• 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.

• Genomics & Informatics
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• 제13권4호
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• pp.152-155
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• 2015

Recently, the Encyclopedia of DNA Elements (ENCODE) Analysis Working Group converted data from ChIP-seq analyses from the Broad Histone track into 15 corresponding chromatic maps that label sequences with different kinds of histone modifications in promoter regions. Here, we publish a frequency profile of the three ChromHMM promoter states, at 200-bp intervals, with particular reference to the existence of sequence patterns of promoter elements, GC-richness, and transcription starting sites. Through detailed and diligent analysis of promoter regions, researchers will be able to uncover new and significant information about transcription initiation and gene function.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권3호
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• pp.167-172
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• 2001

Staphylokinase (SAK) was produced in B. subtilis using two different promoter systems, i.e. the P43 and sacB promoters. To maximize SAK expression in B. subtilis, fermentation control strategies for each promoter were examined. SAK, under P43, a vegetative promoter transcribed mainly by $\sigma$(sup)B containing RNA polymerase, was overexpressed at low dissolved oxygen (D.O.) levels, suggesting that the sigB operon is somewhat affected by the energy charge of the cells. The expression of SAK at the 10% D.O. level was three times higher than that at the 50% D.O. level. In the case of sacB, a sucrose-inducible promoter, sucrose feeding was used to control the induction period and induction strength. Since sucrose is hydrolyzed by two sucrose hydrolyzing enzymes in the cell and culture broth, the control strategy was based on replenishing the loss of sucrose in the culture. With continuous feeding of sucrose, WB700 (pSAKBQ), which contains the SAK gene under sacB promoter, yielded ca. 35% more SAK than the batch culture. These results present efficient promoter-dependent control strategies in B. subtilis host system for foreign protein expression.

• Biotechnology and Bioprocess Engineering:BBE
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• 제2권2호
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• pp.82-85
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• 1997

The nar promoter as an inducible promoter was characterized for the process development for the gene expression and the protein production under anaerobic condition. The LB medium was selected as a main culture medium showing the enzyme activity of 18,000 units/min/g cell in the flask cultivation. The optimum concentration of nitrate was 1%. Under anaerobic conditions, the gene expression was fully induced in the presence of nitrate.

• Journal of Microbiology and Biotechnology
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• 제3권3호
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• pp.146-155
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• 1993

A DNA fragment from an alkali-tolerent Bacillus sp., conferring strong promoter activity, was subcloned into the promoter probe plasmid pPL703 and the nucleotide sequence of this promoter region was determined. The sequence analysis suggested that this highly efficient promoter region containing the complex clustered promoters comprised three kinds of promoters (P1, P2 and P3), which are transcribed by $\sigma^B (formerly \sigma^{37}), \sigma^E(formerly \sigma^{29}) and \sigma^A (formerly \sigma^{43})$ RNA polymerase holoenzymes which play major rules at the onset of endospore formation, during sporulation and at the vegetative phase of growth, respectively. S1 nuclease mapping experiments showed that all three promoters had staggered transcription initiation points. The results of chloramphenicol acetyltransferase assay after the subcloning experiments also indicated that the expression of these clustered promoters was correlated with the programs of growth and endospore development. Promoter P1, P2 and P3 were preceded by 75% AT, 79% AT and 81% AT regions, respectively, and a partial deletion of AT-rich region prevented transcription from promoter P1 in vivo. Two sets of 5 -AGTGTT-3 sequences and inverted repeat sequences located around the promoter P1 were speculated as the possible cis acting sites for the catabolite repression in B. subtilis. In vivo transcripts from these sequence regions may be able to form a secondary structure, however, the possibility that a regulatory protein induced by the excess amount of glucose could be bound to such a domain for crucial action remains to be determined.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권4호
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• pp.247-252
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• 2000

The strength and regulatory characteristics of the heat-inducible HSA1, HSA2 and TPS1 promoters were compared with those of the well-established, carbon source-regulated FMD promoter in a Hansenula polymorpha-based host system in vivo. In addition, the Saccharomyces cerevisiae-derived ADH1 promoter was analysed. While ADH1 promoter showed to be of poor activity in the foreign host, the strength of the heat shock TPS1 promoter was found to exceed that of the FMD promoter, which at present is considered to be the strongest promoter for driving heterologous gene expression in H. polymorpha.

• KSBB Journal
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• 제21권6호
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• pp.461-465
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• 2006

본 연구에서는 저온에서 발현이 유도되고 지속적으로 발현된다고 알려진 대장균의 6개의 유전자 (frdA, glpB, hypE, katG, nupG, ompT)에 대한 promoter의 단백질 생산성을 알아보기 위하여 GFP를 reporter 단백질로 이용하여 각각의 promoter들에 대한 $37^{\circ}C$와 $15^{\circ}C$에서의 발현도와 저온 유도성에 대하여 고찰하였다. nupG promoter의 경우 $37^{\circ}C$와 $15^{\circ}C$ 모두에서 지속적인 유전자 발현도를 보였으나 promoter에 의한 저온 유도성은 없는 것으로 판별되었다.

• 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.

• Molecules and Cells
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• 제26권2호
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• pp.131-139
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• 2008

Expression of the seven open reading frames (ORFs) of single-stranded DNA Curtoviruses such as Beet curly top virus (BCTV) and Beet severe curly top virus (BSCTV) is driven by a bi-directional promoter. To investigate this bidirectional promoter activity with respect to viral late gene expression, transgenic Arabidopsis plants expressing a GUS reporter gene under the control of either the BCTV or BSCTV bi-directional promoter were constructed. Transgenic plants harboring constructs showed higher expression levels when the promoter of the less virulent BCTV was used than when the promoter of the more virulent BSCTV was used. In transgenic seedlings, the reporter gene constructs were expressed primarily in actively dividing tissues such as root tips and apical meristems. As the transgenic plants matured, reporter gene expression diminished but viral infection of mature transgenic plants restored reporter gene expression, particularly in transgenic plants containing BCTV virion-sense gene promoter constructs. A 30 base pair conserved late element (CLE) motif was identified that was present three times in tandem in the BCTV promoter and once in that of BSCTV. Progressive deletion of these repeats from the BCTV promoter resulted in decreased reporter gene expression, but BSCTV promoters in which one or two extra copies of this motif were inserted did not exhibit increased late gene promoter activity. These results demonstrate that Curtovirus late gene expression by virion-sense promoters depends on the developmental stage of the host plant as well as on the number of CLE motifs present in the promoter.

• 한국미생물·생명공학회지
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• 제47권4호
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• pp.662-666
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• 2019

본 연구에서는 Saccharomyces cerevisiae를 이용해서 neoagarobiose hydrolase (NABH)를 효율적으로 생산하기 위한 NABH558 유전자 발현시스템을 구축하였다. ADH1 promoter와 GAL10 promoter 하류에 NABH558 유전자를 가진 pAMFα-NABH plasmid와 pGMFα-NABH plasmid는 S. cerevisiae 2805 균주에 형질전환되었다. 2805/pAMFα-NABH 균주는 YPD (2% dextrose) 배지에서 가장 높은 NABH 효소 활성(0.069 unit/ml/DCW)을 보였고, 2805/pGMFα-NABH 균주의 경우는 배지의 조성과 상관없이 비슷한 수준의 NABH 활성(0.02-0.027 unit/ml/DCW)을 보였다. RT-PCR을 통한 NABH558 유전자의 transcription level은 NABH 활성 증가에 따라 비슷한 수준으로 증가되었음을 확인할 수 있었다. 또한 재조합균주에서 생산된 NABH는 agarose를 galactose와 AHG로 분해하였다. 따라서 NABH558 유전자의 발현에는 ADH1 promoter를 사용하는 것이 더 효율적이며 GAL10 promoter와 비교해서 최대 3배정도 높은 활성의 재조합 NABH를 생산할 수 있음을 알 수 있었다.