• BMB Reports
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• 제32권4호
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• pp.345-349
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• 1999

We fused the promoter region of an H3.2 chicken histone gene, whose expression is dependent on the cell cycle, to the 5' coding region of an H3.3 chicken histone gene, which is expressed constitutively at a low level throughout the cell cycle. This fusion gene showed a cell cycle-regulated pattern of expression, but in a different manner. The mRNA level of the fusion gene increase during the S phase of the cell cycle by about 3.7-fold at 6 h and 2.7-fold at 12 h after the serum stimulation. The mRNA level of the intact H3.2 gene, however, increased by an average of 3.6-fold at 6 h and 8.7-fold at 12 h. This different expression pattern might be due to the differences in their 3' end region that is responsible for mRNA stability. The 3' end of the H3.2 mRNA contains a stem-loop structure, instead of a poly(A) tail present in the H3.3 mRNA. We also constructed a similar fusion gene using a H3.3 histone gene whose introns had been eliminated to rule out the possibility of involvement of the introns in cell cycle-regulated expression. The expression of this fusion gene was almost identical to the fusion gene made previously. These results indicate that the promoter region of the H3.2 gene is only partially responsible for its expression during the S phase of the cell cycle.

• Journal of Nutrition and Health
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• 제35권2호
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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.

• BMB Reports
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• 제37권6호
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• pp.648-656
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• 2004

This paper deals with the development of a predictive probabilistic model, a composite dependency-reflecting model (CDRM), which was designed to detect core promoter regions and transcription start sites (TSS) in vertebrate genomic DNA sequences, an issue of some importance for genome annotation. The model actually represents a combination of first-, second-, third- and much higher order or long-range dependencies obtained using the expanded maximal dependency decomposition (EMDD) procedure, which iteratively decomposes data sets into subsets on the basis of dependency degree and patterns inherent in the target promoter region to be modeled. In addition, decomposed subsets are modeled by using a first-order Markov model, allowing the predictive model to reflect dependency between adjacent positions explicitly. In this way, the CDRM allows for potentially complex dependencies between positions in the core promoter region. Such complex dependencies may be closely related to the biological and structural contexts since promoter elements are present in various combinations separated by various distances in the sequence. Thus, CDRM may be appropriate for recognizing core promoter regions and TSSs in vertebrate genomic contig. To demonstrate the effectiveness of our algorithm, we tested it using standardized data and real core promoters, and compared it with some current representative promoter-finding algorithms. The developed algorithm showed better accuracy in terms of specificity and sensitivity than the promoter-finding ones used in performance comparison.

• International Journal of Industrial Entomology and Biomaterials
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• 제25권1호
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• pp.111-114
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• 2012

In previous studies we have shown that a sw17255 gene was expressed in hemocyte-specific tissues of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). It was verified that the sw17255 core promoter region contains elements that regulate the expression of this gene in hemocyte tissue; the selected promoter region spans nucleotides -1 to -2,112 upstream of the start codon. Each of the luciferase reporter gene expression vectors under the control of 4 different kinds of promoter candidates, (-2,112/-1), (-1,640/-1), (-1,169/-1) and (-579/-1), and the control reporter plasmid DNA, were introduced into B. mori larval coelom by direct injection using a syringe. The promoter candidate [E] (-579/-1) showed more than 1.67 fold transcriptional activity compared to control promoter activity. Higher productivity of an expressed gene in the transgenic silkworm by this promoter combination could be achieved in the near future. The foreign recombinant protein could be easily harvested from the blood of the transgenic silkworm.

• 미생물학회지
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• 제30권5호
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• pp.403-409
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• 1992

STA1 유전자의 promoter 와 분비신호서열을 이용하여 B. subtilis 의 CMSase 를 분비하는 재조합 효모균주를 육성하였다. STA1A 유전자의 promoter, 분비신호서열, TS region 및 mature glucoamylase N-말단부위의 아미노산 98개와 B. subtilis 의 CMCase 구조유전자가 차례로 연결된 재조합플라스미드 pYESC24 를 제작한후 효모에 형질전환하였으나 CMCase 가 세포외로 분비되지 않았다. 반면에 STA1 의 TS region 및 mature glucoamylase N-말단 아미노산 98 개를 제거하여 CMMase 구조유전자갸 STA1 의 분비신호서열에 바로 연결된 재조합 플라스미드 pYESC11 에 의한 효모형질전환 균주는 CMCase 분비능이 아주 우수하였다. 이 형질전환 균주를 YPD 배지에서 4 일간 배양한 후 세포부위 별 CMCase 역가를 측정한 결과 배양액 1 m/당 총역가 44.7 unit 존재하였으며 이중 93% 이상이 배양상등액에서 관찰되었다.

• International Journal of Industrial Entomology and Biomaterials
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• 제20권2호
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• pp.107-114
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• 2010

For stable germline transformation, the promoter of Bombyx mori cytoplasmic actin gene (BmA3) has been used for ubiquitous expression of transgenes. So far, no strong promoter is available for ubiquitous expression in B. mori, excluding BmA3 promoter. To identify more powerful promoter than previously reported BmA3 promoter, we isolated 9 clones that show stronger signal compared to BmA3 by a dot blot hybridization. Among these 9 clones, we focused on one clone which has high amino acid homology (85%) with hypothetical protein 32 gene of Lonomia obliqua. This clone, named bHp32 (B. mori hypothetical protein 32) was ubiquitously expressed in all tissues and developmental stage of fifth instar B. mori larvae. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-1,200/+220) in the 5'-flanking region of bHp32 gene, which has 42-fold more intensive promoter activity than BmA3 promoter. Moreover, the bHp32 promoter was normally regulated in Bm5, Sf9, and S2 cells. Therefore, we suggest that bHp32 promoter may be used more powerful and effectively for transgene expression in various insects containing B. mori as a universal promoter.

• Journal of Microbiology
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• 제38권3호
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• pp.176-179
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• 2000

A challenge phage system was used to study the DNA-protein interaction between C4-dicarboxylic acid transport protein D(DCTD) or $\sigma$54, and a $\sigma$54 -dependent promoter, dctAp. R. meliloti dctA promoter regulatory region replaced the Omnt site on the phage. S. typhimurium strains overproducing either DCTD or $\sigma$54 directed this challenge phage towards lysogency, indicating that DCTD or E$\sigma$54 recognized the dctA promoter on the phage and repressed transcription of the ant gene. These challenge phage constructs will be useful for examining interactions between DCTD(or $\sigma$54) and the dctA promoter region.

• Journal of Plant Biology
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• 제38권3호
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• pp.259-266
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• 1995

To investigate how the dyad symmetry region (DSR) and the distance between vir box and -35 sequence of the virE promoter plays a role in virE gene expression, two mutants were constructed by base substitution and insertional mutagenesis. The base substitutional mutation, a AAlongrightarrowCG substitution at positions -39 and -40 on the DSR, showed the level of $\beta$-galactosidase activity approximately 91% of the wild type virE promoter activity. Therefore, the native structure of the DSR seems to be not essential for virE expression. The insertional mutation, constructed by inserting 8 bp ClaI linker between -49 and -50, displayed the $\beta$-galactosidase activity at 12% of the native virE promoter activity. However, this striking reduction appears to be not caused by destruction of the native DSR structure, but by shifting the vir box far from putative -35 sequence.

• Asian-Australasian Journal of Animal Sciences
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• 제31권3호
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• pp.449-456
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• 2018

Objective: Nanog, a homeodomain protein, has been investigated in humans and mice using embryonic stem cells (ESCs). Because of the limited availability of ESCs, few studies have reported the function and role of Nanog in porcine ESCs. Therefore, in this study, we investigated the location of the porcine Nanog chromosome and its basal promoter activity, which might have potential applications in development of ESCs specific marker as well as understanding its operating systems in the porcine. Methods: To characterize the porcine Nanog promoter, the 5'-flanking region of Nanog was isolated from cells of mini-pig ears. BLAST database search showed that there are two porcine Nanog genomic loci, chromosome 1 and 5, both of which contain an exon with a start codon. Deletion mutants from the 5'-flanking region of both loci were measured using the Dual-Luciferase Reporter Assay System, and a fluorescence marker, green fluorescence protein. Results: Promoter activity was detected in the sequences of chromosome 5, but not in those of chromosome 1. We identified the sequences from -99 to +194 that possessed promoter activity and contained transcription factor binding sites from deletion fragment analysis. Among the transcription factor binding sites, a Sp1 was found to play a crucial role in basal promoter activity, and point mutation of this site abolished its activity, confirming its role in promoter activity. Furthermore, gel shift analysis and chromatin immunoprecipitation analysis confirmed that Sp1 transcription factor binds to the Sp1 binding site in the porcine Nanog promoter. Taken together, these results show that Sp1 transcription factor is an essential element for porcine Nanog basal activity the same as in human and mouse. Conclusion: We showed that the porcine Nanog gene is located on porcine chromosome 5 and its basal transcriptional activity is controlled by Sp1 transcription factor.

• Reproductive and Developmental Biology
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• 제32권1호
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• pp.33-38
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• 2008

DNA 메틸화는 조직특이적인 유전자 조절에 관여하고, 정상적인 배 발달에 필수적이다. POU5F1은 octamer-binding transcription factor 4 (Oct-4)를 encode하며, 초기 분화에 중요한 전사인자이다. 본 실험에서 소의 Oct-4가 조직특이적이고 발달의존적인 epigenetic 표지 인지를 검토하고자, 착상 전 수정란에서 Oct-4 전사산물과 상류 promoter 영역의 CpGs의 메틸화를 조사하였다. Oct-4 전사산물은 정자 그리고 2-cell에서 8-cell 수정란까지 낮은 수준으로 존재하지만, 상실배와 배반포에서 높게 검출되었다. 이러한 결과는 배 발달 과정의 상실배 단계에서 Oct-4의 de novo 발현이 시작됨을 의미한다. Oct-4 상류 promoter 영역에는 메틸화 가변 영역 (tissue-dependent differentially methylated region, T-DMR)이 존재한다. Oct-4 메틸화 가변 영역의 메틸화 상태는 정자, 성체 체조직과 난자에서 서로 다르고, 수정란으로부터 배반포 단계까지 변화하였는데, 이는 착상 전 초기 배 발달 과정에 active 메틸화와 탈메틸화가 일어남을 의미한다. 이상의 결과, Oct-4 유전자 상류 promoter 영역은 DNA 메틸화의 타깃이고, 그 메틸화 상태는 소 수정란 발달 동안에 다양하게 변화한다.