• Microbiology and Biotechnology Letters
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• v.16 no.5
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• pp.343-347
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• 1988

The promoters of alkali-tolerant Bacillus sp. had been cloned in the promoter probe vector pPL703 and recombinant plasmid p-12 had been constructed. As a result of subcloning, two different promoters were found to exist in the cloned 2.9 kb promoter fragment and two recombinant plasmids p-l2B1 and p-l2B2, each harboring different promoter, were constructed. The promoter activity, which was expressed in the CAT specific activity, of p-l2B1 was 7 times higher than that of p-l2B2. The promoter activity as a function of growth revealed that both promoters of p-l2B1 and p-l2B2 were expressed after the late logarithmic growth phase and repressed in the presence of 1.0％ (w/v) glucose.

• Journal of Life Science
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• v.9 no.2
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• pp.153-159
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• 1999

To express constitutively the inulinase gene (INUl) of Kluyveromyces marxianus in Saccharomyces cerevisiae, three yeast promoters such as GAPDH, ADH1 and ENO1 were connected upstream of INUl. The resulting plasmids, pYIGP, pADHl-INU, and pENO-INU were introduced to S. cerevisiae SEY2102 host strain, respectively, and then each transformants were selected by staining of colonies on sucrose-agar plate. When the yeast transformants were cultivated on 2$\%$ dextrose media, the total expression levels of inulinase reached to 1.11 unit/mL, 0.88 unit/mL, and 0.69 unit/mL for respective GAPDH, ADH1, and ENO1 promoter systems. On 4% dextrose media, however, the inulinase activities were observed at 2.00 unit/mL for pYIGP, 0.71 unit/mL for pADH1-INU, and 1.40 unit/mL for pENO-INU. This result indicates that the constitutive expression of INUl was significantly affected by the initial concentration of dextrose and the promoter strength was in the order GAPDH, ENO1, and ADH1 promoter at high dextrose concentration. Taking into account the plasmid stability, however, it is suggested that the ENO1 promoter system is more suitable for the INU1 expression on high dextrose medium or in the fed-batch cultivation accumulating ethanol at high level.

• BMB Reports
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• v.36 no.6
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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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• v.11 no.4
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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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• v.29 no.5
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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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• v.11 no.4
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• pp.445-452
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• 1996

To investigate the promoter effect on heterologous gene expression in S. cerevisiae, the recombinant plasmids pYI11, pYI12, pYI10-2, and pYIGP were constructed to contain the inulinase gene (INUI) as a reporter under the control of GAL10, GAL7, GAL1, and GAP promoters, respectively. When the yeasts transformants were cultivated on galactose-containing rich media, the cell growth reached to 36-39 OD600 at 72 hours of cultivation. The specific growth rates of the cells harboring the four different plasmids decreased similarly : they dropped from $0.24 h^{-1}$ during the glucose-consuming period to 0.04 -$0.10 h^{-1}$ during the galactose-consuming period (gene expression phase for GAL promoter system). After the depletion of glucose, the expression of inulinase gene was started and reached to maximal levels of 4.3(GAL1 promoter), 4.0(GAL10 promoter), 3.8(GAL7 promoter), and 1.6(GAP promoter) unit/mL at 72 hours of cultivation. Based on the maximal expression level and activity staining on the plate, the promoter strength was in the order of GAL1, GAL10, GAL7 and GAP promoter. While the GAL-promoter systems showed a high plasmid stabilities of more than 78%, the GAP-promoter plasmid revealed a lower plasmid stability of 55%. Most of inulinase activity (98%) was found in the extracellular medium, indicating that the secretion efficiency of inulinase is independent on the type of promoter.

• Molecules and Cells
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• v.27 no.5
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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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• v.38 no.1
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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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• v.5 no.1
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• pp.49-58
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• 1990

We constructed hybrid plasmids to allow controlled and extracellular production of human alpha-interferon in Escherichia coli. The hybrid plassmids were constructed by transferring alpha-lFN gene from plasmid Hif-2h which has the alpha-lFN gene at PstI restriction site of pBR322, to plasmids pIN -IIIB3 and pIN-IIIC3 at restriction sites between HindIII and BamHI. Plasmids pIN-IIIB3 and pIN-IIIC3 carry E. coli lipoprotein promoter, lac promoter and operator in tandem. The plasmids also have lacl genes which encode for lac repressors, which allows controlled expression of genes cloned to the plasmids by using of inducer IPTG. Lipoprotein signal sequence is located just ahead of cloning sites of the plasmids, which helps cells to excrete or secrete cloned gene products. Plasmid pUC9 was used as a intermediate vector for transferring of alpha-lFN gene from Hif-2h to pIN vectors in order to solve the problem of different restriction sites between Hif-2h and pIN vectors.

• Journal of Microbiology and Biotechnology
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• v.7 no.1
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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.