• Asian-Australasian Journal of Animal Sciences
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• 제25권3호
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• pp.421-427
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• 2012

The production of therapeutic proteins from transgenic animals is one of the most important successes of animal biotechnology. Milk is presently the most mature system for production of therapeutic proteins from a transgenic animal. Specifically, ${\beta}$-casein is a major component of cow, goat and sheep milk, and its promoter has been used to regulate the expression of transgenic genes in the mammary gland of transgenic animals. Here, we cloned the porcine ${\beta}$-casein gene and analyzed the transcriptional activity of the promoter and intron 1 region of the porcine ${\beta}$-casein gene. Sequence inspection of the 5'-flanking region revealed potential DNA elements including SRY, CdxA, AML-a, GATA-3, GATA-1 and C/EBP ${\beta}$. In addition, the first intron of the porcine ${\beta}$-casein gene contained the transcriptional enhancers Oct-1, SRY, YY1, C/EBP ${\beta}$, and AP-1, as well as the retroviral TATA box. We estimated the transcriptional activity for the 5'-proximal region with or without intron 1 of the porcine ${\beta}$-casein gene in HC11 cells stimulated with lactogenic hormones. High transcriptional activity was obtained for the 5'-proximal region with intron 1 of the porcine ${\beta}$-casein gene. The ${\beta}$-casein gene containing the mutant TATA box (CATAAAA) was also cloned from another individual pig. Promoter activity of the luciferase vector containing the mutant TATA box was weaker than the same vector containing the normal TATA box. Taken together, these findings suggest that the transcription of porcine ${\beta}$-casein gene is regulated by lactogenic hormone via intron 1 and promoter containing a mutant TATA box (CATAAAA) has poor porcine ${\beta}$-casein gene activity.

• Korean Journal of Veterinary Service
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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.

• Clinical and Experimental Pediatrics
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• 제51권2호
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• pp.150-155
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• 2008

Purpose : It has been known that breast milk cause prolonged unconjugated hyperbilirubinemia. UGT1A1 is a important gene of uridine diphosphate glucuronosyltransferase (UGT) which has a major role of bilirubin metabolism. These findings suggest that there is a relationship between UGT1A1 gene mutation and prolonged jaundice of breast feeding infant. The aim of study was to investigate whether a polymorphism of the UGT1A1 gene exist in prolonged hyperbilirubinemia of breast milk feeding Korean infant. Methods : The genomic DNA was isolated from 50 full term Korean neonates, who had greater than a 10 mg/dL of serem bilirubin after 2 weeks of birth with no significant cause, and the other genomic DNA was isolated from 162 full term Korean neonates of the control population. Both group fed breast milk. We performed direct sequencing of TATA box and Gly71Arg polymorphism of the UGT1A1 gene. Results : Two of the 50 neonates with hyperbilirubinemia had AA polymorphism, and 40 had GA polymorphism. Five of the 129 neonates of the control group had AA polymorphism, and 4 had GA polymorphism. The allele frequency of G>A polymorphism in the hyperbilirubinemia group was 44.0%; it was significantly higher than 5.4% of the control group. TATA box polymorpism was not different both group significantly. Conclusion : Our result indicated that Gly71Arg polymorphism is associated with the prolonged hyperbilirubinemia of breast milk-feeding infant in Korean, while TATA box polymorphism is not associated with the prolonged hyperbilirubinemia of breast milk-feeding infant in Korean.

• Journal of fish pathology
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• 제8권1호
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• pp.37-46
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• 1995

The amino acid analysis of polyhedrin protein and nucleotide sequence of polyhedrin gene in H. cunea nuclear polyhedrosis virus (HcNPV) genome have been studied. Polyhedrin had three polypeptide bands in SDS - polyactylamide gel electrophoresis. The major polypeptide had a molecular weight of 25 kd. The polyhedrin was composed of 17 different amino acids. HcNPV DNA was digested with EcoRI restriction enzyme and hybridized with ($\alpha^{32}P$) -labelled AcNPV polyhedrin gene cDNA. The polyhedrin gene was located on the fragment of EcoRI-H. The EcoRI - H fragment containing polyhedrin gene was cloned into the EcoRI site of pUC8 vector which was confirmed with southern blotting, and the recombinant plasmid containg polyhedrin gene was designated as hPE-H. The promoter region of polyhedrin genomic DNA was sequenced. The sequences identified as the TATA box was found at the 5' flanking region of the polyhedrin genomic DNA approximately -79 bp upstream from the transcriptional start site. But CAAT-like box was not shown near the TATA-like box in the polyhedrin gene. Four tandem repeats with the sequence 5' -CTAATAT-3' and 5'-TAAATAA-3' were found between -141 and -108 or -83 upstream and -52 bp downstream from the translation start site. About -141 bp region upstream from the translational start site was highly AT (78%) rich. The coding region for the polyhedrin starts and ends with ATG and TAA, respectively.

• Korean Journal of Plant Tissue Culture
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• 제24권1호
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• pp.1-35
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• 1997

Fertilized egg, by successive cell divisions, differentiates into different tissues and organs with various structures and functions. Different cells and tissues contain different proteins, products of selective gene expression. Not all the genes in any genomes are equally active, temporal and spatial gene expression being the general rule. Present paper attempts to review the tanscriptional mechanisms or the initiations of transcription from several angles. In some of the organisms the genes in the process of transcription or the genes in the inactive state can be seen under the light microscope. Some bands of Drosophila polytene chromosomes may exhibit a swollen or puff appearance under certain conditions. A puff, unfolded or decondensed form of chromomere, represents sets of intense transcriptional activity or RNA synthesis. The heterochromatic X chromosome whose genes remain inactive in the female mammals can be visualized as a dark staining structure called Barr body, Configuration of chromatin differs between transcribed and nontranscribed chromatin. Modification to the chromatin facilitates RNA synthesis. The movement of large polymerase molecule along the DNA would probably be facilitated if some modifications of the chromatin configuration is effected. Methylation of cytosines in CG sequences is associated with inactive genes. Methylation can play a role in determination of mammalian cells during embryogenesis. Demethylation is necessary for the gene to be expressed during development A histone modification that is also known to be correlated with transcriptional capacity of chromatin is acetylation of the lysine residues of the core histones. Chromatin containing a high level of histone acetylation is very sensitive to DNase 1. For the transcription to occur TBP must first bind to the TATA box. Another TF, TF IIB, then binds to the promoter-TBP complex, facilitating the access of RNA polymerase to the transcription initiation site. As recently as eight years ago researchers assumed that histones were irrelevant to the regulation of gene expression. Histones combine with the DNA to form nucleosome of the chromatin. Histones are vital participant in gene regulation. Histone and basal factors compete for access to TATA box. When DNA is exposed to basal factors before histones are introduced, the basal factors assemble on TATA boxes preventing the access of histones, allowing transcription to occur, for transcription to begin, activator protein at the upstream activation sequence or enhancer must interact with the tail of histone H4 at TATA box and cause the histone role particle to dissociate from the TATA box leading to partial breakup of the histone core particle and allowing the basal factors to bind to the TATA box. New concept of genomic flux in contrast to the old concept of static genome has been developed based on the powerful new molecular techniques. Genomic changes such as repetitive DNAs and transposable elements, it is assumed but not yet proved, may affect some of the developmental patterns that characterize particular cells, tissues, organs, and organisms. In the last decade or so remarkable achievement have been made in the researches of the structures and functions of TFs and the specific target sequences located in promoters or enhancers where these TFs bind. TFs have independent domains that bind DNA and that activate transcription. DNA binding domain of TFs serves to bring the protein into the right location. There are many types of DNA binding domains. Common types of motifs can be found that are responsible for binding to DNA. The motifs are usually quite short and comprise only a small part of the protein structure. Steroid receptors have domains for hormone binding, DNA binding, and activating transcription. The zinc finger motif comprises a DNA binding domain. Leucine zipper consist of a stretch of amino acids with a leucine residue in every seventh position Two proteins form a dimer because they interact by means of leucine zippers on similar α-helical domain. This positions their DNA binding basic domains for interaction with the two halves of a DNA sequence with dyad symmetry of TGACTCA, ACTGAGT.

• Korean Journal of Plant Tissue Culture
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• 제28권5호
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• pp.255-261
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• 2001

Vitis vinifera thaumatin-like protein (VVTL1) is a fruit-specific and ripening-related protein in grape. In order to isolate VVTL1-homolog gene and fruit-specific promoter from Campbell cultivar, we isolated a genomic clone containing VVTL1-homolog gene from grape genomic library through plaque hybridization. VVTL1-homolog gene has an intronless genomic structure, which the pattern is matched with those of other PR5 genes such as osmotin and osmotin-like protein genes. Transcription start site was determined by primer extension analysis. The promoter region of VVTL1-homolog gene contains a sequence or structure, especially the location and number of TCA box and ABRE (abscisic acid-responsive element), distinct from other reported plant PR5 genes, though with several known functional elements such as a TATA box and CAAT box. These results suggested that VVTL1-homolog gene may be regulated by a plant hormone, abscisic acid, and one or several stresses such osmotic pressure and pathogen infection. The isolation of fruit-specific promoter may be helpful to breed a genetically modified grape with valuable phenotype or materials in fruits.

• IMMUNE NETWORK
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• 제2권3호
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• pp.150-157
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• 2002

Background: Although Tat plays a role as a potent transactivator in the viral gene expression from the Human Immunodeficiency Virus type 1 long terminal repeat (HIV-1 LTR), it does not function efficiently in rodent cells implying the absence of a human specific factor essential for Tat-medicated transactivation in rodent cells. In previous experiments, we demonstrated that one of chimeric forms of TAR (transacting responsive element) of HIV-1 LTR compensated the restriction in rodent cells. Methods: To characterize the nature of the compensation, we tested the effects of several upstream binding factors of HIV-1 LTR by simple substitution, and also examined the role of the configuration of the upstream binding factor(s) indirectly by constructing spacing mutants that contained insertions between Sp1 and TATA box on Tat-mediated transactivation. Results: Human Sp1 had no effect whereas its associated factors displayed differential effects in human and rodent cells. In addition, none of the spacing mutants tested overcame the restriction in rodent cells. Rather, when the secondary structure of the chimeric HIV-1 TAR construct was destroyed, the compensation in rodent cells was disappeared. Interestingly, the proper interaction between Sp1 and TATA box binding proteins, which is essential for Tat-dependent transcription, was dispensable in rodent cells. Conclusion: This result suggests that the human-specific Tat cofactor acts to allow Tat to interact effectively in a ribonucleoprotein complex that includes Tat, cellular factors, and TAR RNA, rather than be associated with the HIV-1 LTR upstream DNA binding factors.

• BMB Reports
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• 제39권4호
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• pp.355-360
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• 2006

To gain a better understanding on the function of the potato Solanum tuberosum Multiprotein Bridging Factor 1 protein (StMBF1) its interaction with the TATA box binding protein (TBP) was demonstrated. In addition we reported that StMBF1 rescues the yeast mbf1 mutant phenotype, indicating its role as a plant co-activator. These data reinforce the hypothesis that MBF1 function is also conserved among non closely related plant species. In addition, measurement of StMBF1 protein level by Western blot using anti-StMBF1 antibodies indicated that the protein level increased upon $H_2O_2$ and heat shock treatments. However, the potato $\beta$-1,3-glucanase protein level was not changed under the same experimental conditions. These data indicate that StMBF1 participates in the cell stress response against oxidative stress allowing us to suggest that MBF1 genes from different plant groups may share similar functions.

• The Korean Journal of Mycology
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• 제28권1호
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• pp.26-31
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• 2000

We isolated genomic clone of FVFD16 and FVFD30 gene specifically expressed during fruit body formation of Flammulina velutipes [(Curt: Fr.) Sing] and determinated the sequences. The FVFD16 gene is including two introns in open reading frame, and FVFD30 gene is including four introns. The introns were matched GT/AG rule. The FVFD16 and FVFD30 genes contained CAAT box with similarity arrange and TATA box. CT-rich region was presented before the transcription start point. FVFD30 gene is investigated that expected the most activity of CCACC arrange. The result of FVFD16 gene analysis showed 80% homology by cDNA clone that is gene family. From the results of genomic southern blot analysis, we presumed more than two copy number gene family of FVFD16 and FVFD30 gene.

• Bulletin of the Korean Chemical Society
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• 제35권5호
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• pp.1279-1284
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• 2014

The binding of TATA-binding protein (TBP) to the TATA-box containing promoter region is aided by many other transcriptional factors including TFIIA and TFIIB. The mechanistic insight into the assembly of RNA polymerase II preinitation complex (PIC) has been gained by either directly altering a function of target protein or perturbing molecular interactions using drugs, RNAi, or aptamers. Aptamers have been found particularly useful for studying a role of a subset of PIC on transcription for their ability to inhibit specific molecular interactions. One major hurdle to the wide use of aptamers as specific inhibitors arises from the difficulty with traditional assays to validate and determine specificity, affinity, and binding epitopes for aptamers against targets. Here, using a technique called the bio-layer interferometry (BLI) designed for a label-free, real-time, and multiplexed detection of molecular interactions, we studied the assembly of a subset of PIC, TBP binding to TATA DNA, and two distinct classes of aptamers against TPB in regard to their ability to inhibit TBP binding to TFIIA or TATA DNA. Using BLI, we measured not only equilibrium binding constants ($K_D$), which were overall in close agreement with those obtained by electrophoretic mobility shift assay, but also kinetic constants of binding ($k_{on}$ and $k_{off}$), differentiating aptamers of comparable KDs by their difference in binding kinetics. The assay developed in this study can readily be adopted for high throughput validation of candidate aptamers for specificity, affinity, and epitopes, providing both equilibrium and kinetic information for aptamer interaction with targets.