• BMB Reports
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• v.35 no.3
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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.

• Molecules and Cells
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• v.26 no.2
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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.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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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.

• Journal of Microbiology
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• v.34 no.1
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• pp.68-73
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• 1996

An nprX gene of Bacillus subtilis NS15-4 encoding a neutral protease was cloned and its molecular characteristics were analyzed. The complete nucleotide sequence indicated that there is an open reading frame (0RF) possibly encoding 521 amino acid polypeptide. The ORF used all codons expected two cysteine and a proline having a codon bias index (CBI) of 0.09 in Escherichia coli. There were homologous sequences to the consensus sequence of -35 and -10 regions of E. coli promoters and to a Shine-Dalgarno (SD) sequence located 25 bp downstream of a mojor transcription initiation site. Moreover, there were also five minor transcription initiation sites at 6. 7. 8. 14 and 15 nt downstream of the major site. Northern blot analysis revealed the presence of about 1.8 kb mRNA transcript in E. coli having the nprX gene. The nucleotide sequence was identified in GenBank to be a gene for a neutral protease of B. sutilis with six nucleotide difference in the ORF region. The flanking regions of the NprX ORF showed much more differences form those of other neutral protease genes except the nprE gene of B. subtilis, which has the most homology to the nprX gene, and of which the flanking regions were identical to those of the nprX gene.

• Korean journal of applied entomology
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• v.34 no.2
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• pp.100-105
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• 1995

A whole body extract (WBE), a crude nuclear fraction, of aphids was prepared and used to identify the proteins which bound specifically to 5'-upstream regions of the transcription initiation site of the aphid ribosomal RNA gene (rDNA). While DNA fragment (-263/-195) was bound by only one specific 53 kDa protein, two DNA fragments, A(-194/23) and B(-393/-264), were commonly bound by three proteins (52 kDa, 50 kDa and 40 kDa). It was also revealed that the formation of he DNA-protein complex requires a cation.

• Journal of Microbiology and Biotechnology
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• v.3 no.4
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• pp.224-231
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• 1993

The nucleotide sequence of the xylanase gene (xynY) from alkalophilic Bacillus sp. YC-335 was determined and analyzed. An open reading frame of 1, 062 base pairs for xynY gene was observed and encoded for a protein of 354 amino acids with a molecular weight of 38, 915. S1 nuclease mapping showed that the transcription initiation sites of the xynY gene were different in Bacillus sp. YC-335 and Escherichia coli HB101 (pYS55). S1 mapping also showed that -10 region of the xynY gene recognized by RNA polymerases of E. coli and Bacillus sp. YC-335 were TACAGT and TATGAT , respectively. A ribosome binding site sequence with the free energy of -17.0 Kcal/mol was observed 9 base pairs upstream from the unusual initiation codon, TTG. The proposed signal sequence consisted of 27 amino acids, 2 of which were basic amino acid residues and 21 were hydrophobic amino acid residues. When the amino acid sequences of xylanases were compared, Bacillus sp. YC-335 xylanase showed more than 50% homology with xylanases from B. pumilus, B. subtilis, and B. circulans.

• Journal of Life Science
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• v.14 no.5
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• pp.732-740
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• 2004

MCM proteins are essential for eukaryotic DNA replication, playing roles in the initiation and elongation of DNA replication. MCM proteins expression is much higher in malignant tissues than normal tissues. Several reports have indicated the usefulness of MCM proteins as markers of cancer cells in histopathological diagnosis. However, the cause of enhanced expression of MCM proteins in cancer cells remain to be clarified. The purpose of this study is to examine the relative transcriptional activities of human mcm gene promoters in cancer and normal cells. The minimal promoter region required for transcription of a luciferase reporter gene was contained an E-box and one E2F site. In addition, luciferase activities from mcm7 and mcm2 promoter/luciferase gene reporter constructs were significantly increased in cancer cells at 8 times compared with normal cells. E-box and E2F binding site in the promoter of mcm genes are responsible for different mechanism of transcription regulation on the cellular environment.

• Journal of Life Science
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• v.9 no.2
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• pp.52-56
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• 1999

The Raf, a cytoplasmic serine/thereonine protein kinase, acts as an important mediator of signals involving cell proliferation, differentiation and development. Multiple regulatory elements should participate in the expression of D-raf, Drosophila homolog of human c-raf-1. In order to search regulatory factors involved in the D-raf promoter activation, we accomplished the yeast one-hybrid screening using D-raf promoter region from bp-330 to -309 with respect to the transcription initiation site as bait. After screening, sixteen independent positive clones of ${\beta}$-galactosidase activties were identified and sequenced. Two clones having 94-98% identity with daughterless and one clone having 93% identity with escargot by Blast search among these clones were screened.

• Journal of Microbiology and Biotechnology
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• v.3 no.3
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• pp.139-145
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• 1993

The nucleotide sequence of the xylanase gene (xynS) from alkali-tolerant Bacillus sp. YA.14 was determined and analyzed. A 639 base pairs open reading frame for xynS gene was observed and encoded for a protein of 213 amino acids with a molecular weight of 23, 339. S1 nuclease mapping showed that the transcription initiation site of the xynS gene did not exist in the cloned DNA. Ribosome binding site sequence with the free energy of -18.8 Kcal/mol was observed 8 base pairs upstream from the initiation codon, ATG. The proposed signal sequence consisted of 28 amino acids, of which 3 were basic amino acid residues and 21 were hydrophobic amino acid residues. When the amino acid sequences of xylanases were compared, Bacillus sp. YA-14 xylanase showed 48% homology with Bacillus sp. YC-335 xylanase and 96% homology with xylanases from B. subtilis and B. circulans.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.111-116
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• 2003

The xylA gene of Bacillus stearothermophilus No. 236 encoding $\beta$-xylosidase was cloned and sequenced previously. The transcriptional start site of the xylA gene cloned in E. coli was identified to be the guanine (G) by primer extension analysis. This supports that the expression of xylA gene is also directed in the E. coli cells by the previously determined transcription initiation signals, -10 sequence (CATAAT) and -35 sequence (TTGTTA) separated by 12 bp. To increase the expression of $\beta$-xylosidase, firstly the spacer region of xylA promoter was extended from 12 to 17 bp, and then the -10 and -35 elements were converted into their respective consensus sequences. The mutant promoters thus obtained were tested for their activities in both the E. coli and B. subtilis host cells. The change of the length of the spacer region from 12 to 17 bp resulted in a 1.6- and 2.5-fold increase in promoter strength in comparison with the wild type promoter in E. coli and B. subtilis cells, respectively. Also, strength of the promoter with the fourth T to A transversion on its -35 element increased in the transcription level by about 35 times compared with that of wild-type promoter. However, surprisingly the 5' end C-to-T transition of the -10 hexamer showed a 5- to 15-fold reduction in $\beta$-xylosidase activity in both E. coli and B. subtilis. Together, the present data demonstrated that the 5' end nucleotide C of the -10 sequence CATAAT and the fourth nucleotide A of the -35 hexamer are two most critical nucleotides for the promoter activity in the context of the xylA promoter.