• Journal of Microbiology and Biotechnology
• /
• v.5 no.1
• /
• pp.14-17
• /
• 1995

Endoglucanase gene of Pseudomonas sp. LBC505 was previously cloned in pUC19 to yield plasmid pLCl. The Pseudomonas sp. LBC505 endoglucanase gene was subcloned in a temperature-regulated Es-cherichia coli expression vector, pAS1, containing the leftward promoter $P_L$ of bacteriophage lambda. The level of gene expression was controlled by the thermal inactivation of the heat-sensitive lambda cI857 repressor. Best yield of endoglucanase was obtained by lowering the incubation temperature to $37^{\circ}C$ after induction at $42^{\circ}C$ for 1h. Under these conditions enzyme production continued for about 5h at a gradually decreasing rate. Ecoli harboring recombinant plasmid pASC10 expressed 4.3 times as much CMCase activity as E.coli containing pLCl. To enhance the expression level of endogl, ucanase gene, we have also changed the presumptive Shine-Dalgamo sequence (AGAGGT) of the gene to consensus sequence (AGGAGGT) by site-directed mutagenesis. The genes mutated were subcloned in pASl resulting in the formation of recombinant plasmid pASS50. E.coli harboring the plasmid pASS50 expressed 6.2-fold higher levels of CMCase activity than that of E.coli harboring pLC1.

• Microbiology and Biotechnology Letters
• /
• v.19 no.5
• /
• pp.433-438
• /
• 1991

The plasmid pSY130-14 for the high production of phenylalanine is a temperaturecontrollable expression vector composed of the $P_R$ and the $P_L$ promoter and a temperature sensitive repressor, $cI_{857}$ of bacteriophage lambda. Strain AT2471 harbouring plasmid pSY13O- 14 is induced the phenylalanine production by shifting up the incubation temperaure to $38.5^{\circ}C$. Plasmid stability of E. coli AT2471 harbouring pSY130-14 was very low, it was about 30% after 48 h cultivation at $38.5^{\circ}C$ without kanamycin. The plasmid disappeared immediately at $40^{\circ}C$ without kanamycin, and at $40^{\circ}C$ adding kanamycin, the plasmid stability decreased at the beginning, but rose with the extension of the culture time. For the improvement of plasmid stability, the plasmid obtaind was designated as pSY15O-1 by changing origin region (ori) pACYC 177 of pSY130-14 for ori pSC101. E. coli AT2471 harbouring pSY150-1 was stable at $38.5^{\circ}C$ without tetracycline, and the plasrnid stability was about 40% after 48 h cultivation at $40^{\circ}C$.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1997.04a
• /
• pp.81-81
• /
• 1997

We previously demonstrated an enhancer-like positive regulatory element within a 259-bp sequence (-2352 to-2094 bp) of the human CYP1A2 gene in HepG2 cells. Three protein binding sites were identified by DNase I footprint analyses within the 259-bp sequence: protected region A PRA ( -2283 to-2243 bp), PRB (-2218 to-2187 bp), and PRC (-2124 to-2098 bp) (I. Chung and E. Bresnick, Mol. Pharmacol. 47, 677-685, 1995). In the present study, the functional significance of those protected regions was examined. Transfection experiments with deletion and substitution mutants defined the PRB and PRC as containing positive and negative regulatory elements, respectively. Human breast carcinoma MCF-7 cells were cotransfected with a hepatocyte nuclear factor-1 (HNF-1) expression vector and CYP1A2 promoter-or thymidine kinase promoter-luciferase remoter gene constructs. HNF-1, which contributes to the liver specificity of genes, enhanced reporter gene activity in a PRC sequence-dependent manner. These results suggested that PRC could exist bound to a repressor which was displaceable by other transcription factors such as HNF-1. Results obtained by transfection of HepG2 hepatoma cells with various PRB substitution mutant-luciferase gene fusion constructs indicated that the entire sequence of PRB was necessary for promoter activity. Consequently, the regulation of CYP1A3 expression is very complex, requiring a number of both positive and negative regulatory factors.

• Journal of the Korea Society of Computer and Information
• /
• v.12 no.6
• /
• pp.105-113
• /
• 2007

A gene regulatory network is a network of genes representing how genes influence the activities of other genes. Nowadays from microarray experiments, a large number of measurements on the expression levels of genes are available. One of typical data is the so-called "steady-state model" data measuring the expression levels of other genes after knocking out a particular gene. This paper shows how to reverse engineer a parsimonious gene regulatory network, using these measurement data. Our model considers auto-regulation, which forms a cycle in a genetic network. We also model repressor and enhancer roles of genes. which are not considered in previous known methods.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.4
• /
• pp.639-647
• /
• 2008

The heat-inducible expression vectors for Corynebacterium glutamicum and C. ammoniagenes were constructed by using the ${\lambda}O_L1$ and the cryptic promoters, CJ1 and CJ4 that express genes constitutively in C. ammoniagenes. Although the promoters were isolated from C. ammoniagenes, CJ1 and CJ4 were also active in C. glutamicum. To construct vectors, the $O_L1$ from the ${\lambda}P_L$ promoter was isolated and fused to the CJ1 and CJ4 promoters by recombinant PCR. The resulting artificial promoters, CJ1O and CJ4O, which have one ${\lambda}O_L1$, and CJ1OX2, which has two successive ${\lambda}O_L1$, were fused to the green fluorescent protein (GFP) gene followed by subcloning into pCES208. The expression of GFP in the corynebacteria harboring the vectors was regulated successfully by the temperature-sensitive cI857 repressor. Among them, C. ammoniagenes harboring plasmid pCJ1OX2G containing GFP fused to CJ1OX2 showed more GFP than the other ones and the expression was tightly regulated by the repressor. To construct the generally applicable expression vector using the plasmid pCJ1OX2G, the His-tag, enterokinase (EK) moiety, and the MCS were inserted in front of the GFP gene. Using the vector, the expression of pyrR from C. glutamicum was tried by temperature shift-up. The results indicated that the constructed vectors (pCeHEMG) can be successfully used in the expression and regulation of foreign genes in corynebacteria.

• Proceedings of the Microbiological Society of Korea Conference
• /
• 2003.05a
• /
• pp.51-54
• /
• 2003

Analysis of the Neurospora crassa genome data reveals at least 14 proteins that contain tetratricopeptide repeat (TPR) motifs. One of them shows over $60\%$ homology with SSN6 of Saccharomyces cerevisiae, a global repressor that mediates repression of genes involved in various cellular processes. Sequence analysis of its cDNA shows that it encodes a putative 102kDa protein. Mutant strains generated by RIP (repeat induced point mutation) process show four distinctive patterns of vegetative growth at various rates. They are male-fertile, yet all female-sterile and produced little or no perithecium. These results indicate that this gene is pleiotropic and involved in several cellular processes of vegetative growth, conidiation and sexual cycle. It is designated rcm-1(regulation of conidiation and morphology).

• Journal of Life Science
• /
• v.27 no.10
• /
• pp.1225-1231
• /
• 2017

Disruptive expression patterns of the circadian clock genes are highly associated with many human diseases, including cancer. Cell cycle and proliferation is linked to a circadian rhythm; therefore, abnormal clock gene expression could result in tumorigenesis and malignant development. The molecular network of the circadian clock is based on transcriptional and translational feedback loops orchestrated by a variety of clock activators and clock repressors. The expression of 10~15% of the genome is controlled by the overall balance of circadian oscillation. Among the many clock genes, Period 1 (Per1) and Period 2 (Per2) are clock repressor genes that play an important role in the regulation of normal physiological rhythms. It has been reported that PER1 and PER2 are involved in the expression of cell cycle regulators including cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors. In addition, correlation of the down-regulation of PER1 and PER2 with development of many cancer types has been revealed. In this review, we focused on the molecular function of PER1 and PER2 in the circadian clock network and the transcriptional and translational targets of PER1 and PER2 involved in cell cycle and tumorigenesis. Moreover, we provide information suggesting that PER1 and PER2 could be promising therapeutic targets for cancer therapies and serve as potential prognostic markers for certain types of human cancers.

• Journal of Microbiology
• /
• v.38 no.1
• /
• pp.18-23
• /
• 2000

Streptomyces coelicolor produces three kinds of catalases to cope with oxidative stress and to allow normal differentiation. Catalase A is the major vegetative catalase which functions in removing hydrogen peroxide generated during the process of aerobic metabolism. To understand the regulatory mechanism of response against oxidative stress, hydrogen peroxide-resistant mutant (HR4O) was isolated from S. coelicolor J1501 following UV mutagenesis. The mutant overproduced catalase A more than 50-fo1d compared with the wild type. The mutation locus catRI was mapped closed to the mthB2 locus by genetic crossings. An ordered cosmid library of S. coelicolor encompassing the mthB2 locus was used to isolate the regulator gene (catR) which represses catalase overproduction when introduced into HR4O. A candidate catR gene was found to encode a Fur-like protein of 138 amino acids (15319 Da).

• Journal of Applied Biological Chemistry
• /
• v.66
• /
• pp.128-137
• /
• 2023

Transcriptome analysis revealed that the sinR gene encoding a transition-state regulator of Bacillus pumilus, genetically close to B. subtilis, was expressed at high levels during growth. The sinR gene is the second gene of the sinIR operon consisting of three promoters and two structural genes in B. subtilis. This study used the sinIR promoter of B. subtilis DB104 to construct a recombinant protein expression system. First, the expression ability depending on the number of sinIR promoter was investigated using enhanced green fluorescent protein (eGFP). The expression level of eGFP was slightly higher when using two promoters (Psin2) than using original promoters. The Psin2 promoter was further engineered by modifying the repressor binding site and -35 and -10 regions. Shine-Dalgarno (SD) sequence of the sinI gene was modified to the consensus sequence. Finally, combining the engineered Psin2 promoter with the modified SD sequence increased the expression level of eGFP by about 13.4-fold over the original promoter. Our results suggest that the optimized sinIR promoter could be used as a novel tool for recombinant protein expression in B. subtilis.

• Microbiology and Biotechnology Letters
• /
• v.34 no.1
• /
• pp.15-22
• /
• 2006

A 4,971 bp chromosomal DNA fragment containing the pqrA, paraquat resistance gene, was cloned from Ochrobactrum anthropi JW-2, and the complete nucleotide sequence was determined. Nucleotide and deduced amino acid sequences of the fragment revealed the presence of 4 complete ORFs (orf2, pqrA, orf3, orf4) and two incomplete ORFs(orf1, orf5). Orf1, pqrA, orf4 and orf5 exists at the direct strand but orf2 and orf3 exists at the reverse complementary strand. Orf1 which of incomplete sequences without start codon shares homology with ATP binding region of the response regulator receiver. Orf2 shares high homology with members of the tetR family of transcriptional repressor which have a helix-turn-helix (H-T-H) motif. Therefore, the orf2 is predicted as a transcriptional repressor of pqrA and is designated as pqrR2. Orf3 shares high homology with the members of the lysR family acting as a transcriptional activator which have both of a H-T-H motif at the N-terminal region and substrate binding domain at the C-terminal region. Therefore, the orf3 is predicted as a transcriptional activator of pqrA and is designated as pqrR1. Orf4 shows homology with the periplasmic substrate-binding protein of amino acid ABC transporter. Orf5 which of incomplete sequences without stop codon revealed the homology with the permeases protein of amino acid ABC transporter.