• Molecules and Cells
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• 제26권2호
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• pp.212-215
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• 2008

The GC-rich discriminator sequence between the -10 region and the transcription start site of the rnpB promoter is responsible for stringent control of M1 RNA synthesis. The rnpB promoter also contains a G nucleotide at the previously identified transcription start site. In this study, we examined by mutagenesis of G to A whether this +1G nucleotide is involved in the stringent response. We found that the change did not alter the stringent response. Since the +1 mutation might alter transcription initiation, we compared the transcription start sites of the wt and mutant promoters by primer extension analysis. Surprisingly, we found that wild type rnpB transcription starts at both the +1G position (70%) and the -1C position (30%), and that the +1A mutation led to transcription initiation exclusively at the -1C position. We also generated two transversion mutations at the -1 position, both of which led to transcription starting exclusively at that position. The -1G mutant promoter gave a stringent signal similar to the wild-type, whereas the -1A mutant generated a significantly less stringent signal. Base on these results, we propose that a short sequence, up to 7 bp downstream of the -10 region, is involved in the stringent response of the rnpB promoter.

• 한국동물위생학회지
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• 제27권1호
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• pp.95-101
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• 2004

Babesia bovis rap-1 and B equi ema-1 intergenic(IG) nucleotides were analyzed and compared for identifying putative promoter sites using computer programs. The reason to initiate this research was to determine if IG nucleotides of Babesia genes that are predicted to be involved in erythrocyte invasion have functions regulating gene transcription and translation, which can be applied to functional gene knockout. Four IG sequences used included BbIG5(B bovis rap-1 5' IG), BblG3(B bovis rap-1 3' IG), BeIG5(B equi ema-1 5' IG) and BeIG3(B equi ema-1 3' IG). BbIG5 contained a putative promoter at nucleotide 197-246 with a predicted TATA-box and a transcription start site. BbIG3 had a putative promoter at nucleotide 270-320 with two predicted TATA-boxes and a transcription start site. BeIG3 had a putative promoter at nucleotide 155-205 with a predicted TATA-box and a transcription start site. Putative promoter sites in these three sequences mentioned above were identified with score cutoff 0.8, which means detection of about 40% recognized promoters with 0.1-0.4% false positives. In contrast, BeIG5 had a putative promoter at nucleotide 163-213 with score cutoff 0.8, but neither TATA-box nor transcription start site were recognized. However, BeIG5 had a putative promoter at nucleotide 388-438 with a predicted TATA-box and a transcription start site when score cutoff was decreased to 0.18, which means detection of about 70% recognized promoters with 2.2-5.3% false positives. These sequences with putative promoters can be tested if they have functions regulating gene transcription and translation.

• BMB Reports
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• 제42권12호
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• pp.788-793
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• 2009

TLX2 is an orphan homeodomain transcription factor whose expression is mainly associated with tissues derived from neural crest cells. Recently, we have demonstrated that PHOX2A and PHOX2B are able to enhance the neural cell-type specific expression of human TLX2 by binding distally the 5' -flanking region. In the present work, to deepen into the TLX2 transcription regulation, we have focused on the proximal 5'-flanking region of the gene, mapping the transcription start site and identifying a minimal promoter necessary and sufficient for the basal transcription in cell lines from different origin. Site-directed mutagenesis has allowed to demonstrate that the integrity of this sequence is crucial for gene expression, while electrophoretic mobility shift assays and chromatin immunoprecipitation experiments have revealed that such an activity is dependent on the binding of a PBX factor. Consistent with these findings, such a basal promoter activity has resulted to be enhanced by the previously reported PHOX2-responding sequence.

• BMB Reports
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• 제38권6호
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• pp.695-702
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• 2005

The groESL bicistronic operon of a restricted facultative methylotrophic bacterium Methylovorus sp. strain SS1 DSM 11726 was cloned and characterized. It was found to consist of two ORFs encoding proteins with molecular masses of 11,395 and 57,396 daltons, which showed a high degree of homology to other bacterial GroES and GroEL proteins. The genes were clustered in the transcription order groES-groEL. Northern blot analyses suggested that the groESL operon is transcribed as a bicistronic 2.2-kb mRNA, the steady-state level of which was markedly increased by temperature elevation. Primer extension analysis demonstrated one potential transcription start site preceding the groESL operon, which is located 100bp upstream of the groES start codon. The transcription start site was preceded by a putative promoter region highly homologous to the consensus sequences of Escherichia coli ${\sigma}^{32}$-type heat shock promoter, which functioned under both normal and heat shock conditions in E. coli. Heat shock mRNA was maximally produced by Methylovorus sp. strain SS1 approximately 10min after increasing the temperature from 30 to $42^{\circ}C$. The groESL operon was also induced by hydrogen peroxide or salt shock.

• Molecules and Cells
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• 제38권11호
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• pp.1007-1012
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• 2015

EphA7 is a key molecule in regulating the development of the dien- and mesencephalon. To get insight into the mechanism of how EphA7 gene expression is regulated during the dorsal specification of the dien- and mesencephalon, we investigated the cis-acting regulatory sequence driving EphA7 to the dorsal midline of the dien- and mesencephalon. Transgenic LacZ reporter analysis, using overlapping EphA7 BACs, was used to narrow down the dorsal midline-specific enhancer, revealing the 25.3 kb genomic region as the enhancer candidate. Strikingly, this genomic DNA was located far downstream of the EphA7 transcription start site, +302.6 kb to +327.9 kb. Further enhancer mapping, using comparative genomic analysis and transgenic methods, showed that the 187 bp genomic DNA alone, approximately 305 kb downstream of the EphA7 transcription start site, was sufficient to act as the dorsal midline-specific enhancer of EphA7. Importantly, our results indicate that the 187 bp dorsal midline-specific enhancer is critically regulated by homeobox transcription factors during the development of the dien- and mesencephalon.

• BMB Reports
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• 제35권3호
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• pp.273-282
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• 2002

Sp3 is a bifunctional transcription factor that has been reported to stimulate or repress the transcription of numerous genes. Although the size of Sp3 mRNA is 4.0kb, the size of the known Sp3 cDNA sequence is 3.6kb. Thus, Sp3 functional studies have been performed with an artificially introduced start codon, and thus an amino-terminus that differs from the wild-type. Ideally, full-length cDNA expression vectors with the appropriate start codon should be utilized for these studies. Using 5'rapid amplification of cDNA ends, a full-length Sp3 cDNA clone was generated and the sequence verified in nine cell lines. No AUG initiation codon was present. However, stop codons were present in all three frames 5' to the known coding sequence. In vitro translation of this full-length cDNA clone produced the expected three isoforms-one at 100 kDa and two in the mid 60 kDa range. Electrophoretic mobility shift assays showed that the protein products had the ability to bind to the Sp1/3 consensus sequence. In vitro studies, using our Sp3 clone and site directed mutagenesis, identified the translation initiation site for the larger isoform as AUA. AUA has not been previously described as an endogenous initiation codon in eukaryotes.

• Tuberculosis and Respiratory Diseases
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• 제48권6호
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• pp.879-886
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• 2000

연구배경: 폐특이 전사조절 유전자인 Thyroid Transcription Factor-1 (TTF-1)유전자는 폐에 선택적인 유전자의 표현의 조절에 중요한 전사인자로 작용하고 폐의 발생에서 morphogenic protein으로서 작용한다. 그러나 현재까지 이 TTF-1 유전자의 전사인자에 대한 연구는 거의 미미하다. DNase 1 hypersensitive(DH) regions은 활동적인 염색체에 대한 중요한 표식자이며 유전자를 조절하는 많은 DNA sequences와 밀접한 관계가 있다. 방법 : 추정적인 distal regulatory elements를 밝혀 내기 위해서 TTF-1을 표현하는 인간의 폐선암 세포주인 NCI-H441을 사용해 DNase 1 hypersensitive site assay를 이용하였다. 결과 : TTF-1 유전자에는 전사의 시작부위에서 +150, -450, -800, 그리고 -1500 base pair부위에 4곳의 DH sites가 있음을 할 수 있었다. 결론 : 이상의 결과로 전사 조절부위가 TTF-1 유전자 내에 그리고 5' prime부위에 위치함을 추정할 수 있었다.

• 한국미생물·생명공학회지
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• 제23권2호
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• pp.131-137
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• 1995

The alkaline phosphatase (K-ALPase) gene of Kluyveromyces fragilis has been cloned (1) and determined its base sequences (2) previously in our laboratory. When the K-ALPase gene was expressed in Escherichia coli and Saccharomyces cerevisiae, it showed a constitutive activity in E. coli, and a derepressed activity in S. cerevisiae in phosphate-limited medium. Northem hybridization experiment was performed to elucidate the transcription level of the K-ALPase gene. Northern experiment showed that transcription level of K-ALPase gene in S. cerevisiae was higher in phosphate depletion, but it was higher in high phosphate medium than in phosphate limited medium in K. fragilis. The transcription initiation site of the K-ALPase gene was determined by primer extension analysis. It matched nucleotide position - 169 in relation to the putative trnslational start site.

• 한국정보과학회:학술대회논문집
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• 한국정보과학회 1999년도 가을 학술발표논문집 Vol.26 No.2 (2)
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• pp.12-14
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• 1999

Promoter는 transcript start site 앞부분에 위치하여 RNA polymerase가 높은 친화성을 보이며 바인당하는 DNA상의 특별한 부위로서 여기서부터 DNA transcription이 시작된다. function이나 tissue-specific gene들의 그룹별로 그 promoter들의 특이한 패턴들의 조합을 발견함으로써 Specific한 transcription을 조절하는 것으로 알려져 있어 promoter로 인한 그 gene의 정보를 어느 정도 알 수가 있다. 사람의 housekeeping gene promoter들을 EPD(eukaryotic promoter database)와 EMBL nucleic acid sequence database로부터 수집하여 이것들 간에 의미 있게 나타나는 모든 패턴들을 optimization algorithm으로 알려진 genetic algorithm을 이용해서 찾아보았다.

• Journal of Microbiology and Biotechnology
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• 제5권5호
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• pp.245-249
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• 1995

Expression of the adjacent but divergently transcribed glpD and glpE gene is positively regulated by cAMP-CRP. In this study, we constructed several mutants in which a CRP-binding site is placed at different distances upstream of the glpD promoter. The effect of the spacer length on transcription activation by cAMP-CRP was tested in vivo by $\beta$-galactosidase. The cAMP-CRP complex activated transcription from glpD when bound at a number of positions, all of which lay on the same face of the DNA helix, although the degree of activation varied with the length of the spacer. By contrast, the insertion of spacer length with non-integral turns of the DNA helix extremely inhibited the activation of transcription. The observed transcription activation by cAMP of the glpD promoter was influenced by the distance between the CRP binding site and the transcription start point.