• Journal of Life Science
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• v.19 no.9
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• pp.1289-1293
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• 2009

Proto-oncogene human cervical cancer oncogene (HCCR) functions as a negative regulator of p53 and contributes to tumorigenesis in various human tissues. However, it is unknown how HCCR contributes to the cellular and biochemical mechanisms of human tumorigenesis. In this study, we showed how the expression of HCCR is modulated. The luciferase activity assay indicated that the HCCR 5'-flanking region at positions -370 to -406 plays an important role in the promoter activity. Computational analysis of this region identified one consensus sequence for the TG-interacting factor (TGIF) located at -390 to -366 (TG). Mobility shift assays (EMSA) revealed that nuclear proteins from K562 bind to the TG site, but not to the mutated TG site. The reporter activity assay with promoter constructs carrying mutated TGIF sequences pGL3-mTGIF significantly increased reporter activities compared to wild type constructs pGL3-$406{\sim}+30$. In this study, we characterized the HCCR promoter and found that HCCR expression was partially regulated by the transcription repressor TGIF, which bound the promoter at positions -390 to -366.

• 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.431-437
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• 1996

The nar promoter of Escherichta coli, which is maximally induced under anaerobic conditions in the presence of nitrate, was characterized to see whether the nar promoter cloned onto pBR322 can be used as an expression promoter. The modified nar promoter, in which several bases in the -10 region was mutated to the consensus sequence by site-directed mutagenesis, was characterized in E. coli, on which chromosome the fnr gene affecting expression of the nar promoter according to dissolved oxygen level was mutated. The E. coli lacZ gene was used as a reporter gene. The following effects were investigated to find optimal conditions to induce the modified nar promoter: induction methods, optimal nitrate concentrations, the amount of ${\beta}$-galactosidase expressed at the different growth conditions, and induction characteristics. The following results were obtained from the experiments : expression of ${\beta}$-galactosidase from the modified nar promoter was not affected much by nitrate concentrations. The maximal specific ${\beta}$-galactosidase activity was obtained when E. coli was grown under aerobic conditions, and then the modified nar promoter was induced at OD600=2.2 under microaerobic conditions (DO=1∼2%), under which conditions the maximal specific ${\beta}$-galactosidase activity was 13,000 Miller units. However, the specific ${\beta}$-galactosidase activity was approximately 6,000 Miller units even before the modified nar promoter was induced. Therefore, the modified nar promoter seemed to be useful when the cloned gene wants to be expressed in E. coli constitutively.

• Genomics & Informatics
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• v.1 no.2
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• pp.113-118
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• 2003

Among a number of antigens characterized in M leprae, an etiological agent of Leprosy, the 18 kDa antigen, is unique to M leprae. We have previously determined a sequence specific element in the 18 kDa gene of M leprae, which confers transcriptional repression. In this report, we have examined if the element could be applied to genes other than the 18 kDa gene of M leprae. To identify the roles of the regulatory sequence in heterologous promoter, we have constructed pB3 vector series, which contains BCG hsp65 promoter and the M leprae 18 kDa transcription repression responsive element in tandem using LacZ gene as a reporter gene. Cloning of hsp65 promoters of M bovis BCG or M smegmatis in front of LacZ gene resulted in normal $\beta$­galactosidase activity as expected. However, when the sequence element was placed between the promoter and the LacZ gene, $\beta$-galactosidase activity was reduced 10-fold less. Also we have examined with pB3(-) vector, that harbors the transcription repression responsive element in a reversed orientation, the $\beta$-galactosidase activity was found to be similar to pB3(+) vector. Thus, these results further confirm that M leprae 18 kDa transcription repression responsive element could regulate BCG hsp65 heterologous promoter and that the element could act as an operator for the transcription of mycobacteria.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.38 no.2
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• pp.101-109
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• 2012

Objectives: The inactivation of the tumor suppressor gene $p16^{INK4a}$ plays an important role in the development of malignant tumors, including oral squamous cell carcinoma. The p16 gene is involved in the p16/cyclin-dependent kinase/retinoblastoma (Rb) gene pathway of cell cycle control. The p16 protein is considered a negative regulator of this pathway. The p16 gene encodes an inhibitor of cyclin-dependent kinases 4 and 6 which regulate the phosphorylation of the retinoblastoma gene and G1 to S phase transition in the cell cycle. However, the p16 gene can lose its functionality through point mutations, loss of heterozygosity or methylation of its promoter region. Materials and Methods: In this study, the authors analyzed the correlation between various clinicopathological findings- patient age, gender and smoking, disease recurrence, tumor size, stage, and differentiation- and p16 protein expression or p16 promoter hypermethylation in 59 cases of head and neck squamous cell carcinoma. Results: The results revealed p16 protein expression and p16 promoter hypermethylation in 28 cases (47.5%) and 21 cases (35.6%), respectively, of head and neck squamous cell carcinoma. However, neither p16 protein expression nor p16 promoter hypermethylation had any statistical influence on clinicopathological findings or survival rate. Conclusion: This data, and a review of the literature, suggest that p16 promoter hypermethylation cannot yet be used as an independent prognostic factor influencing carcinogenesis, but must be considered as an important factor along with other genetic alterations affecting the pRb pathway.

• Journal of Life Science
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• v.31 no.11
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• pp.995-1003
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• 2021

Chromatin remodeling regulates gene expression through epigenetic mechanisms. Aberrations in histone modification have been associated with depression-like behaviors in animal models. Additionally, growing evidence also indicates that epigenetic modification is associated with depression. p11 (S100A10) has been implicated in the pathophysiology of depression both in human and rodent models. In the present study, we investigated alterations in histone acetylation and methylation at the promoter of the p11 gene in the hippocampus of mice subjected to chronic unpredictable stress (CUS). C57BL/6 mice were exposed to CUS daily for 3 weeks. Depression-like behaviors were measured with the forced swimming test (FST). The levels of hippocampal p11 expression were analyzed by quantitative real-time polymerase chain reaction (PCR) and Western blotting. The levels of acetylated and methylated histone H3 at the promoter of p11 were measured by chromatin immunoprecipitation followed by real-time PCR. CUS-exposed mice displayed depression-like behaviors with prolonged immobility in FST. CUS led to significant decreases in the expression of p11 at both protein and mRNA levels. Meanwhile, there was a decrease in histone H3 acetylation (Ac-H3) and H3-K4 trimethylation (H3K4met3) and an increase in H3-K27 trimethylation (H3K27met3) at the p11 promoter. These results indicate that chronic stress causes the epigenetic suppression of p11 expression in the hippocampus.

• Journal of Nutrition and Health
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• v.35 no.2
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• pp.207-212
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• 2002

Acetyl-CoA carboxylase (ACC) is the enzyme that controls no devo fatty acid biogynthesis, and this enzyme catalyzes the carboxylation pathway of acetyl-CoA to malonyl-CoA. Acetyl-CoA carboxylase gene expression was regulated by nutritional and hormonal status. The present study was performed to identify the regulation mechanism of ACC gene promoter I. The fragments of ACC promoter I -1.2-kb region wert recombined to pGL3-Basic vector with luciferase as a reporter gene. The primary hepatocytes from the rat were used to investigate the hormonal regulation of ACC promoter I activity. ACC PI (-1.2)/Luc plasmid was trtransferred into primary hepatocytes using lipofectin. Activity of luciferase was increased two-fold by 10-9M, three-fold by 10-8M, 10-6M, 3.5-fold by 10-6M, and 4.5-fold by 10-7M insulin treatment, respectively. In the presence of dexamethasone (1 $\mu$M), the effects of insulin increased about 1.5-fold, showing the additional effects of dexamethasone. Moreover, the activity of luciferase increased with insulin+dexamethasone, insulin+T3, dexamethasone+T3, and dexamethasone+insulin+T3 treatment approximately 6-, 4-, 6.5-, and 10-fold, respectively. Therefore it can be postulated that 1) these hormones coordinately regulate acetyl-CoA caroxylase gene expression via regulation of promoter activity, 2) the -1.2-kb region of ACC promoter I may have the response element sequences for insulin, dexamethasone, and T3.

• Journal of Life Science
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• v.24 no.1
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• pp.1-7
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• 2014

Eukaryotic gene expression is an important process, which is initiated by several transcription factors and RNA polymerases that occupy the promoter region of genomic DNA. Although there are many experiments to identify the promoter region in a gene, it is time and labor consuming to finalize it. In this study, we utilized bioinformatic programs, including Ensembl, NCBI, and CpG plots, to identify the cloning promoter region in SETDB1 genomic DNA. We performed PCR amplification to obtain the SETDB1 promoter on an approximately 2 kb region upstream from the TSS named SETDB1-P1. The PCR product was ligated with TA cloning vectors, and we confirmed the insert size using restriction enzyme digestion. Sequentially, the insert was subcloned into a pGL3-luc vector to produce pGL3-SETDB1- P1-luc and then confirmed by DNA sequencing. We also obtained a fragmented PCR product called P2 and P3 and performed a luciferase assay using pGL3-SETDB1-P1-luc transfection. We found that several anticancer drugs, including taxol, 4-FU, and doxorubicin, decreased the promoter activity of SETDB1. We obtained consistent data on the regulation of SETDB1 gene expression after anticancer drug treatment using Western blot analysis and RT-PCR. Our results suggest that promoter cloning of the human SETDB1 gene utilizing bioinformatics is a very useful and timesaving approach to study gene expression.

• Journal of Aquaculture
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• v.10 no.1
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• pp.33-37
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• 1997

construct containing reporter gene(pFV4CAT) regulated by carp $\beta$-actin promoter was microinjected into the one-cell stage egg of mud loach (Misgurnus mizolepis), and was successfully expressed, possibly by the integration into the genome. Both mean hatching success and early survival of the microinjected groups were not significantly different with those of control groups (P>0.05). The incidence of transgene was ranged from 7 to 48% based on the PCR and/or Southern blot analyses with the DNA prepared from fin or blood tissue. The spatial and temporal patterns of expression of the pFV4CAT gene, measured by in situ immunohistochemical analysis peroxidase-conjugated anti-CAT antibody, were variable among the experimental individuals. These results suggest that carp $\beta$-actin promoter is effective to express other transgene in mud loach, such that this promoter can be useful in the generation of valuable transgenic mud loach.

• Genomics & Informatics
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• v.8 no.4
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• pp.201-205
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• 2010

The H1 histone family, member N, testis-specific (H1FNT) is exclusively expressed in the testis, and had its possible role for sperm chromatin formation. The purpose of this study is to investigate any genetic association of H1FNT gene with male infertility, especially at the promoter region. We examined the promoter single nucleotide polymorphisms (SNP) of H1FNT gene which is located within transcription factor binding site for its association with male infertility. The statistical analysis showed that the -1129A>T polymorphism was present at a statistically significance in male infertility (p=0.0059 and 0.0349 for hetero and risk type, respectively). The dual-luciferase promoter assay was performed to examine the polymorphic effect of this promoter SNP by the cloning of promoter region (1700bp fragment) into pGL3-basic vector. In our plasmid based reporter system, there is no big difference between wild and risk type. In conclusion, H1FNT -1129A>T promoter SNP is statistically significant with male infertility, especially with subfertile (non-azoospermia) group. Further analysis of its functional polymorphic effect in vivo may provide the biological significance of testis-specific histone with spermatogenesis.