• The Plant Pathology Journal
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• v.17 no.1
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• pp.22-28
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• 2001

Barley yellow dwarf virus PAV (BYDV-PAV) has a 5.7-kb positive-sense single-stranded RNA genome that contains six open reading frames (ORFs). BYDV-PAV produces three subgenomic RNAs (sgRNAs). The largest of which encodes the coat, 17-kDa, and readthrough proteins from two initiation codons. To investigate the role of intergenic and 3'-proximal noncoding regions (NCRs) in coat protein (CP) expression and BYDV-PAV replication, a full-length infectious cDNA of the RNA genome of an Illinois isolate of BYDV-PAV was constructed downstream of the Cauliflower mosaic virus-35S promoter. Linear DNA molecules of these cDNAs were infectious, expressed the 22-kDa CP, and produced both genomic RNA sgRNAs in ratios similar to those observed in protoplasts inoculated with viral RNA. The portion of 5'NCR of sgRNA1 between ORFs 2 and 3 was not required for, but enhanced translation of CP from ORF3. Mutants containing deletions in the NCR downstream of ORF5 failed to replicate in oat protoplasts. These results indicate that an intact 3$^1$NCR is required for BYDV-PAV replication.

• Korean Journal of Plant Tissue Culture
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• v.27 no.3
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• pp.239-243
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• 2000

Helicobacter pylori, an etiologic agent of gastritis and peptic ulceration, produces urease which elicits a powerful immunoglobulin response in H. pylori-infected individuals. To establish a model plant vaccine agains H. pylori, 750 bp -ureA DNA amplified by polymerase chain reaction from pH 808 plasmid harboring urease gene cluster was cloned and manipulated to be expressed in tobacco plants. From the regenerated transgenic tobacco plants, ureA DNA integration,m its mRNA expression and protein synthesis were analyzed and confirmed by standard molecular techniques. The CaMV 35S promoter-driving ureA construct was expressed to produce a 30 kDa protein which was identical with bacterial UreA in size when detected on immunoblot of SDS polyacrylamide gel electrophoresis.

• Korean Journal of Plant Tissue Culture
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• v.21 no.5
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• pp.315-318
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• 1994

To obtain transformed plants, we cocultured cotyledonary explants of Codonopsis lanceolata with Agrobacterium tumefaciens LBA4404, a disamed strain harboring a binary vector pBI121 carrying the CaMV35S promoter-$\beta$-glucuronidase (GUS) gene fusion used as a reporter gene and NOS promoter-neomycin phosphotransferase gene as a positive selection marker in MS liquid medium with 1mg/L BA. After 48 h of culture, explants were transferred onto MS solid medium with Img/L BA, 250mg/L carbenicillin, and 100mg/L kanamycin sulfate and cultured in the dark. Numerous adventitious buds formed on the cut edges of the explants after 2 weeks of culture. When subjected to GUS histochemical assay buds showed a positive response at a frequency of 15%. Explants formed adventitious shoot at a frequency of 56.7%, after 6 weeks of culture. Upon transfer onto the basal medium, most of the shoots were rooted and subsequently the regenerants were transplanted to potting soil. Southern blot analysis confirmed that the GUS gene was incorporated into the genomic DNA of the GUS-positive regenerants.

• Molecules and Cells
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• v.27 no.4
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• pp.475-480
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• 2009

The transcription of soybean (Glycine max) calmodulin isoform-4 (GmCaM-4) is dramatically induced within 0.5 h of exposure to pathogen or NaCl. Core cis-acting elements that regulate the expression of the GmCaM-4 gene in response to pathogen and salt stress were previously identified, between -1,207 and -1,128 bp, and between -858 and -728 bp, in the GmCaM-4 promoter. Here, we characterized the properties of the DNA-binding complexes that form at the two core cis-acting elements of the GmCaM-4 promoter in pathogen-treated nuclear extracts. We generated GUS reporter constructs harboring various deletions of approximately 1.3-kb GmCaM-4 promoter, and analyzed GUS expression in tobacco plants transformed with these constructs. The GUS expression analysis suggested that the two previously identified core regions are involved in inducing GmCaM-4 expression in the heterologous system. Finally, a transient expression assay of Arabidopsis protoplasts showed that the GmCaM-4 promoter produced greater levels of GUS activity than did the CaMV35S promoter after pathogen or NaCl treatments, suggesting that the GmCaM-4 promoter may be useful in the production of conditional gene expression systems.

• Microbiology and Biotechnology Letters
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• v.17 no.6
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• pp.600-605
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• 1989

For the purpose of isolation of the Zymomonas mobilis DNA fragments showing promoter activity in Escherichia coli, a promoter screening vector, PCMT215 was constructed by transferring a promoterless chloramphenicol acetyltransferase (CAT) gene of pYEJ001 into pMT21 which contains $\beta$-lactamase gene and multiple cloning sites. A library of Z, mobilis Sau3AI DNA fragments was constructed in E. coli using the newly constructed pCMT215. Fourteen clones showing resistance to chloramphenicol ranging in concentration from 30 to 750 $\mu$g/$m\ell$ were selected. From five clones of them, the Z. mobilis DNA fragments expressing CAT gene of the recombinant plasmids were sequenced and then sites of transcriptional initiation were identified. The nucleotide sequences of the cloned DNA shared AT rich regions, poly A's or T's stretches and palindromic regions. The positions of transcriptional initiation for CAT gene occurred at more than one site spaced over by 4 to 190 base pairs on the cloned fragments in E. coli.

• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.952-960
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• 2006

As a unicellular green alga that possesses many of the metabolic pathways present in higher plants, Chlorelia offers many advantages for expression of heterologous proteins. Since strong and constitutive promoters are necessary for efficient expression in heterologous expression systems, the development of such promoters for use in the Chlorella system was the aim of this study. Proteins encoded by the early genes of algal viruses are expressed before viral replication, probably by the host transcriptional machinery, and the promoters of these genes might be useful for heterologous expression in Chlorella. In this study, putative promoter regions of DNA polymerase, ATP-dependent DNA ligase, and chitinase genes were amplified from eight Korean Chlorella virus isolates by using primer sets designed based on the sequence of the genome of PBCV-1, the prototype of the Phycodnaviridae. These putative promoter regions were found to contain several cis-acting elements for transcription factors, including the TATA, CAAT, NTBBF1, GATA, and CCAAT boxes. The amplified promoter regions were placed into Chlorella transformation vectors containing a green fluorescence protein (GFP) reporter gene and the Sh ble gene for phleomycin resistance. C. vulgaris protoplasts were transformed and then selected with phleomycin. The GFP fluorescence intensities of cells transformed with chitinase, DNA polymerase, and DNA ligase gene promoter-GFP fusion constructs were 101.5, 100.8, and 95.8%, respectively, of that of CaMV 35S-GFP-transformed Chlorella cells. These results demonstrate that these viral promoters are active in transformed Chlorella.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.38 no.2
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• pp.101-109
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• 2012

Objectives: The inactivation of the tumor suppressor gene $p16^{INK4a}$ plays an important role in the development of malignant tumors, including oral squamous cell carcinoma. The p16 gene is involved in the p16/cyclin-dependent kinase/retinoblastoma (Rb) gene pathway of cell cycle control. The p16 protein is considered a negative regulator of this pathway. The p16 gene encodes an inhibitor of cyclin-dependent kinases 4 and 6 which regulate the phosphorylation of the retinoblastoma gene and G1 to S phase transition in the cell cycle. However, the p16 gene can lose its functionality through point mutations, loss of heterozygosity or methylation of its promoter region. Materials and Methods: In this study, the authors analyzed the correlation between various clinicopathological findings- patient age, gender and smoking, disease recurrence, tumor size, stage, and differentiation- and p16 protein expression or p16 promoter hypermethylation in 59 cases of head and neck squamous cell carcinoma. Results: The results revealed p16 protein expression and p16 promoter hypermethylation in 28 cases (47.5%) and 21 cases (35.6%), respectively, of head and neck squamous cell carcinoma. However, neither p16 protein expression nor p16 promoter hypermethylation had any statistical influence on clinicopathological findings or survival rate. Conclusion: This data, and a review of the literature, suggest that p16 promoter hypermethylation cannot yet be used as an independent prognostic factor influencing carcinogenesis, but must be considered as an important factor along with other genetic alterations affecting the pRb pathway.

• Applied Biological Chemistry
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• v.39 no.5
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• pp.355-360
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• 1996

In vitro-tested ribozyme against synthesized cucumber mosaic virus (CMV) RNA (Agric. Chem. & Biotech. 37:56-63(1994)) was expressed in tobacco plant to develop virus resistant plants. The ribozyme sequence was linked to cauliflower mosaic virus 35S promoter and nopaline synthase(nos) terminator and this chimeric 35S-ribozyme-nos gene was sequenced. The sequenced chimeric gene was transferred to Agrobacterium tumefaciens LBA4404 using tri-parental mating system. The E. coli HB101 containing chimeric gene was incubated with E. coli HB101(pRK2073) as a helper and Agrobacterium tumefaciens LBA4404. Then Agrobacterium cells containing the ribozyme construct was cocultivated with tobacco leaf pieces. Ten different plants were regenerated from kanamycin containing MS medium. The presence of the ribozyme construct in the transgenic tobacco plants was confirmed by polymerase chain reaction (PCR). Seven different transgenic plants in ten different kanamycin resistant plants showed the expected size (570 base pairs) of 35S-ribozyme-nos gene fragment. Total RNAs were isolated from four different transgenic plants and separated on a 1% agarose gel containing formamide. Northern hybridization with 35S-ribozyme-nos gene fragment as a probe indicated that ribozyme transcripts may be degraded tv nuclease. Therefore, nuclease-resistant ribozymes are needed for the development of virus-resistant transgenic plants using ribozymes.

• Journal of Plant Biotechnology
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• v.6 no.1
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• pp.9-13
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• 2004

Transient uidA expression was used to optimize parameters required for biolistic transformation of suspension cells of Easter lily, Lilium longiflourm. Maximum uidA expression occurred following bombardment with gold particles as compared to tungsten. A 3hr pre-treatment of suspension cells with 0.125M osmoticum resulted in a 1.5X increase in uidA expression. A helium pressure of 1550 psi combined with a particle travelling distance of 6cm resulted in maximum uidA expression as compared to either 1100, 1200, or 1800 psi. Transient transformation resulted in up to 493 uidA expressing cells/Petri plate. For stable transformation suspension cells of Lilium longiflorum, were co-bombarded with plasmid DNA containing cucumber mosaic virus (CMV) replicase under the rice actin (Act1) promoter and either the bar or PAT genes under the cauliflower mosaic virus (CaMV 355) promoter. Ten regenerated plants contained the transgene as analyzed by PCR, and two of the ten plants were confirmed to contain the transgene by Southern hybridization. The two transgenic plants were independent transformants, one containing the bar gene and the other both the CMV replicase and bar genes. Plants were sprayed at the rosette stage and found to be resistant to 1000 mg/L of phosphinothricin (Trade name-Ignite) indicating expression of the bar gene throughout the leaves when bar was under control of the CaMV 35S promoter.

• Korean Journal of Plant Tissue Culture
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• v.22 no.4
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• pp.195-200
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• 1995

The mammalian adenosine deaminase(ADA) gene was stably expressed in transgenic tobacco plane. The chimeric ADA gene 35S/35S/AMV/ADA/Tnos, has been constructed. This chimeric gene was introduced into the binary vector pRD400, which was thereafter mobilized into Agrobacterium tumefaiens strain MP90 harboring disarmed Ti-plasmid. The resulting strains were used to transform Nicofiana tabacum L. using the leaf disc. Incorporation of the chimeric gene into plant were confirmed by PCR and Northern blot analyses. Immunoblot analysis showed that ADA protein was successfully synthesized in the transgenic tobacco plants.