• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2003년도 춘계 학술발표대회
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• pp.121-121
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• 2003

Expressed sequence tags (EST) are help to quickly identify functions of expressed genes and to understand the complexity of gene expression. To assist genetic study of the root development in Panax ginseng, which is one of the most important medicinal plant, expressed sequence tags (EST) analysis was carried out. We constructed a CDNA library using the 4-year Chunpoon root. Partial sequences were obtained from 3,841 clone. The ESTs could be clustered into 2,056 (64%) non-redundant groups. Similarity search of the non-redundant ESTs against public non-redundant databases of both protein and DNA indicated that 1,498 groups show similarity to genes of known function. These ESTs clones were divided into eighteen categories depending upon gene function. The most abundant transcripts were major latex protein (41), ribonuclease 2 (36), metallothionein 2(35). Our extensive EST analysis of genes expressed in 4-year Chunpoong root not only contributes to the understanding of the dynamics of genome expression patterns in root organ development but also adds data to the repertoire of all genomic genes.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2003년도 춘계 학술발표대회
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• pp.125-125
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• 2003

To assist genetic study of the root development in Panax ginseng, which is one of the most important medicinal plant, expressed sequence tags (EST) analysis was carried out. We constructed a cDNA library using the 14-year ginseng root. Partial sequences were obtained from 2,975 clone. The ESTs could be clustered into 1,991 (70.2%) non-redundant groups. Similarity search of the non-redundant ESTs against public non-redundant databases of both protein and DNA indicated that 1,553 groups show similarity to genes of blown function. These ESTs clones were divided into sixteen categories depending upon gene function. The most abundant transcripts were ribonuclease 1 (67) and ribonuclease 2 (65). Our extensive EST analysis of genes expressed in 14-year ginseng root not only contributes to the understanding of the dynamics of genome expression patterns in root organ but also adds data to the reperoire of all genomic genes.

• Archives of Pharmacal Research
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• 제24권4호
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• pp.316-322
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• 2001

Arylsulfate sulfotransferase (ASST) transfers a sulfate group from a phenolic sulfate ester to a phenolic acceptor substrate. In the present study, the gene encoding ASST was cloned from a genomic library copy of Citrobacter freundii, subcloned into the vector pGEM3Zf(-) and sequenced. Sequencing revealed two contiguous open reading frames (ORF1 and ORF2) on the same strand and based on amino acid sequence homologyl they were designated as astA and dsbA, respectively. The amino acid sequence of astA deduced from C. freundii was highly similar to that of the Salmonella typhimurium, Enterobacter amnigenus, Klebsiella, Pseudomonas putida, and Campylobacter jejuni, encoded by the astA genes. However, the ASST activity assay revealed different acceptor specificities. Using p-nitrophenyl sulfate (PNS) as a donor substrate, $\alpha$-naphthol was found to be the best acceptor substrate, followed by phenol, resorcinol, p-acetaminophen, tyramine and tyrosine.

• Journal of Microbiology
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• 제37권1호
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• pp.35-40
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• 1999

Sporulation-specific glucoamylase (SGA) gene was isolated from genomic library of Saccharomyces diastaticus 5114-9A by using glucoamylase non-producing mutant of S. diastaticus as a recipient. When the glucoamylase activities of culture supernatant, periplasmic, and intracellular fraction of cells transformed with hybrid plasmid containing SGA gene were measured, the highest activity was detected in culture supernatant. SGA produced by transformant and extracellular glucoamylase produced by S. diastaticus 5114-9A differed in enzyme characteristics such as optimum temperature, thermostability, and resistance to SDS and urea. But the characteristics of SGA produced by sporulating yeast cells and vegetatively growing transformants were identical.

• 한국미생물·생명공학회지
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• 제18권1호
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• pp.98-102
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• 1990

본 실험에서는 채소 등에서 연부병을 유발시키는 원인균의 일종인 Erwinia herbicola의 생육을 저해시키는 bacteriocin 생산균주를 경작지로부터 분리하여 몇 가지 특성을 조사하였다. 분리균주가 생산하는 bacteriocin은 배양 48시간안에서 가장 많은 축적량을 보였고 bacteriocin 생산유전자는 chromosome 상에 있음을 확인하였다. 또한 Erwinia 속의 chromosomal DNA 를 분리하여 EcoRI으로 절단하고 pLAFR3 vector의 EcoRI site에 cloning 하여 bacteriocin을 생성하는 형질전환체, 두 개체를 얻었다. 이들 중 bacteriocin 생산능이 우수한 CH 49 clone의 제한효소 지도를 작성하였다. Vector 내에 삽입된 3.0kb insert 내에 BamHI과 NglII site가 각각 한 개씩 있음을 밝혔다.

• 한국식물병리학회지
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• 제14권2호
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• pp.177-183
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• 1998

A DNA fragment which is involved in tolassin production was cloned to obtain a molecular marker of Pseudomonas tolaasii, a casual agent of bacterial brown blotch disease of oyster mushroom (Pleurotus ostreatus). Tolaasin is a lipodepsipeptide toxin and known as a primary disease determinant of the P. tolaasii. It is responsible for formation of white line in agar when P. tolaasii were cultured against white line reacting organisms (WLROs). White line negative mutants (WL-) were generated by conjugation between rifampicin resistant strain of P. tolaasii and E. coli carrying suicidal plasmid pSUP2021 : : Tn5. The ability of tolaasin production of the WL- mutants was examined by hemolysis test, pathogenicity test, and high pressure liquid chromatography (HPLC) analysis of culture filtrate. All of the WL- mutants were lost the ability of tolaasin production (Tol-). Genomic library of the Tol- mutant was constructed in pLAFR3 and the cosmid clone containing Tn5 was selected. DNA fragment fro franking region of Tn5 was cloned from the plasmid and used as a probe in Southern blot. DNA-DNA hybridization with the probe to total DNA from group of bacteria ecologically similar to P. tolaasii including WLORs, fluorescent Pseudomonads isolated from oyster mushroom, P. agarici, P. gingeri, and some of other species of Psedomonas showed that some of the tested bacteria do not have any hybridized band and others have bands sowing RFLP. The cloned DNA fragment or its nucleotide sequence will be useful in detection and identification of the P. tolaasii.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.80.1-80
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• 2003

Several plant and microbial genes that could confer disease resistance in transgenic rice plants are being cloned and characterized. We are currently constructing transgenic rice lines that overexpress the gene products, such as a galactinol synthase, a defensin, and a bacterial ACC deaminase. Subtractive hybridization of a rice cDNA library constructed from the Xanthomonas oryzae-infected ice leaves resulted in isolation of many inducible cDNA clones including a elongation factor EF2, a oryzain alpha, a catalase, a aldehyde dehydrogenase, a S-adenosylmethionine synthetase, a caffeic acid O-methyltransferase, a glyceraldehyde-3-phosphate dehydrogenase, a light-regulated protein, nKY transcription factors, and a nucleotide diphosphate kinase. Some genes among those may be useful genetic sources for construction of disease resistant transgenic rice. Full lengths of the rice OsFIERG and a rice oryzain genomic clones were cloned, and serial deletion fragments of the promoter regions of these genes were fused with GUS reporter gene in pCAMBIA1201, respectively. Promoter activities of these constructs will be examined upon various stresses and Pathogen infections to obtain the pathogen specific inducible-promoter. This work was supported by a grant from BioGreen 21 Program, Rural Development Administration, Republic of Korea.

• 미생물학회지
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• 제35권1호
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• pp.7-12
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• 1999

Pseudomons sp. PY002의 exotoxin A 의 발현 양상을 관찰하기 위하여 P.aeruginosa PAO1의 anti-exotoxin A와 immunoblot hybridization을 실시한 결과 배지내에 유용 가능한 철이 고갈됨에 따라 exotoxin A의 발현양은 점차적으로 증가하는 양상을 보였으며, CAS 배지에 점적한 배양 상층액에서 siderophore의 발현양도 증가함을 보였다. P.sp.PY002 의 genomix library를 제조하여, exotoxin A를 분비하는 2개의 클론을 선별하려 pETA23과 pETA42 로 명명한 후, 반응성이 강한 Peta42를 선발하였다. pETA 42는 약 1.7kb 크기의 insert를 가지며, 양쪽 말단에 cloning site 인 pstI site 가 존재하며 2개의 NcoI, 1개의 PvuII, 1개의 SstI , 3개의 SmaI, 1개의 KpnI, 3개의 HaeII, 1개의 EcoRI site가 존재하였다.

• Applied Biological Chemistry
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• 제38권4호
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• pp.313-319
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• 1995

본 실험은 알팔파(Medicago sativa) 근류균인 Rhizobium meliloti TAL1372 균주에서 섬유소분해효소의 활성을 확인하고 이 유전자를 크로닝하기 위해 코스미드 벡타인 pLAFR3를 사용하여 유전자 은행을 만들었다. 이 유전자 은행으로 부터 분리한 1,000개의 형질 도입체에서 CM-cellulase(carboxymethylcellulase) 활성이 있는 클론(pRC8-71) 하나를 분리하였으며 30kb 크기의 R. meliloti DNA 단편을 함유하고 있는 것을 확인하였다. 이 CM-cellulase 유전자의 발현 산물을 native PAGE 법에 의하여 분자량을 측정한 결과 약 45kD 정도의 크기로 추정되었으며 pRC8-71로 부터 Tn5 변이법에 의해 조사한 결과 30kb 단편 내에서 CM-cellulase 유전자의 위치는 제한효소 KpnI에 의해 절단되는 약 3kb 단편에 존재하였다. 표시교환 변이법에 의해 야생균주 R. meliloti TAL1372로부터 cellulase 무생산 변이체를 얻어 근류형성 정도를 실험한 결과 CM-cellulase유전자가 근류형성에 관련될 것으로 추정되었다.

• 생명과학회지
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• 제17권9호통권89호
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• pp.1197-1203
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• 2007

세균인 Paenibacillus sp. DG-22의 유전체 DNA library가 제조되었으며, ${\beta}-xylosidase-$양성 클론이 형광기질인 $4-methylumbelliferyl-{\beta}-D-xylopyranoside$ $({\beta}MUX)$를 사용하여 확인되었다. 이 클론으로부터 재조합 플라스미드가 분리되었고 삽입된 4.3-kb 크기 DNA의 염기서열이 결정되었다. ${beta}-xylosidase$ 유전자는 분자량이 78.710 dal-ton이고 pI가 5.0인 701개의 아미노산을 암호화하는 2,106 염기쌍의 열린해독틀(ORF)로 구성되어있었다. xylA 유전자산물의 추론된 아미노산 서열은 과(family) 52에 속하는 클리코실 가수분해효소로 분류된 ${beta}-xylosidase$들과 상당한 유사성을 가지고 있었다. 이 xylA 유전자에 6개의 히스티딘-꼬리표를 붙이기 위해 pQE60 발현벡터에 다시 클로닝하였다. 재조합 ${beta}-xylosidase$ $(XylA-H_6)$가 열처리와 고정화금속친화성 크로마토그래피(IMAC)에 의해 순수하게 정제되었다. $XylA-H_6$ 효소의 최적 pH와 온도는 각각 pH 5.5-6.0과 $60^{\circ}C$이었다.