine에 의해 유도되는 apoptotic DNA fragmentation 및 apoptotic sub-$G_1$ peak를 비교하여 본 바, $p56^{lck}$-negative JCaM1.6 세포가 $p56^{lck}$-positive E6.1 세포에 비해 L-canavanine의 apoptotis 유도활성에 훨씬 더 민감한 것으로 나타났다. 이러한 $p56^{lck}$-negative JCaM1.6 세포의 민감성은 JCaM1.6 세포에 $p56^{lck}$ 유전자를 transfection시켜 발현시키면 현저히 감소되었다. L-Canavanine에 의해 유도되는 apoptosis관련 현상들을 $p56^{lck}$-stable transfectant인 JCaM1.6/lck 세포와 empty vector-transfectant 인 $p56^{lck}$-negaive JCaM1.6/vector 세포에서 Western blot analysis로 비교한 결과, L-canavanine에 의해 유도되는 mitochondrial membrane potential (${\Delta\Psi}m$)의 감소, caspase-9, -8, -7 및 -3의 활성화, 그리고 PARP 및 $PLC{\gamma}$-1의 분해가 JCaM1.6/vector 세포에 비해 JCaM1.6/lck 세포에서 더 약하게 나타났다. JCaM1.6/lck 세포를 2.5 mM L-canavanine으로 처리한 다음 세포 내 $p56^{lck}$ kinase 활성의 변화를 $\alpha$-casein을 기질로 하여 시간 별로 측정한 결과, L-canavanine의 처리 후 15분만에 $p56^{lck}$ kinase의 활성이 약 1.6배 증가되었으며 이후 6시간 동안은 약 1.3~1.4 배정도 증가된 수준으로 kinase 활성이 유지되는 것으로 확인되었다. L-Canavanine에 의한 apoptosis의 개시에 Fas/FasL 상호작용이 관련되는지를 규명하기 위해 FADD-negative Jurkat T 세포주 I2.1, caspase-8-negative Jurkat T 세포주 I9.2 및 wild-type Jurkat T 세포주 A3에 대한 L-canavanine의 세포독성을 비교한 결과, A3와 I2.1 세포의 경우는 L-canavanine의 세포독성이 동일하게 나타났고, 특히 caspase-8가 결손된 I9.2 세포의 경우는 L-canavanine의 세포독성에 대한 민감성이 A3와 I2.1 세포에 비해 단지 미약하게만 완화되는 것으로 나타나, L-canavanine의한 apoptosis에는 Fas/FasL 상호작용이 관련되어 있지 않으며, 또한 caspase-8의 역할이 필수적이지 않음을 시사하였다. Jurkat T 세포에 있어서 L-canavanie에 의해 유도되는 sub-$G_1$ peak 및 caspases 활성화에 미치는 pan-caspase inhibitor (z-VAD-fmk), caspase-9 inhibitor (z-LEHD-fmk), caspase-3 inhibitor (z-DEVD-fmk), caspase-4 inhibitor (z-LEVD-fmk) 및 caspase-12 inhibitor (z-ATAD-fmk)의 영향을 조사한 결과, L-canavanie에 의한 apoptosis는 ${\Delta\Psi}m$의 감소, caspase-9 및 caspase -3의 활성화에 뒤따른 caspase-8 및 caspase-7의 활성화, 그리고 PARP의 분해의 순서로 유도되는 것으로 나타났으며, 아울러 caspase-9의 활성화와 함께 caspase-12의 활성화가 L-canavanine 처리에 따른 caspase-3의 활성화에 요구되는 것으로 확인되었다. 결론적으로, L-canavanine 처리에 의한 Jurkat T 세포의 apoptosis는 ${\Delta\Psi}m$ 감소, caspase-9, caspase-3 및 caspase-7의 활성화에 의해 유도되며, 이들 apoptosis 현상들은 $p56^{lck}$에 의해 negative regulation되었다.

질소시용, 예초 및 재식밀도가 한국잔디(Zoysia Japonica Steud)의 생육에 미치는 영향 (Studies on the growth of Korea Lawn Grass (Zoysia japonica Steud.)in Reponse to Nitrogen Application, Clipping Treatment and Plant Density)

  • 심재성
    • 자연과학논문집
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    • 제1권
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    • pp.61-113
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    • 1987
  • The increasing emphasis placed on the production of fine turf for lawns, golf courses, parks, and other recreational sites has led to many unsolved problems as to how such turf could be best established and mainteined. For this purpose, a series of experiments were conducted under con ditions of pot and field. The results obtained were as follows EXPERIMENT I. The effect of nitrogen fertilizer and clipping interval on Zoysia japonica. 1. Increasing the rate of nitrogen and frequent clipping increased tiller number of Zoysis japonica and the maximum number of tillers were obtained from 700 kg N application and freqnent clippings (10 days interval ) in October. Treatment of 350kg N with 10 days clipping interval increased tillers much more than those of 700 kgN with 20 and 30 days clipping intervals. 2. The average number of green leaves occurred during the growth period maximized by applying 700 kg N and clipping 10 days interval. 3. Increasing tiller numbers significantly decreased tops DM weight per tiller by clipping plants at interval of 10 and 20 days, irrespective of nitrogen applied, and with nil N, at the interval of 30 days. By applying 700 kg N, however, top DM weight per tiller increased as the number of tillers increased consistently. 4. The highest top DM weight was achieved from late August to early September by applying 350 and 700kgN. 5. During the growth period, differences in unders ( stolon + root ) DM weight occurred bynitrogen application were found between nil N and two applied nitrogen levels, whereas, at the same level of nitrogen applied, the increase in stolon DM weight enhanced by lengthening the clipping interval to 30 days. 6. Nitrogen efficiency to green leaves, stolon nodes and DM weight of root with high nitrogen was achieved as clipping interval was shortened. 7. By increasing fertilizer nitrogen rate applied, N content n the leaves and stems of Zoysiajaponica was increased. On the other hand, N content in root and stolon had little effect onfertilizer nitrogen, resulting in the lowest content among plant fractions. The largest content of N was recorded in leaves. Lengthening the clipping interval from 10 or 20 to 30 days tends to decrease the N content in the leaves and stems, whereas this trend did not appeared in stolon androot. 8. A positive correlations between N and K contents in tops and stolon were established andthus K content increased as N content in tops and stolon increased. Meanwhile, P content was not affected by N and clipping treatments. 9. Total soluble carbohydrate content in Zoysia japonica was largest in stolon and stem, and was reduced by increasing fertilizer nitrogen rate. Reduction in total soluble carbohydrate due to increased nitrogen rate was severer in the stolons and stems than in the leaves. 10. Increasing the rate of nitrogen applied increased the number of small and large vascular bundles in leaf blade, but shortened distance among the large vascular bundles. Shortening the clipping interval resulted in increase of the number of large vascular bundles but decrease ofdistance between large vascular bundles.EXPERIMENT II. Growth response of Zoysia japonica imposed by different plant densities. 1. Tiller numbers per unit area increased as plant density heightened. Differences in num ber between densities at higher densities than 120 D were of no significance. 2. Tiller numbers per clone attained by 110 days after transplanting were 126 at 40D,77 at 80D, 67 at 120D, 54 at 160D, and 41 at 200D. A decreasing trend of tiller numbers per clone with increasing density was noticable from 100 days after transplanting onwards. 3. During the growth period, the greatest number of green leaves per unit area were attainedin 90days after transplanting at 160D and 200D, and 100 days after transplanting at 40D, 80Dand 120D. Thus the period to reach the maximum green leaf number with the high plantdensity was likely to be earlier that with the low plant density. 4. Stolon growth up to 80 days after transplaning was relatively slow, but from 80 daysonwards, the growth quickened to range from 1.9 m/clone at 40D to 0.6m/clone at 200Din 200 days after transplanting, these followed by the stolon node produced. 5. Plant density did not affect stolon weight/clone and root weight/clone until 80 daysafter transplanting. 6. DM weight of root was heavier in the early period of growth than that of stolon, butthis trend was reversed in the late period of growth : DM weight of stolon was much higherthan that of root.EXPERIMENT Ill. Vegetative growth of Zoysia japonica and Zoysia matrella as affected by nitrogen and clipping height. 1. When no nitrogen was applied to Zoysia japonica, leaf blade which appeared during theAugust-early September period remained green for a perid of about 10 weeks and even leavesemerged in rate September lived for 42 days. However, leaf longevity did not exceed 8 weeks asnitrogen was applied. In contrast the leaf longevity of Zoysia matrella which emerged during the mid August-earlySeptember period was 11 weeks and, under the nitrogen applied, 9 weeks, indicating that thelife-spen of individual leaf of Zoysia matrella may be longer than that of Zoysia japorica. Clipping height had no effect on the leaf longevity in both grasses. 2. During the July-August period, tiller number, green leaf number and DM weightof Zoysia japonica were increased significantly with fertilizer nitrogen, but were not with twolevel of clipping height. This trend was reversed after late September ; no effect of nitrogen wasappeared. Instead, lax clipping increased tiller number, green leaf number and DM weight. Greenleaves stimulated by lax clipping resulted in the occurrance of more dead leaves in late October. 3. Among the stolons outgrown until early September, the primary stolon was not influencedby nitrogen and clipping treatments to produce only 2-3 stolons. However, 1st branch stoIon asaffected by nitrogen increased significantly, so most of stolons which occurred consisted of 1st branch stolons. 4. Until early September, stolon length obtained at nil nitrogen level was chiefly caused bythe primary stolons. By applying nitrogen, the primary stolons of Zoysia japonica waslonger than 1st branch stolons when severe clipping was involved and in turn, shorter than 1stbranch stolons when lax clipping was concerned. In Zoysia matrella, 1st branch stolons were muchlonger than the primary stolon when turf was clipped severely but in conditions of lax clippingthere was little difference in length between primary and 1st branch stolons. 5. Stolon nodes of both Zoysia japonica and Z. matrella were positively influenced by nit rogen, but no particular increase by imposing clipping height treatment was marked in Zoysiamatrella. Although the stolon of Zoysia japonica grew until late October, the growthstimulated by nitrogen was not so remarkable as to exceed that by nil N.

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상처와 효모추출물 처리조건에서 유발되는 야생벼 유전자 스크린 (Genes of Wild Rice (Oryza grandiglumis) Induced by Wounding and Yeast Extract)

  • Shin, Sang-Hyun;Im, Hyun-Hee;Lee, Jai-Heon;Kim, Doh-Hoon;Chung, Won-Bok;Kang, Kyung-Ho;Cho, Sung-Ki;Shin, Jeong-Sheop;Chung, Young-Soo
    • 생명과학회지
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    • 제14권4호
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    • pp.650-656
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    • 2004
  • 야생벼의 일종인 Oryza grandiglumis (CCDD, 2n=48)는 도열병, 잎집무늬마름병, 흰빛잎마름병, 그리고 벼멸구와 같은 병충해에 저항성을 가지는 것으로 알려져 있다. 이와 같은 곰팡이와 해충에 반응하여 차별 발현하는 유전자를 클로닝 하기 위하여 상처처리와 yeast extract를 Oryza grandiglumis에 0시간과 24시간 각각 처리하였다. 유전자의 클로닝을 위하여 희귀 발현유전자의 클로닝에 효율적인 것으로 알려진 Suppression Subtractive Hybridization (SSH) 방법이 처리 후 24시간 된 식물을 재료로 사용되었다. 그 결과, 776개의 cDNA clones이 확보되었으며, 유전자 발현의 진위여부를 빠르게 스크린하기 위하여 colony array가 수행되었다. 115개의 colony가 positive로 판명되었고, 이들의 평균 insert size는 400 bp에서 700 bp에 이르렀고, 이들에 대한 염기서열 분석이 수행되었다. 염기서열 분석 결과, 68개 clone들이 알려진 기능의 유전자와 homology를 나타냈으며, 이중에서 16개 clone이 일차대사에 관련된 것과 유사성을, 5개가 plant retrotransposon과 유사성을, 5개가 식물 방어기작 관련 metallothionein-like gene과 염기서열 유사성을 보였다. 이외에 다양한 유전자들이 아미노산 합성관련, membrane transport, signal transduction등에 관여하는 유전자들과 상동성을 나타내었다. 이들 유전자중에서 4 개의 클론(ogwfi-161, ogwfi-646, ogwfi-663, ogwfi-695)들이 선발되었고 이들에 대한 Northern 분석이 수행되었다. Northern 분석 결과 ogwfi-161, ogwfi-646, ogwfi-663, ogwfi-695는 wounding과 yeast extract처리 에 의한 차별 발현이 확인되었다. 이상의 결과를 종합하여 볼 때, SSH방법은 병충해등과 같은 조건에 의해 차별 발현되는 유전자들을 빠른 시간 내에 다량으로 발굴할 수 있는 매우 효율적인 방법이라고 생각된다.

p-Coumaric acid에 의해 유도되는 인체 Jurkat T 세포의 에폽토시스 기전 (Activation of Pro-Apoptotic Multidomain Bcl-2 Family Member Bak and Mitochondria-Dependent Caspase Cascade are Involved in p-Coumaric Acid-Induced Apoptosis in Human Jurkat T Cells)

  • 이제원;김영호
    • 생명과학회지
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    • 제21권12호
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    • pp.1678-1688
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    • 2011
  • 다양한 식용식물에 함유되어 있는 것으로 알려진 phenolic acids의 일종인 p-coumaric acid의 항암활성을 규명하고자, 인체 급성백혈병 T 세포주인 Jurkat T 세포에 대한 p-coumaric acid의 에폽토시스 유도기전을 조사하였다. Jurkat T 세포를 p-coumaric acid (50-$150{\mu}M$)로 처리한 결과, 세포독성, 에폽토시스-관련 DNA fragmentation, 및 pro-apoptotic multidomain Bcl-2 family member인 Bak의 활성화, ${\Delta}{\psi}m$ loss, caspase-9, -3, -7, 및 -8의 활성화, 그리고 PARP 분해 등의 여러 에폽토시스-관련 생화학적 현상들이 농도의존적으로 나타났다. 그러나 이러한 에폽토시스-관련 생화학적 현상들은 Jurkat T 세포에 anti-apoptotic Bcl-2 단백질을 과발현할 경우에는 나타나지 않았다. 또한 p-coumaric acid처리에 의해 유도되는 Jurkat T 세포의 에폽토시스에는 necrosis가 수반되지 않는 것으로 확인되었다. Jurkat T 세포를 pan-caspase inhibitor인 z-VAD-fmk를 전처리할 경우, p-coumaric acid 처리에 의해 유도되는 apoptotic sub-$G_1$ peak는 차단되어 나타나지 않았으나 ${\Delta}{\psi}m$ loss는 여전히 나타났는데, 이는 p-coumaric acid처리에 의한 에폽토시스의 유도에 caspase cascade 활성화가 필수적이며 ${\Delta}{\psi}m$ loss의 downstream 현상임을 나타낸다. 한편, FADD 및 caspase-8을 함께 발현하는 Jurkat T 세포주 A3, FADD-결손 Jurkat T 세포주 I2.1, 그리고 caspase-8-결손 Jurkat T 세포주 I9.2의 p-coumaric acid의 세포독성에 대한 감수성은 서로 유사하게 나타났는데, 이는 p-coumaric acid처리에 의한 에폽토시스의 유도가 Fas와 FasL간의 상호작용에 의해 개시되지 않음을 시사한다. p-Coumaric acid의 세포독성은 Jurkat T 세포에 비해 인체 정상 말초혈액 T 세포에서 훨씬 낮게 나타났다. 이러한 결과들은 p-coumaric acid 처리에 의해 유도되는 Jurkat T 세포의 에폽토시스가 Bak 활성화, ${\Delta}{\psi}m$ loss, caspase-9, -3, -7, 및 -8로 이루어진 caspase cascade의 활성화, 그리고 PARP 분해에 의해 유도되며, 또한 anti-apoptotic 단백질인 Bcl-2의 과발현에 의해서 음성적으로 조절됨을 나타낸다.

Molecular Cloning and Characterization of Mn-Superoxide Dismutase Gene from Candida sp.

  • Hong, Yun-Mi;Nam, Yong-Suk;Choi, Soon-Yong
    • Journal of Microbiology
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    • 제35권4호
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    • pp.309-314
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    • 1997
  • The manganese-containing superoxide dismutase (MnSOD) is a major component of the cellular defence mechanisms against the toxic effects of the superoxide radical. Within the framework of studies on oxidative stress=responsible enzymes in the Candida sp., the gene encoding the MnSOD was isolated and examined in this study. A specific primer was designed based on conserved regions of MnSOD sequences from other organisms, and was used to isolate the gene by PCR on reverse-transcribed Candida poly($A^{+}$) RNA. The PCR product was used to screen a Candida genomic lambda library and the nucleotide wequence of positive clone was determined. The deduced primary sequence encodes a 25kDa protein which has the conserved residues for enzyme activity and metal binding. The 28 N-terminal amino acids encoded by the Candida cDNA comprise a putatice mitochondrial transit peptide. Potential regulatory elements were identified in the 5' flanking sequences. Northern blot analysis showed that the transcription of the MnSOD gene is induced 5-to 10-fold in response to mercury, cadmium ions and hydrogen peroxide.

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Molecular characterization and functional analysis of a protease-related protein in Chang-liver cells

  • Wang, Congrui;Zhang, Huiyong;Feng, Huigen;Yang, Baosheng;Pramanik, Jogenananda;Guo, Zhikun;Lin, Juntang
    • BMB Reports
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    • 제43권5호
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    • pp.375-381
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    • 2010
  • In this study, the cDNA library of Chang-liver cells was immunoscreened using common ADAMs antibody to obtain ADAM related genes. We found one positive clone that was confirmed as a new gene by Blast, which is an uncharacterized helical and coil protein and processes protease activity, and named protease-related protein 1 (ARP1). The submitted GenBank accession number is AY078070. Molecular characterizations of ARP1 were analyzed with appropriate bioinformatics software. To analyse its expression and function, ARP1 was subcloned into glutathione S-transferase fusion plasmid pGEX-2T and expressed by E. coli system. The in vitro expression product of ARP1 was recognized by common ADAMs antibody with western blot. Interestingly, ARP1 cleaves gelatine at pH9.5, which suggests it is an alkaline protease. Semi-quantitative RT-PCR result indicates that ARP1 mRNA is strongly transcribed in the liver and the treated Chang-liver cells.

Screening and Characterization of an Esterase from a Metagenomic Library

  • KIM JEONG-NYEO;SEO MYUNG-JI;CHO EUN-AH;LEE SANG-JAE;KIM SEONG-BO;CHEIGH CHAN-ICK;PYUN YU-RYANG
    • Journal of Microbiology and Biotechnology
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    • 제15권5호
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    • pp.1067-1072
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    • 2005
  • A metagenomic library was constructed using a fosmid vector, and total genomic DNA was extracted directly from soil at Cisolok (hot spring area, Indonesia). This library was composed of 10,214 clones and screened for lipolytic enzyme on tributyrin agar plates. An esterase gene (estMa) was subcloned and sequenced from a positive lipolytic active clone. Esterase EstMa was encoded by a 954-bp open reading frame and showed low ($11-33\%$) amino acid similarity to known esterases. The amino acid sequence analysis demonstrated that the enzyme is a new member of lipolytic enzyme family VI. The estMa gene encodes a preprotein of 317 amino acids with a predicted molecular mass of 34,799 Da. The purified enzyme exhibited optimal activity at $50^{\circ}C$ and pH 6.5. The $K_m,\;and\;V_{max}$ values of EstMa for the hydrolysis of p-nitrophenyl valerate were $45.3\;{\mu}M$ and 4.45 U/mg, respectively.

Construction of Infectious cDNA Clone of a Chrysanthemum stunt viroid Korean Isolate

  • Yoon, Ju-Yeon;Cho, In-Sook;Choi, Gug-Seoun;Choi, Seung-Kook
    • The Plant Pathology Journal
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    • 제30권1호
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    • pp.68-74
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    • 2014
  • Chrysanthemum stunt viroid (CSVd), a noncoding infectious RNA molecule, causes seriously economic losses of chrysanthemum for 3 or 4 years after its first infection. Monomeric cDNA clones of CSVd isolate SK1 (CSVd-SK1) were constructed in the plasmids pGEM-T easy vector and pUC19 vector. Linear positive-sense transcripts synthesized in vitro from the full-length monomeric cDNA clones of CSVd-SK1 could infect systemically tomato seedlings and chrysanthemum plants, suggesting that the linear CSVd RNA transcribed from the cDNA clones could be replicated as efficiently as circular CSVd in host species. However, direct inoculation of plasmid cDNA clones containing full-length monomeric cDNA of CSVd-SK1 failed to infect tomato and chrysanthemum and linear negative-sense transcripts from the plasmid DNAs were not infectious in the two plant species. The cDNA sequences of progeny viroid in systemically infected tomato and chrysanthemum showed a few substitutions at a specific nucleotide position, but there were no deletions and insertions in the sequences of the CSVd progeny from tomato and chrysanthemum plants.

Serratia marcescens Chitinase 유전자의 대장균에로의 클로닝 (Molecular Cloning of Serratia marcescens Chitinase Gene into Escherichia coli)

  • 장규일;김기석;조무제;이상열;신용철
    • 한국미생물·생명공학회지
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    • 제20권2호
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    • pp.129-135
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    • 1992
  • 본 연구에서는 Serratia marcescens ATCC 27117 균주로부터 키나아제 유전자를 대장균으로 클로닝 하고 발현시켰다. pUC 19 플라스미드를 이용하여 S.marcescens의 genomic library를 만들고 팽화된 키틴이 포함된 한천배지에서 키티나아제 활성을 가지는 클론을 선별하였다. 약 1x10 transformant들 중에서 키티나아제 활성을 보이는 하나의 클론을 선발하였으며 이것은 pUC 19 플라스미드속에 8.9Kb 염색체 DNA 삽입단편을 가지고 있었다.

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