• International Journal of Oral Biology
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• v.41 no.2
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• pp.97-103
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• 2016

Mammals have 3 pairs of major salivary glands i.e., the parotid, submandibular, and sublingual glands. Saliva secretion of these glands is modulated by taste perception. Salivary glands are composed mainly of acinar and ductal cells. Primary saliva is secreted by acinar cells and modified during ductal flow. Recently, of the murine 35 bitter taste receptors, Tas2r108 was expressed at highest levels in the submandibular gland by qPCR. Further, Tas2r108-transfected cells respond to a range of bitter compounds, such as denatonium, quinine, colchicine, diphenidol, caffeine and dapson. The objective of the present study was to characterize the expression of Tas2r108 mRNA in acinar and/or ductal cells of the submandibular gland using in situ hybridization (ISH). Male 42-60 days old DBA2 mice were used in the study. Messenger RNAs were extracted from the submandibular gland for generating digoxigenin (DIG) labeled-cRNA probes. These probes were transcribed in anti-sense and sense orientation using T7 RNA polymerase. Dot blot hybridization was performed using DIG labeled-cRNA probes, in order to estimate integrity and optimal diluting concentration of these probes. Subsequently, ISH was performed on murine submandibular gland to detect Tas2r108 mRNA. Dot blot hybridization data demonstrated that Tas2r108 DIG labeled-cRNA anti-sense probes specifically detected Tas2r108 cDNA. ISH results showed that the anti-sense probes labeled acinar and ductal cells in the submandibular gland, whereas no staining was visible in sense controls. Interestingly, the Tas2r108 expression levels were higher in acinar than ductal cells. These results suggested that Tas2r108 might be more associated with primary saliva secretion than with ductal modification of saliva composition.

• Fisheries and Aquatic Sciences
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• v.3 no.1
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• pp.64-70
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• 2000

DNA probes for the l6S rRNA have been designed for the detection of Edwardsiella tarda. In order to accomplish this purpose, the l6S rRNA gene from E. tarda has been cloned and sequenced. Two highly feasible oligonucleotide probe sites have been determined by the database analysis programs presented by PCGENE and BLAST. These two probes have been evaluated by slot blot hybridization analysis. Hetero- and homo-trimeric templates have been synthesized using these two probe sites. The templates have been further multimerized by PCR to generate between 150 and 300 bp long DIG-11-dUTP labeled probes. Unlike 3' end labeled oligonucleotide probes or templates, multimerized probes showed no cross­hybridization in the given experimental condition. Furthermore, a significant increase in sensitivity has been observed with these probes. This method, we presented here, may be useful for the designing of probes for the detection of other fish pathogenic microorganisms also.

• 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.

• Plant Resources
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• v.7 no.2
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• pp.116-122
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• 2004

We tried to isolating a noble gene from cucumber library with heterogeneouse RNA probe labeled DIG of Arabidopsis PIN3 gene. Two kinds of RNA probes which had no significant homology each others, were designed from the 5'- and 3'- prime nucleotides of the AtPIN3 gene. In the first and second screenings of the cDNA library of cucumber with the probes, two positive clones were identified with specific duplicate signals. However, we isolated cDNA fragments homologous with putative nucleases from Nicotiana, Arabidopsis, Cordialis, and Oryza sativa, there was no significant homology with any other PIN family genes.

• Journal of Microbiology and Biotechnology
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• v.28 no.11
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• pp.1823-1833
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• 2018

Fluorescence in-situ hybridization (FISH) is a common and popular method used to investigate microbial communities in natural and engineered environments. In this study, two specific 16S rRNA-targeted oligonucleotide probes, CLZ and KCLZ, were designed and verified to quantify the genus Clostridium and the species Clostridium kluyveri. The optimal concentration of hybridization buffer solution for both probes was 30% (w/v). The specificity of the designed probes was high due to the use of pellets from pure reference strains. Feasibility was tested using samples of Chinese liquor from the famed Luzhou manufacturing cellar. The effectiveness of detecting target cells appears to vary widely in different environments. In pit mud, the detection effectiveness of the target cell by probes CLZ and KCLZ was 49.11% and 32.14%, respectively. Quantitative analysis by FISH technique of microbes in pit mud and fermented grains showed consistency with the results detected by qPCR and PCR-DGGE techniques, which showed that the probes CLZ and KCLZ were suitable to analyze the biomass of Clostridium spp. and C. kluyveri during liquor fermentation. Therefore, this study provides a method for quantitative analysis of Clostridium spp. and C. kluyveri and monitoring their community dynamics in microecosystems.

• Korean Journal of Microbiology
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• v.29 no.3
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• pp.189-194
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• 1991

The degree of the genetic variations among Clostridium thermocellum ATCC 27405 and the wild type strains was investigated by the mehtod of GC ratio, DNA-DNA hybridization and RFLP (Restriction Fragment Length Polymorphism) patterns by ribosomal RNA and M13 probe. GC ratio and KNA homology values of th three isolates were approximately equal to those of ATCC type strain. The RFLP patterns by the rRNA and M13 probe showed some differences among C. thermocellum ATCC 27405, wild type strains and Clostridium thermohydrosulfuricum ATCC 33223, indicating that the two probes can be useful in subspecies- and apecies-identification.

• Korean Journal Plant Pathology
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• v.11 no.4
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• pp.367-373
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• 1995

The cDNAs derived from the coat protein (CP) genes of six plant RNA viruses, tobacco mosaic virus-pepper strains (TMV-P) and -ordinary strain (TMV-OM), potato virus Y (PVY), turnip mosaic virus (TuMV), cucumber mosaic virus (CMV) and potato leafroll virus (PLRV), were subcloned into the transcription vector, pSPT18, containing SP6 and T7 promoters. The digoxigenin (DIG)-labeled RNA polymerase after linearlization of the cloned pSPTs with XbaI or SacI, and were tested for their sensitivities for the detection of the six viruses. In slot-blot hybridization, dilution end points for the detection of TMV-P and TMV-OM were 10-4, while those of PVY, TuMV and CMV were 10-3. PLRV was detected at the dilution of 10-2. When each RNA probe was applied for the detection of the viruses in the preparations from the leaf disks (8 mm in diameter, and 12 to 15 mg in weight) of infected natural host plants, TMV-P, TMV-OM and TuMV could be detected from one disk, while PVY from 1 or 2 disks. CMV was detected in the preparation from two disks, and PLRV from three disks. With DIG-labeled RNA probe, PVY was detected at 5 days after inoculation, but with ELISA the virus was detected at 8 days after inoculation to tobacco (Nicotiana tabacum cv. Xanthi nc) plants on which symptoms appeared at 9 days after inoculation. No difference was observed in cross reaction between the RNA probes for the detection of TMV-P and TMV-OM.

• Bulletin of the Korean Chemical Society
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• v.34 no.11
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• pp.3471-3473
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• 2013

• Journal of the Korean Chemical Society
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• v.42 no.2
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• pp.209-213
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• 1998

The higher order structure of Pseudomonas alcaligenes 5S rRNA has been investigated by using ($\eta^{6}$-mesitylene) manganese (Ⅰ) tricarbonyl hexafluorophosphate[MTH-Mn (Ⅰ)], dimethylpyrocarbonate, potassium permanganate as chemical probes. The sequences cleaved strongly by MTH-Mn (Ⅰ) on the tertiary structure of the 5S rRNA are $G_{12}AUGG_{16}$ of loop a, $G_{51}AAGUGAAGC_{60}$ of the region b-C, $U_{65}-AGCG_{69}$. of the region B-a, and $G_{72}AUGG_{76}$ of loop d. Based on such cleavage patterns of 5S rRNA by MTH-Mn(Ⅰ) and other chemical probes, we presume that the sequences strongly cleaved form pocket-like structure as in the the corner of L structure of $tRNA^{Phe}$. We also presume that the region b-C and loop d together play a role of hinge in forming the pocket-like structure in the 5S rRNA.

• Proceedings of the Korean Society of Life Science Conference
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• 2000.09a
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• pp.27-27
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• 2000