• Proceedings of the Korean Society of Plant Biotechnology Conference
• /
• 2003.04a
• /
• pp.151-151
• /
• 2003

• Journal of Microbiology and Biotechnology
• /
• v.25 no.8
• /
• pp.1241-1245
• /
• 2015

The aim of the present work was to examine the putative promoter region of the operon ansPAB and to determine the general elements required for the regulation of transcriptional activity. The transcriptional start site of the ansPAB promoter was determined by using highresolution S1-nuclease mapping. Sequence analysis of this region showed -10 and -35 elements, which were consistent with consensus sequences for R. etli promoters that are recognized by the major form of RNA polymerase containing the σ⁷⁰ transcription factor. Mutation studies affecting several regions located upstream of the transcriptional start site confirmed the importance of these elements on transcriptional expression.

• Journal of Microbiology
• /
• v.39 no.1
• /
• pp.37-41
• /
• 2001

The fission yeast Schizosaccharomyces pombe contains two distinct superoxide dismutase (SOD) activities, one in the cytosol encoded by the $sod2^{+}$ gene and the other in mitochondria. The $sod2^{+}$ gene encoding putative mitochondrial manganese superoxide dismutase (MnSOD) was isolated from the S. pombe genomic library using a PCR fragment as the probe. The nucleotide sequence of the $sod2^{+}$ gene and its flanking region (4051 bp HindIII fragment) was determined. An intron of 123 nt in size was predicted and confirmed by sequencing the cDNA following reverse transcription PCR. The predicted Sod2p consists of 218 amino acid residues with a molecular mass of 24,346 Da. The deduced amino acid sequence showed a high degree of homology with other MnSODs, especially in the metal binding residues at the active site and their relative positions. The transcriptional start site was mapped by primer extension at 231 at upstream from the ATG codon. A putative TATA box(TATAAAA) was located 58 nt upstream from the transcriptional start site and putative polyadenylation sites were located at 1000, 1062, and 1074 nt downstream from the ATG start codon.

• Journal of Microbiology and Biotechnology
• /
• v.15 no.4
• /
• pp.749-755
• /
• 2005

The ccpA gene encoding catabolite control protein A (CcpA) of Leuconostoc mesenteroides SYl, a strain isolated from kimchi, was cloned, sequenced, analyzed for transcript, and overexpressed in Escherichia coli. The ccpA ORF (open reading frame) is 1,011 bp in size, which can encode a protein of 336 amino acid residues with a molecular mass of 36,739 Da. The transcription start site was mapped at a position 49 nucleotides upstream of the start codon, and promoter sequences were also identified. The putative cre site overlapped with the -35 promoter sequence. The deduced amino acid sequence of the CcpA contained the helix-turn-helix motif found in many DNA-binding regulatory proteins. CcpA from 1. mesenteroides SY1 had $54.6\%$ identity with CcpA from Lactobacillus casei. The Northern blot experiment showed that ccpA was transcribed as a single 1.1 kb transcript, and transcription was repressed when grown on media containing glucose. CcpA was overproduced in E. coli BL21(DE3) cells using the pET expression vector, and purified to an apparent homogeneity. Gel Mobility Shift Assay with purified CcpA and a DNA fragment containing the ere sequence of the $\alpha$-galactosidase gene (aga) from L. mesenteroides SY1 revealed that CcpA bound specifically to the cre site of aga.

• Asian-Australasian Journal of Animal Sciences
• /
• v.31 no.3
• /
• pp.449-456
• /
• 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.

• Korean Journal of Plant Tissue Culture
• /
• v.25 no.4
• /
• pp.237-243
• /
• 1998

To understand the tissue specific expression pattern of S RNase genes associated with self-incompatibility in L. peruvianum, two promoter regions of $S_{11}$ and $S_{12}$ RNase genes were compared. Homologous sequences between two S RNase gene promoters were found within 300 bp upstream of transcription start site. Moreover short direct repeat sequences within $S_{11}$ RNase gene promoter existed in the vicinity of 350-500 bp upstream of transcription start site. To identify whether the unique promoter sequences of $S_{11}$ RNase gene confer the tissue specific expression, six deletion fragments for $S_{11}$ genomic gene promoter constructed by PCR were fused to $\beta$-glucuronidase gene, and introduced into various tissues of L. peruvianum by microprojectile bombardment. Transient expression assays indicated that $S_{11}$ RNase gene promoter contained the positive and negative regulatory sequences, which can control the floral tissue-specific expression in L. peruvianum.

• Journal of Life Science
• /
• v.14 no.6 s.67
• /
• pp.1009-1017
• /
• 2004

Sialyltransferases cloned so far show the remarkable tissue-specific expression, which is correlated with the existence of cell type-specific sialylated sugar structure in glycoconjugates. In the previous studies, we found various mRNA isoforms of human sialyltransferases generated by alternative splicing and alternative promoter utilization. To understand the regulatory mechanisms for specific expression of human sialyltransferase genes and for production of their mRNA isoforms, in this study, we have isolated and characterized five kinds of human sialyltransferase genes: hST3Gal II, hST8Sia II, hST8Sia III, hST8Sia IV, and hST8Sia V. The hST3Gal II gene is composed of six exons, which span over 17kb, with exons ranging in size from 46 to over 1017 bp. The hST8Sia III gene comprises over 10 kb, and consists of only four exons, which is much smaller and simpler than other human sialyltransferase genes. In contrast, three genes (hST8Sia II, hST8Sia IV and hST8Sia V) span more than 70 kb, and comprise five or more exons. All exon-intron boundaries follow the GT-AG rule. In particular, the sialylmotif L, which is a highly conserved region in all cloned sialyltransferases, was found in one exon of hST8Sia III, whereas this motif is encoded by discrete exons in the other human sialyltransferases. Exon structures of these sialyltransferase genes show the structural diversity, as found in other human sialyltransferase genes reported so far. We determined the transcription start site of hST3Gal II gene by the 5'-RACE and cap site hunting experiments.

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

• Korean Journal of Microbiology
• /
• v.31 no.4
• /
• pp.267-273
• /
• 1993

We screened promoters inducible by superoxide radical from Escherichia coli. For this. we constructed random promoter library from E. coli MG 1655 using a promoter-probing plasmid. pJAC4. Six hundred and sixty clones in this library were classified based on their promoter strength by ampicillin gradient plate assay. Three hundred and eighty three clones with relatively weak to medium promoter strength were selected and then screened for their inducibility by superoxide radical on ampicillin gradient plate containing paraquat. Three clones (clones 5. 15 and 34) were detected to be induced by paraquat treatment and the level of induction were between 1.4 and 4 folds. Comparison of nucleotide sequences of the cloned promoter fragment with registered sequences in GENBANK and EMBL databases suggests that the cloned DNA fragments have not been yet characterized in E. coli. Transcription start sites in these clones were determined by rrimer extension and S I nuclease protection analysis. S 1 analysis of clones 5 and IS indicated that the mRNA levels were increased by paraquat treatment. Especially. clone 5 \vas found to have two transcription start sites. the upstream start site of which was selectively used by paraquat treatment. Searching for promoter clements. we found that only the downstream promoter of clone 5 has -10 and - 35 promoter elements recognized by RNA polymerase ($E\sigma^{70}$) and the others have no conserved promoter elements. This suggests that these superoxideinducible promoters may require transcription initiation protein(s) other than $E\sigma^{70}$.

• BMB Reports
• /
• v.43 no.2
• /
• pp.110-114
• /
• 2010

The yeast three-hybrid system (Y3H), a powerful method for identifying RNA-binding proteins, still suffers from many false positives, due mostly to RNA-independent interactions. In this study, we attempted to efficiently identify false positives by introducing a tetracycline operator (tetO) motif into the RPR1 promoter of an RNA hybrid expression vector. We successfully developed a tight tetracycline-regulatable RPR1 promoter variant containing a single tetO motif between the transcription start site and the A-box sequence of the RPR1 promoter. Expression from this tetracycline-regulatable RPR1 promoter in the presence of tetracycline-response transcription activator (tTA) was positively controlled by doxycycline (Dox), a derivative of tetracycline. This on-off control runs opposite to the general knowledge that Dox negatively regulates tTA. This positively controlled RPR1 promoter system can therefore efficiently eliminate RNA-independent false positives commonly observed in the Y3H system by directly monitoring RNA hybrid expression.