• Journal of Microbiology and Biotechnology
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• 제1권1호
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• pp.37-44
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• 1991

A shuttle promoter-probe vector, pEB203, was derived from pBR322, pPL703 and pUB110. Using the vector, a useful DNA fragment, 319 bp EcoRI fragment, having strong promoter activity has been cloned from Bacillus subtills chromosomal DNA. Selection was based on chloramphenicol resistance which is dependent upon the introduction of DNA fragments allowing expression of a chloramphenicol acetyl transferase gene. The nucleotide sequence of the 319 bp fragment has been determined and the putative -35 and -10 region, ribosome binding site, and ATG initiation codon were observed. This promoter was named EB promoter and the resultant plasmid which can be used as an expression vector was named pEBP313. The crystal protein gene from B. thuringiensis subsp. kurstaki HD-73 was cloned downstream from the EB promoter without its own promoter. When the resultant plasmid, pBT313, was introduced into Escherichia coli and B. subtilis, efficient synthesis of crystal protein was observed in both cells, and the cp gene expression in B. subtilis begins early in the vegetative phase. The cell extracts from both clones were toxic to Hyphantria cunea larvae.

• 한국미생물·생명공학회지
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• 제16권2호
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• pp.126-130
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• 1988

토양에서 분리한 알카리 내성 Bacillus sp. YA-14 의 promoter를 Bacillus promoter probe vector인 pPL703을 이용하여 Bacillus subtilis내에 cloning 하였다. 얻어진 형질전환체 중 promoter 활성이 가장 높은 균주의 CAT 비활성은 8.07로 expression vector인 pPL708의 CAT 비활성보다 2.5배 이상 높았으며 대수 증식기가 끝난 이후에 그 활성이 급격하게 증가하였다. 재조합 plasmid내의 삽입 DNA 단편은 그 크기가 2.8kb이고 제한효소 BamHI, Sal I 인식부위가 각각 한군데 존재하였다.

• Journal of Microbiology and Biotechnology
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• 제1권4호
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• pp.240-245
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• 1991

We have constructed a promoter-probe vector pKU20 using pKT230, a derivative of broad-host-range plsmid RSF1010, as a base. The pKU20 contains structural gene for aminoglycoside phos-photransferase (aph), without promoter, and a multiple cloning site upstream the aph. Using this vector, a 412base pairs (bp) PstI fragment showing strong promoter activity both in Escherichia coli LE392 and Pseudomonas putida KCTC1644 has been cloned from Pseudomonas fluorescens chromosomal DNA on the basis of streptomycin resistance. The nucleotide sequence of the 412 bp fragment has been determined and the putative - 35 and -10 region was observed. Insecticidal protein gene of Bacillus thuringiensis subsp. kurstaki HD-73 inserted on downstream of the promoterlike DNA fragment was efficiently expressed in E. coli and P. putida. The toxin protein was efficiently synthesized in an insoluble form in both strains.

• 생명과학회지
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• 제19권9호
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• pp.1289-1293
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• 2009

원암단백질로 알려진 Human cervical cancer oncogene (HCCR)은 발암억제 단백질인 p53과 작용하여 다양한 암조직에서 암의 유발을 촉진한다. 그러나, 아직 정확한 발암 유도기전이 알려져 있지 않다. 이러한 의문을 해소하기 위한 일환으로 본 연구에서는 HCCR의 발현이 어떻게 조절되는지를 조사하였다. 이를 위해 먼저 HCCR 5' 쪽의 promoter 영역을 분리하여 Luciferase assay법을 이용하여 K562, HEK293, A549 세포에서 분석하였고, Promoter의 -370에서 -406사이 36bp의 첨가로 promoter활성이 의미 있게 억제됨을 관찰하였다. 또한, 36 bp만을 포함하는 probe를 이용한 mobility shift assays (EMSA)에서 핵단백질이 결합함을 관찰하였고, 컴퓨터를 이용한 분석에서 TG-interacting factor (TGIF)에 대한 consensus sequences 존재함을 관찰하였다. TGIF 만을 포함하는 probe (TC)와 돌연변이를 유발한 probe (mTG)를 이용한 EMSA에서 이 자리에 TGIF가 결합함을 보였다. 또한, TGIF 자리에 돌연변이를 유발하면(pGL3-mTGIF) 발현의 억제가 회복됨을 관찰하였다. 본 연구에서는 HCCR promoter의 특성을 분석하였고, 이 과정에서 -390에서 -366 사이에 TGIF 전사인자가 결합하여 전사활성을 조절함을 증명하였다.

• BMB Reports
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• 제30권1호
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• pp.41-45
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• 1997

The present study was performed to analyze the molecular mechanism which dictates the transcription regulation of the $O^6$-methylguanine-DNA methyltransferase (MGMT) gene in Saccharomyces cerevisiae. Previously we identified one possible upstream repressing sequence (URS) in MGMT promoter by promoter deletion and competition analysis. In this paper we report another regulatory element (UAS: upstream activating sequence. -213 to -136) which affects the transcription activity of MGMT promoter. Gel mobility shift assay and Southwestern blot analysis using UAS probe showed several specific proteins which were able to bind to this sequence.

• 미생물학회지
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• 제35권4호
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• pp.266-269
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• 1999

박테리오파지 E3가 만드는 plaque은 그 지름이 약 1㎝정도이고 대단히 빠르게 성장한다. 구조 단백질 중 가장 많은 copy를 가지는 major capsid 단백질을 발현하는 유전자를 조절하는 promoter가 가장 효율적일 것이라 생각되며, 이 promoter를 찾기 위하여 먼저 이 유전자를 mapping하였다. 정제한 파지 입자로부터 major capsid 단백질을 분리하여 그 N-terminal amino acid 서열을 확인하였고, 그에 해당하는 degenerate oligonucleotide probe를 이용하여 E3의 genomic library로부터 major capsid 단백질을 발현하는 유전자를 함유하는 clone을 찾았다. 이 clone의 DNA 서열 분석을 통하여 major capsid 단백질을 발현하는 유전자를 확인하였으며, 이는 E3 genome에서 약 72%에 mapping 되었다. 이 gene을 조절하는 promoter의 성질을 고찰하기 위하여 E3의 성장이 rifampicin에 의하여 영향을 받는지 확인한 결과 E3는 자기 고유의 RNA polymerase를 가지고 있음을 알 수 있었다.

• 한국미생물·생명공학회지
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• 제16권5호
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• pp.343-347
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• 1988

토양에서 분리한 알칼리 내성 Bacillus속의 chromosomal DNA로부터 promoter를 cloning하여 선별된 재조합 plasmid p-12내 의 promoter를 subcloning을 하였다. 그 결과 cloning된 promoter 내에는 서로 다른 두 가지의 promoter가 존재하는 것을 확인할 수 있었고 이로부터 각각의 promoter를 함유한 재조합 plasmid p-l2B1, p-l2B2를 제조하였다. 또한 CAT 비활성 측정에 의해 각 promoter의 활성을 비교해 본 결과 p-l2B1의 promoter는 p-l2B2의 promoter에 비해 상대적으로 높은 활성을 가지고 있었다. CAT 비활성을 생육시기에 따라 측정해 본 결과 p-l2B1과 p-l2B2는 대수증식기 이후 활성이 급증되었으며 배지 중 첨가된 1.0%의 glucose에 의해 활성이 억제되는 효과를 받았다.

• Journal of Microbiology and Biotechnology
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• 제21권11호
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• pp.1179-1183
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• 2011

In this study, the chromosome-encoded pcuRCAXB genes that are required for p-cresol degradation have been identified by using a newly constructed green fluorescent protein (GFP)-based promoter probe transposon in the long-chain alkylphenol degrader Pseudomonas alkylphenolia. The deduced amino acid sequences of the genes showed the highest identities at the levels of 65-93% compared with those in the databases. The transposon was identified to be inserted in the pcuA gene, with the promoterless gfp gene being under the control of the pcu catabolic gene promoter. The expression of GFP was positively induced by p-cresol and was about 10 times higher by cells grown on agar than those in liquid culture. In addition, p-hydroxybenzoic acid was detected during p-cresol degradation. These results indicate that P. alkylphenolia additionally possesses a protocatechuate ortho-cleavage route for p-cresol degradation that is dominantly expressed in colonies.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.789-795
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• 2004

A promoter-probe shuttle vector pSK1Cat was constructed for the isolation of transcriptional signal sequences from Corynebacterium glutamicum. Besides conferring resistance to kanamycin in Escherichia coli and C. glutamicum, the vector carried a promoterless cat gene to confer resistance to chloramphenicol upon insertion of the appropriate transcriptional signals in the multiple cloning site. By utilizing the vector, a series of transcriptionally active fragments were isolated from the genome of C. glutamicum. The clones, ranging from 200 bp to 1 kb in size, were grouped into 3 classes of strong, medium, and weak, based on the chloramphenicol acetyltransferase (CAT) activity and sensitivity to the chloramphenicol of the clone-carrying C. glutamicum cells. C. glutamicum cells carrying the $P_{19}$ clone, a representative in the strong class, were able to grow on minimal agar plates containing over $40 mg/mell$ chloramphenicol, and showed CAT activity of 10 m㏖/mgㆍmin, performing slightly better than the cells carrying $P_{tac}$ , a strong E. coli promoter. Subcloning analysis of the $P_{19}$ clone identified a 180 bp intergenic fragment ($P_{180}$), which was located upstream of a gene encoding a hypothetical membrane protein. The expression conferred by $P_{180}$ was not affected by either the kinds of carbon sources or changes in temperature. These properties make the $P_{180}$ clone useful for the deregulated expression of biosynthetic genes in C. glutamicum during amino acid fermentation.

• Journal of Microbiology
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• 제37권2호
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• pp.86-89
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• 1999

An OTHBVS cell line from HepG2 was established. This cell line stably expresses the human hepatitis B virus (HBV) middle S protein that includes the preS2 region which is important for HBV particle entry into the hepatocyte. To establish this cell line, the middle S open reading frame (ORF), with a promoter located in the 5' region and enhancer located in the 3' region, was cloned downstream from the metallothionine (MT) promoter of the OT1529 vector. In this vector, expression of the middle S protein was constructed to be regulated by its own promoter and enhancer. Expression of the large S protein which contains the preS1 region in addition to the middle S protein was designed to be regulated by the MT promoter. When extracts of OTHBVS cells were examined with an S protein detection kit (RPHA, Korea Green Cross Co.), an S protein was detected. Total mRNA of OTHBVS cell examined by northern blot analysis with an S ORF probe revealed small/middle S transcripts (2.1 kb). When the MT promoter was induced by Zn, large S transcripts (2.4 kb) were detected. The GP36 and GP33 middle S proteins were presumably detected, but large S proteins were not detected by immunostain analysis using anti-preS2 antibody.