• Molecules and Cells
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• v.26 no.6
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• pp.536-547
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• 2008

Anthocyanidin synthase (ANS, leucoanthocyanidin oxygenase), a 2-oxoglutarate iron-dependent oxygenase, catalyzed the penultimate step in the biosynthesis of the anthocyanin class of flavonoids, from the colorless leucoanthocyanidins to the colored anthocyanidins. The full-length cDNA and genomic DNA sequences of ANS gene (designated as GbANS) were isolated from Ginkgo biloba for the first time. The full-length cDNA of GbANS contained a 1062-bp open reading frame (ORF) encoding a 354-amino-acid protein. The genomic DNA analysis showed that GbANS gene had three exons and two introns. The deduced GbANS protein showed high identities to other plant ANSs. The conserved amino acids (H-X-D) ligating ferrous iron and residues (R-X-S) participating in 2-oxoglutarate binding were found in GbANS at the similar positions like other ANSs. Southern blot analysis indicated that GbANS belonged to a multi-gene family. The expression analysis by real-time PCR showed that GbANS expressed in a tissue-specific manner in G. biloba. GbANS was also found to be up-regulated by all of the six tested abiotic stresses, UV-B, abscisic acid, sucrose, salicylic acid, cold and ethylene, consistent with the promoter region analysis of GbANS. The recombinant protein was successfully expressed in E. coli strain with pET-28a vector. The in vitro enzyme activity assay by HPLC indicated that recombinant GbANS protein could catalyze the formation the cyanidin from leucocyanidin and conversion of dihydroquercetin to quercetin, suggesting GbANS is a bifunctional enzyme within the anthocyanidin and flavonol biosynthetic pathway.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.15
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• pp.6769-6772
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• 2015

Background: High risk human papillomaviruses (HPVs) are the leading cause of cervical cancer (CC) and Pap smear screening has not been successful in preventing CC in Tunisia. HPV vaccination that targets HPV16 and 18 offers a new efficient prevention tool. Identification of HPV types in CC is thus essential to determine the impact of HPV vaccine implementation. The aim of this study is to provide specific data from Tunisia. Materials and Methods: A total of 89 histological confirmed paraffin embedded samples isolated from patients with CC diagnosed between 2001 and 2011 were collected from five medical centres from Northern and Southern Tunisia. HPV DNA was detected using a nested PCR (MY09/MY11-GP5+/GP6+) and genotyping was assessed using a reverse blot line hybridisation assay that enables the detection of 32 HPV types. Results: HPV DNA was detected in all samples. Twelve high risk types were detected; HPV16 and/or 18 were predominant, accounting together for 92.1% of all the CC cases (HPV16: 83.1%). Single infections accounted for 48.8% of the cases and were mostly linked to HPV 16 (32.6%) and less frequently to HPV 18 (2.4%). The other high risk HPV single infections were linked to HPV 35 (4.6%), 45 (4.6%), 58 (2.3%) and 59 (2.3%). Multiple infections with mixing of 2 to 4 genotypes predominately featrued HPV16 and/or 18 with HPV 35 and 45 (96.6 %) and less frequently with HPV 59, 40, 66, 73 and 58. There was no statistically significant variation in the relative distribution of HPV types with age. Conclusions: These results strongly indicate that prophylactic HPV vaccines can have a major impact in preventing CC in Tunisia.

• Korean journal of applied entomology
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• v.32 no.3
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• pp.300-306
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• 1993

The CryIIA gene encoding the insecticidal crystal protein of Bacillus thuringiens!s subsp. kurstalri HD-l has been cloned in Escherichia col!, and its nucleotide sequences were determined completely. 5kb Hindlli fragment harboring CryIIA gene was screened in the large ca. 225kb plasmid DNA by southern blot. HindlIT digested 5kb fragment was ligated into pUC19 and transformed in E. coli. The 4kb BamHI-HindlIT fragment containing the CryIIA gene was subcloned and named pSKIIA. DNA sequence analysis demonstrates that pSKIIA is the gene of an operon which is comprised of Lhree open reading frames (designated orn, orf2 and or£3). The CrylIA gene is composed of 3,952bp-long BamHI-Hindill DNA restriction fragment. The orf3 code for a polypeptide of 633 amino acid residues. The protoxin protein has a predicted molecular weight of 70,780. The E. coli derived protoxin gene product is biologICally active against three species of Lepidopteran (Plu.lelia maculipennis, He/iolhis assulta, Spodoptera litura) and a species of Dip Leran( Culex pipines) larvae in bioassay.

• Korean Journal of Veterinary Pathology
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• v.3 no.1
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• pp.15-25
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• 1999

To elucidate the mechanism of age-related development in FGS/NgaKIST mice with spontaneous glomerulosclerotic lesion, we examined expression and localization of various cytokine mRNA in the kidney in the progression of diseases. This mouse model is the first to develop spontanously occuring glomerosclerotic lesion in the kidney. In this study, we detected the up-regulation of local cytokine genes such as IL-1$\beta$, IL-2, IL-6, IL-10, TNF-$\alpha$, TGF-$\beta$, and IFN- $\gamma$ in the kidneys. In RT-PCR and Southern blot analysis, we detected gradual expressions of cytokine mRNA of IL-1$\beta$, IL-2, IL-6, IFN- $\gamma$, and TNF $\alpha$ mRNA during the course of disease. Other cytokines including IL -10 and TGF -$\beta$ were found to be appeared the slightly expressed level at 3 to 12 weeks before onset of inflammatory lesion but they are highly expressed at the end-stage of the disease accompaning high proteinurea and wasting. In situ RT-PCR, each cytokine mRNA were specifically localized in a variety of cells including mesangial, endothelial, parietal epithelial, tubular epithelial, arterial muscle cell, and infiltrated inflammatory cells. In addition, TNF - $\alpha$was detected moderately in the visceral and parietal epithelial cell, but weakly in endothelial and mesangial cells, whereas IL-1 $\beta$ and IL -6 were strong in mesangial regions. IL-6 and TNF- $\alpha$ was highly localized in the damaged proximal and collecting tubules. Especially, TGF -$\beta$ mRNA was highly found in mesangial cells within glomerulus and interstitium during the end-stage of this disease.. These results indicate that pro inflammatory cytokines such as IL-1 $\beta$, IL-2, IL-6, and TNF- $\alpha$ were gradually expressed from the early stage of this disease to the end-stage, and that IL-10 and TGF-$\beta$ may be important in the accumulation of extracellular matrix(ECM) within glomerulus and periglomerular fibrosis in the progression of this disease as well as tissue destruction in end-stage of this disease.

• BMB Reports
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• v.41 no.2
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• pp.112-118
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• 2008

Camptothecin is an anti-cancer monoterpene indole alkaloid. The gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (designated as CaHDR), the last catalytic enzyme of the MEP pathway for terpenoid biosynthesis, was isolated from camptothecin-producing Camptotheca acuminata. The full-length cDNA of CaHDR was 1686 bp encoding 459 amino acids. Comparison of the cDNA and genomic DNA of CaHDR revealed that there was no intron in genomic CaHDR. Southern blot analysis indicated that CaHDR belonged to a low-copy gene family. RT-PCR analysis revealed that CaHDR expressed constitutively in all tested plant organs with the highest expression level in flowers, and the expression of CaHDR could be induced by 100 ${\mu}M$ methyl-jasmonate (MeJA), but not by 100 mg/L salicylic acid (SA) in the callus of C. acuminata. The complementation of CaHDR in Escherichia coli ispH mutant MG1655 demonstrated its function.

• Korean Journal of Medicinal Crop Science
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• v.21 no.1
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• pp.32-38
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• 2013

FPS (farnesyl diphosphate synthase) plays an essential role in organ development in plants. However, FPS has not previously been identified as a key regulatory enzyme in triterpene biosynthesis. In order to investigate the effect of FPS on ginsenosides biosynthesis, we over-expressed FPS of Centella asiatica (CaFPS) in Panax giseng adventitious roots. PCR analysis showed the integrations of the CaFPS and hygromycin phosphotransferase genes and we ultimately selected three lines. The result of Southern blot analysis demonstrated the introduction of the CaFPS gene into genome of ginseng. In addition, the results of RT-PCR analysis revealed that CaFPS gene overexpression induced an accumulation of its transcription in the ginseng adventitious roots. To determine whether or not the overexpression of the CaFPS gene contributes to the downstream gene expression associated with triterpene biosynthesis, the level of mRNAs was analyzed by real-time PCR. The result showed that no differences were detected in any expression of all genes. To determine quantitatively the content of ginsenosides in transgenic ginseng adventitious roots, HPLC analysis was conducted. The content of total 7 ginsenosides was increased to 1.8, 1.4, and 1.7 times than that of the controls, respectively. This indicated that the overexpression of CaFPS in ginseng adventitious roots causes an increase in ginsenoside content, although down stream genes of FPS gene were suppressed by CaFPS overexpression.

• Korean Journal of Microbiology
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• v.45 no.2
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• pp.215-221
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• 2009

Site-specific incorporation of unnatural amino acids into proteins in vivo can be achieved by co-expression of an orthogonal pair of suppressor tRNA and engineered aminoacyl-tRNA synthetase (ARS) that specifically ligates an unnatural amino acid to the suppressor tRNA. As a step to establish this technique, here we generated an Escherichia coli reporter strain DH10B(Tn:lacZam) by integrating amber mutated lacZ gene into the chromosome of E. coli DH10B strain. In vivo expression of E. coli amber suppressor $tRNA^{Gln}$ produced blue colonies in culture plates containing X-Gal as well as dramatically increased $\beta$-galactosidase activity. In addition, expression of an orthogonal pair of Saccharomyces cerevisiae suppressor $tRNA^{Tyr}$ and tyrosyl-tRNA synthetase also produced blue colonies as well as moderate increase of $\beta$-galactosidase activity. These data demonstrate that our reporter strain will provide an efficient method to assess amber suppression in both qualitative and quantitative manners.

• Journal of Plant Biotechnology
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• v.36 no.4
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• pp.384-391
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• 2009

Previously, two events (H15 and B20) of transgenic pepper (Capsicum annuum L.) that enhanced resistance to Cucumber mosaic virus (CMV) by the introduction of CMV coat protein (CP) gene were constructed. Presently, a single copy number of the CP gene was revealed in H15 and B20 by Southern blot. To predict possible unintended effects due to transgene insertion in an endogenous gene, we carried out sequencing of the 5'-flanking region of the CP gene and a Blastbased search. The results revealed that insertion of the transgene into genes encoding putative proteins may occur in the H15 and B20 transgenic event. Mutiplex polymerase chain reaction (PCR) for simultaneous detection and identification of transgenic pepper was conducted with a set of nine primers. Both transgenic event were differentiated from non-transgenic event by the presence of 267 bp and 430 bp PCR products indicative of CP gene specific primer pairs and primer pairs targeting the CP gene and 35S promoter. H15 and B20 uniquely possessed a 390 bp and 596 bp PCR product, respectively. The presence of a 1115 bp product corresponding to intrinsic pepper actin gene confirmed the use of pepper DNA as the PCR template. The primer set and PCR conditions used presently may allow the accurate and simple identification of CMV resistant transgenic pepper.

• Journal of Genetic Medicine
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• v.13 no.1
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• pp.14-19
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• 2016

Purpose: We examined the prevalence and CGG/AGG repeat structure of expanded alleles of the FMR1 gene in preconceptional and pregnant Korean women. Materials and Methods: The CGG repeats in the FMR1 genes of 1,408 women were analyzed by polymerase chain reaction and Southern blot analysis. To estimate the prevalence of expansion alleles, the individuals were divided into low risk and high risk group. Results: Within this population, 98.4% had normal alleles and 1.6% had abnormal alleles including intermediate (0.6%), premutation (0.5%), full mutation (0.1%), and hemizygous (0.4%) alleles. There were 2 premutation alleles (1:666, 95% confidence interval [CI] 1:250-1,776) in the low risk group and 5 premutation alleles (1:15, 95% 1:6-36) in the high risk group. There were 8 intermediate alleles (1:167, 95% CI 1:130-213) in the low risk group and 1 intermediate alleles (1:76, 95% CI 1:11-533) in the high group. Six of the 7 premutation alleles did not contain AGG interruptions within the repeats and 1 had a single AGG interruption. Four of the 9 intermediate alleles contained 2-3 AGG, 4 had a single AGG, and 1 had no AGG interruptions. Conclusion: Our study demonstrates the prevalence and CGG/AGG structure of expansion alleles in Korean women. The identified premutation prevalence is higher than that of other Asian populations and lower than that of Caucasian populations. Although our study is limited by size and population bias, our findings could prove useful for genetic counseling of preconceptional or pregnant women.

• Plant Biotechnology Reports
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• v.3 no.2
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• pp.147-155
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• 2009

Oryza grandiglumis Chitinase IVa (OgChitIVa) cDNA encoding a class IV chitinase was cloned from wild rice (Oryza grandiglumis). OgChitIVa cDNA contains an open reading frame of 867 nucleotides encoding 288 amino acid residues with a predicted molecular weight of 30.4 kDa and isoelectric point of 8.48. Deduced amino acid sequences of OgChitIVa include the signal peptide and chitin-binding domain in the N-terminal domain and conserved catalytic domain. OgChitIVa showed significant similarity at the amino acid level with related monocotyledonous rice and maize chitinase, but low similarity with dicotyledoneous chitinase. Southern blot analysis showed that OgChitIVa genes are present as two copies in the wild rice genome. It was shown that RNA expression of OgChitIVa was induced by defense/stress signaling chemicals, such as jasmonic acid, salicylic acid, and ethephon or cantharidin and endothall or wounding, and yeast extract. It was demonstrated that overexpression of OgChitIVa in Arabidopsis resulted in mild resistance against the fungal pathogen, Botrytis cinerea, by lowering disease rate and necrosis size. RT-PCR analysis showed that PR-1 and PR-2 RNA expression was induced in the transgenic lines. Here, we suggest that a novel OgChitIVa gene may play a role in signal transduction process in defense response against B. cinerea in plants.