• Korean Journal Plant Pathology
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• v.14 no.6
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• pp.636-641
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• 1998

Digoxigenin (DIG) was used to prepare nucleic acid probe for the detection of RNA of potato leafroll virus (PLRV) in the potato leaf extracts. The 0.6 kb coat protein (CP) gene cDNA of PLRV in plasmid pSPT 18 vector was labeled with digoxigenin by in vitro run-off transcription and then used for cRNA probe. In the several buffers tested for increase the total RNA extraction efficiency AMES buffer was the most suitable for this detection method. The RNA extracts from potato leaves shown symptoms of PLRV were dot blotted onto nylon membrane and hybridized with labeled RNA probes. After hybridization, labeled RNA bound to PLRV RNA on membrane was detected with anti-digoxigenin alkaline phosphatase. 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (NBT) salt and CSPD were used as substrate for colorimetric and film exposure detection, respectively. These detection methods were very sensitive allowing for detection of 1/32 diluted total RNA extract from 100 mg leaf tissue.

• Journal of Radiopharmaceuticals and Molecular Probes
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• v.6 no.2
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• pp.165-170
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• 2020

Histone Deacetylases (HDACs) are enzymes that have control gene expression regulation and cell state. In additions, inhibitions of HDACs are associated with growth arrest, differentiation, or apoptosis of tumor cell. Thus HDAC inhibition is one of the interesting biological targets. A variety of HDAC inhibitors has been developed by many scientists, and some of chemical structures related with HDAC inhibitors were modified to give radiolabeled HDAC inhibitors for positron emission tomography (PET) study. In this highlight review, the development of radiolabeled HDAC inhibitors for PET study are described.

• Proceedings of the Zoological Society Korea Conference
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• 1995.10b
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• pp.120-120
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• 1995

The total size of the pF AR1, a genomic clone of Frankia FaCI, was estimated to be about 44Kb by summation of the individual fragment length generated by single or double restriction enzymes. Southern hybridization analyses with Azotobacter vinelandii nif-genes as probes and partial sequencing analyses of the subclones revealed that organization of the nif-gene in the FaCI strain was nifV, H, D, K, E, N, X, W, B. The organization of the structural genes for nitrogenase is the same in this Frankia strain as it is in most other nitrogen-fixing prokaryotes but the positioning of the nifV-like gene relative to the nifHDK cluster differs. A consensus nif-promoter-like sequence, found at 5' of nifH, was not detected upstream of the niJV-like gene. nifV-like gene contained a ORF of 1206 NT encoding 401 amino acids. The nucleotide sequence and deduced amino acid sequence of the gene exhibit homology value of 65% and 41% with that from A vinelandii, respectively. The putative Shine-Dargamo sequences were present preceding nitK, nifH, D, K, and nifE, and in nitK gene putative start codon GTG was detected instead of A TG. The nucleotide and amino acid sequence of niIK of FaCI showed 82% and 76% homolgy with those of Frankia HFPCc 13, respectively. Amino acid sequence of niIK showed 69% and 61% homology with those of A vinelandii, Klebsiella pnewnoniae, respectively, while that of nifE 73% and 71%, respecti vely.i vely.

• Journal of Radiopharmaceuticals and Molecular Probes
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• v.1 no.2
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• pp.118-122
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• 2015

Suicidal gene therapy is based on the transduction of tumor cells with "suicide" genes encoding for prodrug-activating enzymes that render target cells susceptible to prodrug treatment. Suicidal gene therapy results in the death of tumor with the expression of gene encoding enzyme that converts non-toxic prodrug into cytotoxic product. Cytochrome P450 4B1 (CYP4B1) activates 4-ipomeanol (4-IPO) or 2-aminoanthracene (2-AA) to cytotoxic furane epoxide and unsaturated dialdehyde intermediate.In this study, therapeutic effects of suicidal gene therapy with rabbit CYP4B1/2-AA or 4-IPO system were evaluated in HT-29 (human colon cancer cell). pcDNA-CYP4B1 vector was transfected into HT-29 by lipofection and stable transfectant was selected by treatment of hygromycin ($500{\mu}g/mL$) for 3 weeks. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed for confirmation of CYP4B1 expression in CYP4B1 gene transduced cell. The cytotoxic effects of CYP4B1 transduced cell were determined using dye-exclusion assay after treatment of 2-AA or 4-IPO for 96 hrs. Dye-exclusion assay showed that $IC_{50}$ of HT-29 and CYP4B1 transduced HT-29 was 0.01 mM and 0.003 mM after 4-IPO or 2-AA treatment at 96 hrs exposure, respectively. In conclusion, CYP4B1 based prodrug gene therapy probably have the potential for treatment of colorectal adenocarcinoma.

• Journal of Crop Science and Biotechnology
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• v.11 no.2
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• pp.91-100
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• 2008

Melting curve analysis of fluorescently labeled DNA fragments is used extensively for genotyping single nucleotide polymorphism(SNP). Here, we evaluated a SNP genotyping method by melting curve analysis with the two probe chemistries in a 384-well plate format on a Roche LightCycler 480. The HybProbe chemistry is based on the fluorescence resonance energy transfer(FRET) and the SimpleProbe chemistry uses a terminal self-quenching fluorophore. We evaluated FRET HybProbes and SimpleProbes for two SNP sites closely linked to two quantitative trait loci(QTL) for southern root-knot nematode resistance. These probes were used to genotype the two parents and 94 $F_2$ plants from the cross of PI 96354$\times$Bossier. The SNP genotypes of all samples determined by the LightCycler software agreed with previously determined SSR genotypes and the SNP genotypes determined on a Luminex 100 flow cytometry instrument. Multiplexed HybProbes for the two SNPs showed a 98.4% success rate and 100% concordance between repeats two of the same 96 DNA samples. Also, we developed a HybProbe assay for the Rcs3 gene conditioning broad resistance to the frogeye leaf spot(FLS) disease. The LightCycler 480 provides rapid PCR on 384-well plate and allows simultaneous amplification and analysis in approximately 2 hours without any additional steps after amplification. This allowed for a reduction of the potential contamination of PCR products, simplicity, and enablement of a streamlined workflow. The melting curve analysis on the LightCycler 480 provided high-throughput and rapid SNP genotyping and appears highly effective for marker-assisted selection in soybean.

• YAKHAK HOEJI
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• v.38 no.3
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• pp.293-299
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• 1994

Forty nine clinical isolates of S. aureus showing resistance to erythromycin(EM) were selected from 83 strains isolated recently in Korea. Fourteen strains of S. aureus showing inducible resistance to MLS antibiotics were selected by disc agar diffusion method. Colony hydridization was executed using two MLS inducible resistance genes, ermA and ermC, identified previously from S. aureus as probes. S. aureus 375 and S. aureus 507 whose genes were not homologous to those probes were finally selected. It was confirmed that the resistance genes of S. aureus 375 and S. aureus 507 had no homology with those probes in southern hybridization test using ermA, ermC and ermAM as probes. It was determined that S. aureus 375 had a plasmid whose size was about 35 kb. To know if the plasmid may have the genes related to inducible resistance to MLS antibiotics, it was attempted to transform Bacillus subtillis BR151 and S. aureus RN4220 with the plasmid isolated from S. aureus 375. It was shown that the gene related to inducible resistance to MLS antibiotics did not exist in this plasmid. These results indicate that two clinical isolates of S. aureus showing inducible resistance to MLS antibiotics have novel genes that have no homology with MLS resistance genes identified so far. It is assumed that these genes may exist in chromosomal DNA.

• Journal of Food Hygiene and Safety
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• v.22 no.4
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• pp.248-256
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• 2007

In this study, the competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Salmonella spp. in pork meat. After comparing three DNA extraction methods, the modified guanidine thiocyanate-phenol-chloroform method was chosen for Salmonella DNA extraction in artificially inoculated pork meat. The previously reported 284-bp invA gene (Rahn et al. Mol. Cell. Probes 1992) was tested for specificity, and 57 Salmonella strains and 24 non-Salmonella strains were evaluated. All Salmonella strains tested were invA positive, and all non-Salmonella strains produced no false positive amplification products. The detection limit achieved was as low as 1,460 colony-forming units (cfu) per 0.1g of pork meat. For cPCR, the invA gene, which features a 82 bp-deletion, was cloned in the pGEM-4Z vector. A known amount of competitor DNA, which has the same primer binding sites, was co-amplified with Salmonella chromosomal DNA from the artificially inoculated pork meat. The cell-number determined by cPCR was approximately equal to the cfu from the most probable number (MPN) method. Finally, the whole procedure took only 5 hr.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.10
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• pp.4153-4158
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• 2014

Background: This study was aimed to establish a novel method to simultaneously detect expression of four genes, ribonucleotide reductase subunit M1(RRM1), X-ray repair cross-complementing gene 1 (XRCC1), thymidylate synthase (TS) and class III ${\beta}$-tubulin (TUBB3), and to assess their application in the clinic for prediction of response of non-small cell lung cancer (NSCLC) to chemoradiotherapy. Materials and Methods: We have designed four gene molecular beacon (MB) probes for multiplex quantitative real-time polymerase chain reactions to examine RRM1, XRCC1, TUBB3 and TS mRNA expression in paraffin-embedded specimens from 50 patients with advanced or metastatic carcinomas. Twenty one NSCLC patients receiving cisplatin-based first-line treatment were analyzed. Results: These molecular beacon probes could specially bind to their target genes in homogeneous solutions. Patients with low RRM1 and XRCC1 mRNA levels were found to have apparently higher response rates to chemoradiotherapy compared with those with high levels of RRM1 and XRCC1 expression (p<0.05). The TS gene expression level was not significantly associated with chemotherapy response (p>0.05). Conclusions: A method of simultaneously detecting four molecular markers was successfully established and applied for evaluation of chemoradiotherapy response. It may be a useful tool in personalized cancer therapy.

• Microbiology and Biotechnology Letters
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• v.20 no.3
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• pp.301-310
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• 1992

This work was performed to investigate the possibility of using J96 hemolysin(Hly, Hly A) vaccine against urinary tract infecting Escherichia coli. Based on the known sequence of J96 hemolysin which was originally isolated from a pyelonephritis patient, ten 20-mer oligonucleotide probes were synthesized. Radioactive labelled 8 probes showed positive colony blots against most of the hemolysin producing wild type E. coli, while HA484 and HA661 showed 28.3, 71.7% positive blots, respectively. This result means that hemolysin genes are highly conserved. Also, 12 anti-Hly MABs(monoclonal antibodies) showed more than 90% positive immunoblots against secreted hemolysin from wild type E. coli. Especially, the result that MAB132 neutralized hemolysin from all of the wild type E. coli augments the idea that hemolysin will be effective as a vaccine.

• Journal of fish pathology
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• v.6 no.2
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• pp.111-122
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• 1993

The mechanism by which $V_H$ region gene segments is selected in B lymphocyte is not known. Moreover, evidence for both random and nonrandom expression of $V_H$ genes in matured B cells has been presented previously. In this report, the technique of in situ hybridization allowed us to analyze expressed $V_H$ gene families in normal B lymphocyte at the single cell level. The analysis of normal B cells in this study eliminated any posssible bias resulting from transformation protocols used previously and minimized limitation associated with sampling size. Therefore, an accurate measure of the functional and expressed $V_H$ gene repertoire in B lymphocyte could be made. One of the most important controls for the optimization of in situ hybridization is to establish probe concentration and washing stringency due to the degree of nucleotide sequence similarlity between different families which in some cases can be as high as 70%. When the radioactive $C{\mu}$ and $V_{H}J558$ RNA probes are tested on LPS-stimulated adult spleen cells, $2{\sim}4{\times}106cpm$/slide shows low background and reasonable frequency of specific positive cells. For the washing condition. 40~50% formamide at $54^{\circ}C$ is found to be optimum for the $C{\mu}$. $V_{H}S107$ and $V_{H}J558$ probes. The analyzed results clearly demonstrate that the level of each different $V_H$ gene family expression is dependent upon the complexity or size of that family. These findings are also extended to the level of $V_H$ gene family expression in separated bone marrow B cells depend upon the various stage of differentiation and conclude no preferential utilization of specific $V_H$ gene family. Thus, the utilization of VH gene segments in B lymphocyte of adult BALB/c mice is random and is not regulated or changed during the differentiation of B cells.