• 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.

• 생명과학회지
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• 제9권2호
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• pp.153-159
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• 1999

S. cerevisiae의 대표적인 구성적 promoter로 GAPDH, ADH1 및 ENO1를 사용하고 이들 promoter 하류에 INUl의 ORF를 in frame으로 연결한 각각의 plasmid pYIGP, pADHl-lNU 및 pENO-INU를 구축하였다. 이들 plasmid 를 함유한 재조합 S. cerevisiae SEY2102 균주들을 sucrose 함유 평판배지에서 선별한 후, 초기 포도당 농도가 2$\%$ 또는 4$\%$인 배지에서 배양했을 때, 모든 균주들은 12시간 이후부터 정지기에 들어갔으며, 정지기에서도 느리지만 균체증식과 inulinase 발현은 계속되었다. 4% 포도당 배지에서 inulinase 총발현량은 ADH1 promoter 계를 제외하고 GAPDH와 ENO1 promoter의 경우 2$\%$ 포도당 배지 때 보다 약 2배 증가한 2.0 unit/mL과 1.4 unit/mL를 각각 보였다. 단위균체농도당 inulinase 활성 즉, 비활성은 GAPDH와 ENOl promoter계의 경우 포도당 농도가 4$\%$일 때 2$\%$때보다 약 63$\%$ 정도의 비활성 증가를 나타내었다. 하지만 ADH1 promoter의 경우는 오히려 비활성이 약 40$\%$ 감소하였다. 그러나, plasmid 안정성 측면에서는 ADH1과 ENO1 promoter 발현계가 GAPDH promoter 경우의 34$\%$보다 훨씬 뛰어난 80$\%$이상을 보였다. 결론적으로 높은 포도당 농도에서 구성적 promoter의 활성 (발현능)은 GAPDH, ENO1, ADH1 promoter 순으로 나타났지만, 초기 포도당 농도가 높을 때나 에탄을 생산이 심각한 유가식 배양에서는 ENO1 promoter가 inulinase의 구성적 발현ㆍ생산에 더 적합할 것으로 사료된다.

• BMB Reports
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• 제36권6호
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• pp.586-592
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• 2003

Four putative promoters of the temperate mycobacteriophage L1 were cloned by detecting the $\beta$-galactosidase reporter expression in E. coli transformants that carried L1 specific operon-fusion library. All of the four L1 promoters were also found to express differentially in the homologous environment of mycobacteria. Of the four promoters, two were suggested to be the putative early promoters of L1 since they express within 0 to 10 min of the initiation of the lytic growth of L1. One of the putative early promoters showed a relatively better and almost identical activity in both E. coli and M. smegmatis. By a sequence analysis, we suggest that the L1 insert that contained the stronger early promoter possibly carries two convergent E. coli $\sigma^{70}$-like L1 promoters, which are separated from each other by about 300 nucleotides. One of them is the early promoter of L1 as it showed a 100% similarity with the early $P_{left}$ promoter of the homoimmune phage L5. The second promoter, designated P4, was suggested for its appreciable level of reporter activity in the absence of the -10 element of the $P_{left}$ equivalent of L1. By analyzing most of the best characterized mycobacteriophages-specific promoters, including the L1 promoter P4, we suggest that both the -10 and -35 hexamers of the mycobacteriophage promoters are highly conserved and almost similar to the consensus -10 and -35 hexamers of the E. coli $\sigma^{70}$ promoters.

• KSBB Journal
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• 제11권4호
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• pp.504-509
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• 1996

Promoters which are useful for constructing expression vectors for lactic acid bacteria were obtained from the chromosomal DNA of Lactococcus lactis ssp. lactis MG1363. pBV5030, a promoter-selection vector, replicates in L. lactis and Escherichia coli and carries a promoterless chloramphenicol acetyltransferase gene (cat-86). After examining E. coli transformants which grew on LB media containing chloramphenicol (Cm, 20$\mu\textrm{g}$/mL) , many MG1363 derived DNA fragments which encompass promoter sequences were identified. Some recombinant E. coli cells can grow at the Cm concentration of 1,000$\mu\textrm{g}$/mL. When plasmids from those highly resistant E. coli cells were purified and introduced into L. lactis ssp. lactis MG1614 cells by electroporation, lactococcal transformants showing Cm resistance were obtained. So far, five plasmids with different promoter inserts were introduced into L. lactis MGl614 cells. The maximum level of Cm resistance in L. lactis MG1614 transformants was quite low (20$\mu\textrm{g}$/mL) when compared with that observed in recombinant E. coli cells harboring the same plasmids.

• BMB Reports
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• 제29권5호
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• pp.468-473
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• 1996

The P38 promoter of minute virus of mice (MVM) is a very weak promoter which is strongly transactivated by viral nonstructural proteins. To analyze the upstream sequence of the P38 promoter which is responsible for the transactivation by nonstructural proteins in MVM, chloramphenicol acetyltransferase (CAT) reporter plasm ids containing a series of 5' deletion and internal deletion mutants of the P38 promoter were constructed. The wild type and mutant CAT constructs of P38 promoter were cotransfected into murine A92L fibroblast cells with a plasmid expressing viral nonstructural proteins by DEAE-dextran method. Each promoter activity was analyzed by CAT assay. As previously reported (Ahn et al., 1992), the proximal DNA cis-elements required for transactivation of the MVM P38 promoter are GC box and TATA box. However, the analysis of 5' deletion mutants showed that H-l tar like sequence (MVM TAR) which is located between -143 and -122 relative to the transcription initiation site is also required for transactivation of the P38 promoter by nonstructural proteins. Interestingly, even if the MVM TAR was removed by internal deletion, the level of the transactivation is still 70% of wild type level of transactivation. We also found that, in addition to the MVM TAR motif, there are two other motifs which are similar to the MVM TAR sequence. When these TAR like motifs were further deleted, the levels of transactivation were decreased further. Taken together, the MVM TAR sequence and TAR like motifs located upstream of P38 promoter are playing an important role for the transactivation of P38 promoter by nonstructural proteins in minute virus of mice.

• KSBB Journal
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• 제11권4호
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• pp.445-452
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• 1996

본 연구에서는 GALl, GALl, GALlO 및 GAP promoter 하류에 reporter 유전자인 K. marxianus의 inulinase 유전자(lNUl)를 연결하여 각각의 재조합 plasmid들을 구축하고, 이들로 형질전환된 S. cerevrswe를 회분배양(YPOG 배지 )하여 외래 유전자 발현에 미치는 promoter의 영향을 비교.검토하 였다. 재조합 효모의 최종 균체농도는 36-39 00600 값을 보여 promoter에 따른 큰 차이를 보이지 않았으나, 포도당 소모기간 동안 비증식속도는 평균 $0.24 h^{-1}$로 유지되다가 galactose 소모기간 동안에 GAL promoter 함유 효모배양의 경우 $0.04-0.06 h^{-1}$, pYIGP 함유 재조합 효모배양은 $0.10 h^{-1}$로 감소하였다. 포도당 고갈 후 inulinase 발현은 시작되었고 균체외 inulinase의 발현 수준은 배양 72시간에 4.3 (GALl promoter), 4.0 (GAL7 promoter), 3.8 (GAL10 promoter) 및 1.6 (GAP promoter) unit/mL에 도달하였다. 평판배지상에서의 활성염색과 회분배양의 결과(최종발현양 및 초기 inulinase 말현속도), inulinase 발현에 미치는 promoter 세기 는 GALl > GALlO > GAL7 > GAP 순임을 알 수 있었다. GAL promoter가 배양말기까지 78 % 이상의 높은 plasmid 안정성을 보인 반면에, GAP promoter의 경우 55%의 낮은 plasmid 안정성을 보였다. 또한, 재조합 inulinase는 promoter 종류에 상관없이 98% 이상 배양액으로 분비되였다.

• Molecules and Cells
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• 제27권5호
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• pp.583-589
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• 2009

GTP cyclohydrolase I (GTPCH) is a key enzyme in the de novo synthesis of tetrahydrobiopterin. Previously, the Drosophila melanogaster GTPCH gene has been shown to be expressed from two different promoters (P1 and P2). In our study, the 5'-flanking DNA regions required for P1 and P2 promoter activities were characterized using transient expression assay. The DNA regions between -98 and +31, and between -73 and +35 are required for efficient P1 and P2 promoter activities, respectively. The regions between -98 and -56 and between -73 and -41 may contain critical elements required for the expression of GTPCH in Drosophila. By aligning the nucleotide sequences in the P1 and P2 promoter regions of the Drosophila melanogaster and Drosophila virilrs GTPCH genes, several conserved elements including palindromic sequences in the regions critical for P1 and P2 promoter activities were identified. Western blot analysis of transgenic flies transformed using P1 or P2 promoter-lacZ fusion plasmids further revealed that P1 promoter expression is restricted to the late pupae and adult developmental stages but that the P2 promoter driven expression of GTPCH is constitutive throughout fly development. In addition, X-gal staining of the embryos and imaginal discs of transgenic flies suggests that the P2 promoter is active from stage 13 of embryo and is generally active in most regions of the imaginal discs at the larval stages.

• Journal of Plant Biology
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• 제38권1호
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• pp.107-113
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• 1995

pRLYS1 containing intact rbcL gene of maize (Zea mays L. cv Golden X Bantam T-51; Zm-A) was digested with several restriction enzymes to construct subcones carrying promoter region of rbcL. The DNA fragments of 0.20, 0.19, 0.92 and 1.55 kb among the EcoRI digests, the EcoRI-DdeI digests, the AvaI digests and the EcoRI-BamHI digests of pRLYS1 were subcloned into pBluscriptSK＋and named pRLPS2, pRLPS3, pRLPS14 and pRLPS35, respectively. Four subclones contain the 1.92 kb portion from 136 nucleotide downstream to 1780 nucleotide upstream from the ATG initiation codon of rbcL gene. pRLPS2 (-29 to -229) and pRLPS3 (-239 to -420 from the ATG) were sequenced. When nucleotide sequence of Zm-A was compared with sequence of rbcL promoter region of a different cultivar of maize (Zea mays L. cv WFG TMS X BS7; Zm-B), the difference rate between two cultivars was 4.3%. The mean of sequence divergence between Zm-A and three grass species in the same tribe, Andropogoneae, in the upstream region from 29 to 420 of ATG was 1.8%, whereas between Zm-B and above-mentioned three species was 5.4%. Therefore, Zm-A seems to evolutionarily closer to three other species in Andropogoneae tribe than Zm-B is.

• KSBB Journal
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• 제5권1호
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• pp.49-58
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• 1990

대장균으로부터 alpha interferon의 생산과 분비를 유도하기 위해 대장균의 lipoprotein promoter, lactose promoter 및 operator와 lipoprotein의 signal seqquence를 가지는 vector에 alpha-IFN유전자를 cloning하여 발현 vector pIF-III-B와 vector pIF-III-C를 제작하였다.

• Journal of Microbiology and Biotechnology
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• 제7권1호
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• pp.8-12
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• 1997

In order to overproduce D-xylose isomerase, the Escherichia coli D-xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) gene (xylA) was fused to ${\lambda}P_{L}$ promoter. The promoterless xylA gene containing the ribosome binding site and coding region for D-xylose isomerase was cloned into a site 0.3 kb downstream from the ${\lambda}P_{L}$ promoter on a high copy number plasmid. An octameric XbaI linker containing TAG amber codon was inserted between 33rd codon of ${\lambda}N$ and the promoterless xylA gene. The resulting recombinant plasmid (designated as pPX152) was transformed into E. coli M5248 carrying a single copy of the temperature sensitive ${\lambda}cI857$ gene on its chromosomal DNA. When temperature-induced, the transformants produced 15 times as much D-xylose isomerase as that of D-xylose-induced parent strain. The amount of overproduced D-xylose isomerase was found to be about 60% of total protein in cell-free extracts.