• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.85-85
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• 1997

스트레스가 유발된 랫드의 대뇌에서 Vasopressin-catecholamine pathway의 활성도를 알아보기 위해 면역화학염색법으로 vasopressin 호르몬의 분비와 catecholamine의 생성변화를 tyrosine hydroxylase (TH) 효소의 발현변화로 규명하고, arginine vasopressin (AVP)과 V1 vasopressin receptor의 유전자 발현변화를 in situ hybridization 방법을 이용하여 살펴보았다. 수컷 SD rat를 7시간동안 stress cage에 넣어 16$\pm$1$^{\circ}C$의 물에 수침구속 스트레스를 준 후 대조군과 함께 관류고정하여 brain을 적출하였다. Brain의 hypothalamus 부위를 중심으로하여 동결절편하여 면역조직화학 염색과 in situ hybridization을 시행하였다. TH 면역조직화학 염색에서 대뇌의 줄무늬체 부위의 꼬리조가비핵에서와 시상하부 부위의 내측등쪽시상하부와 흑색질부위에서 스트레스군이 대조군에 비해 TH 면역염색성이 증가되어 관찰되었으나 시상하부 부위의 시삭위핵, 뇌실주위핵, 뇌실옆핵에서는 두 군간의 큰 면역염색성의 차이는 보이지 않았다. AVP 면역조직화학 염색에서는 시삭위핵에 많은 수의 AVP 양성 신경세포체들이 밀집되어 있으며 뇌실옆핵에서는 스트레스군에서 AVP 면역염색성이 약간 증가되어 관찰되었으나 신경섬유의 분포양상은 비슷하였다. 중간융기에서는 모두 강한 염색성의 신경섬유들이 관찰되어 두 군간에 큰 차이는 없었다. AVP 유전자에 대한 in situ hybridization 결과 시삭위핵의 신경세포에서 AVP mRNA 양성반응을 관찰할 수 있었으나 다른 시상하부핵에서는 관찰할 수 없었으며, V1 vasopressin receptor에 대한 in situ hybridization 결과는 두 군의 대뇌에서 모두 양성반응을 관찰할 수 없었으며 V1 vasopressin receptor 유전자의 조직별 발현정도와 스트레스에 의한 발현량 조절을 관찰할 필요가 있다고 사료된다.

• Microbiology and Biotechnology Letters
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• v.18 no.6
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• pp.640-646
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• 1990

We have cloned the cdd gene from E. coli C600 using (cdd-) as a host. From the sequenced promoter region of E=, coli cdd gene which has been determined by Valentin-Hansen P. (1985), we synthesized the 23 mer oligonucleotides corresponding to the transcription initiation region and used as a probe for cloning of the cdd gene by Southern blotting. The isolated fragments in the blotting were introduced to the colony hybridization after transforming it into the E. coli JF611 (cdd-, pyr double mutant), and we identified the hybridized band at 27 kb long. From the original insert of 27 kb fragment in theBamHI site of pBR322, the 5.3 kb fragment containing the cdd gene was isolated by subsequent deletion and subeloning. From the derived plasmid pTK509, further deletion and subcloning were performed and clarified that the cdd gene was located in the 2.1 kb of SaZI/DraI segment in the insert of pTK605. The polypeptide encoded by the cloned DNA was appeared to be a molecular mass of 33,000.

• Fisheries and Aquatic Sciences
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• v.21 no.5
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• pp.11.1-11.8
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• 2018

Interspecific hybridization experiments were conducted between the common seahorse Hippocampus kuda (male) and the slender seahorse H. reidi (female) during artificial rearing to develop a new aquarium fish with unique polyandrous mating. Molecular analysis via mitochondrial DNA (mtDNA) cytochrome b and nuclear DNA (ncDNA) ribosomal protein S7 gene supported the hybridization between the two species, and the hybrid also showed morphological characteristics of both species. Juveniles of H. kuda have dense melanophores on the whole body or only on the trunk and tail, whereas juveniles of H. reidi have thin melanophores on the whole body or present in stripes only along their prominent trunk and tail rings. However, all the hybrid juveniles had dense melanophores only on the tail, with the striped trunk rings, thus showing an intermediate pattern, and these patterns were limited to the fairly early stage of development (1-10 days old). In contrast, the two eye spines in the hybrid were apparent after 9 days old, which were not inherited from H. kuda (one eye spine), but from H. reidi (two eye spines). According to LOESS (local regression) analysis, the growth rate increased between 20 and 25 days, and the hybrids grew faster than H. kuda when they entered the explosive second phase of growth between 25 and 45 days for all the seahorses. This study highlights the hybridization between H. kuda and H. reidi may contribute to the improved taxonomic information of young seahorses.

• 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.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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• pp.215-220
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• 2000

Abstract White spot disease (WSD), resulting in more than 90% mortality of aquacultured penaeid shrimp, has been reported off the southern and western coasts of Korea since 1993. The pafuogen of WSD has been identified as being a virion wifu an envelope around a central nucleocapsid, and with an average size of 167 nm in diameter and 375 nm in length. In the present study, an in situ hybridization technique was developed as a rapid. sensitive, and specific diagnostic assay for the WSD viros infection in shrimp. Furthermore. the pathological changes ofWSD, in shrimp experimentally infected with WSD viroses. were investigated. Using a biotinylated 643 bp probe obtained from a peR using primers specific to the rod-shaped virus of Penaeus japonicus (RV-PJ), positive signals were detected in both naturally and experimentally infected shrimps. The in situ hybridization revealed positive reactions in the nuclei of the stromal matrix cells in the lymphoid organ, epithelia of the gills, foregut. epidermis, and hematopoietic cells of the interstitial tissues, suggesting the presence of WSD virus. Tills result indicates that the in situ hybridization method can be useful for a rapid and sensitive detection of WSD viruses in shrimp.shrimp.

• Korean Journal of Microbiology
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• v.39 no.2
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• pp.114-117
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• 2003

In molecular biology, it is necessary to develop an easy and rapid method to identify a specific DNA sequence. Though Southern and Northern blot techniques have been used widely for the analysis of gene structure and function, those methods are inconvenient in the points that we need to control incubation temperature, time, and other parameters to get the final result. In this study, we report a new method for the rapid analysis of specific DNA sequence with the modification of an immunochromatographic method. The lateral flow DNA analysis strip is composed of a sample pad, a nitrocellulose membrane for the separation and propagation of analytes, and an absorption pad for the generation of capillary action. Capture DNA was immobilized on the membrane by UV cross-linking and target DNA was labeled with Cy-5 for signaling. The samples containing target DNA were applied onto the sample pad, incubated for 15 min for separation, and scanned with a GSI fluorescence scanner. Though the hybridization reaction occurs in a short time without any washing steps, there appears to be little cross hybridization between the different sequences. The result showed a possibility that the new method can be used for the rapid identification of specific DNA sequence among the samples.

• Journal of Aquaculture
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• v.18 no.3
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• pp.207-214
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• 2005

We report on the diagnosis, pathology, and taxonomy of Perkinsus sp. infection in Manila clams (Ruditapes philippinarum) from Korean waters. Amplimers were designed from internal portions of the non-transcribed spacer (NTS) of P. atlanticus for molecular diagnosis of Perkinsus infection. PCR-based identification methods and an in situ hybridization assay were developed for detection of Perkinsus sp. in live tissues as well as in histological preparations. Hybridization signals were observed around the nucleus of trophozoites. Positive results from PCR and in situ hybridization indicated that Korean Perkinsus sp. is genetically identical with P. atlanticus reported in Europe, which is currently synonymous with P. olseni reported from Australia. Microscopic morphological features of different lift stages of Perkinsus sp. appeared very similar to those of P. atlanticus. Severely infected clams often exhibited white nodules on their mantles and gills as a consequence of inflammation. In lightly to moderately infected clams, Perkinsus sp. was mainly found in gill tissues, whereas the protozoan parasites were found in digestive tracts, gonadal tissues, and foot tissues of heavily infected clams. It is likely that the gills are the portal of the infection and that P. olseni spreads to other tissues as the infection advances. In conclusion, by considering the taxonomic priority of P. olseni, Korean Perkinsus sp. is accepted as P. olseni. P. olseni appears to be common on tidal flats on the western and southern Korean coasts and is considered to be a pathogen capable of causing mass mortality of clams.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.17
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• pp.7997-8002
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• 2015

Gene amplification is an important mechanism in the development and progression of cancer. Currently, gene amplification status is generally determined by in situ hybridization (ISH). Multiplex ligation-dependent probe amplification (MLPA) is a PCR-based method that allows copy number detection of up to 50 nucleic acid sequences in one reaction. The aim of the present study was to compare results for HER2, CCND1, MYC and ESR1 gene amplification detected by MLPA with fluorescent in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) as clinically approved methods. Tissue samples of 170 invasive breast cancers were collected. All were ER positive. Tissue samples had previously been tested for HER2 using immunohistochemistry. Amplification of the selected genes were assessed using MLPA, FISH and CISH and results were compared. HER2 MLPA and ISH results were also compared with HER2 immunohistochemistry (IHC) which detects protein overexpression. Amplification of HER2, CCND1, MYC and ESR1 by MLPA were found in 9%, 19%, 20% and 2% of samples, respectively. Amplification of HER2, CCND1, MYC and ESR1 by FISH was noted in 7%, 16%, 16% and 1% of samples, respectively. A high level of concordance was found between MLPA/FISH (HER2: 88%, CCND1: 88%, MYC: 86%, ESR1: 92%) and MLPA/CISH (HER2: 84%). Of all IHC 3+ cases, 91% were amplified by MLPA. In IHC 2+ group, 31% were MLPA amplified. In IHC 1+ group, 2% were MLPA amplified. None of the IHC 0 cases were amplified by MLPA. Our results indicate that there is a good correlation between MLPA, IHC and ISH results. Therefore, MLPA can serve as an alternative to ISH for detection of gene amplification.

• Journal of the Institute of Electronics Engineers of Korea SC
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• v.48 no.2
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• pp.86-90
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• 2011

The effects of cations are very important in field-effect transistors (FETs) type DNA sensors detecting the intrinsic negative charge between single-stranded DNA and double-stranded DNA without labeling, because the intrinsic negative charge of DNA is neutralized by cations in electrolyte solution. We consider the Debye length, which depends on the concentration of cations in solution, to detect DNA hybridization based on the intrinsic negative charge of DNA. The Debye length is longer in buffer solution with a lower concentration of NaCl and the intrinsic negative charge of DNA is more effective on the channel surface in longer Debye length solution. The shifts in the gate voltage by DNA hybridization with complementary target DNA are 21 mV in 1 mM NaCl buffer solution, 7.2 mV in 10 mM NaCl buffer solution, and 5.1 mV in 100 mM NaCl buffer solution. The sensitivity of FETs to detect DNA hybridization based on charge detection without labeling depends on the Debye length.

• Journal of Plant Biology
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• v.38 no.2
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• pp.203-209
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• 1995

Tobacco (Nicotiana tahacum L. cv. Wisconsin 38) leaf discs were cocultivated with Agrohacterium carrying a leghemoglobin (Lb) cDNA from Canavalia lineata. Seven plants were regenerated from the transformed leaf discs on MS media supplemented with 0.5 mg/L BAP, 0.1 mg/L ${\alpha}-NAA$, 200 mg/L kanamycin and 500 mg/L carbenicillin. Southern hybridization and PCR of genomic DNA from transgenic plants showed that the Lb cDNA was stably integrated into the genome of the tobacco. Total RNA from the transgenic tobacco showed northern hybridization signal at 1,000 nt and PCR of the first strand cDNA synthesized from the total RNA amplified 0.5 kb Lb cDNA. Furthermore, western hybridization using a polyclonal antibody against soybean Lb showed a 15.8 kD LB-like band on SDS-PAGE of proteins from the transformed tobacco. These results demonstrated that the Lb cDNA of C. lineata was not only incorporated into the genome of tobacco, but also transcribed into mRNA and translated into Lb protein in the transformed tabacco.