• Journal of Photoscience
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• v.8 no.3_4
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• pp.93-97
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• 2001

Heterosigma akashiwo has been reported as red-tide causing phytoplankton in the Korean coastal area during summer when they are exposed to high light. It also shows photosynthetic adaptability to strong light during culture in the laboratory. On the basis of these observations, we tried to find out some genes specifically expressed in Heterosimga akashiwo during exposure to high light, assuming that they might have some resistant mechanisms associated with light adaptation. For this purpose, we carried out DD-PCR to detect differentially expressed mRNAs from cells that had been illuminated under high light for 3 days. We found eight cDNA clones that had been expressed specificically for high light. When they were further screened by reverse Northern hybridization, three of them were identified to be positive cDNA clones. When these cDNA fragments were subjected to DNA sequencing and then their base sequences were compared to GenBank database, one of them showed sequence homology 86% identical to the partial sequence of 16S rRNA gene of eubacterium CRO-18.

• The Korean Journal of Zoology
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• v.27 no.2
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• pp.103-116
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• 1984

The structural gene of rabbit hemoglobin was cloned into Pst I site of pBR322 in E. coli. The complementary DNA (cDNA) was synthesized from rabbit globin mRNA with avian myeloblastosis viral reverse transcriptase, and then RNA was destroyed at pH 11. The double stranded cDNA was synthesized with both Klenow fragment of E. coli DNA polymerase I and reverse transcriptase and then the hairpin loop was opened with Sl nuclease. Double stranded cDNA was subsequently tailed with dCTP and annealed to dGMP-tailed vector DNA. After transformation and initial screening of appropriate clones by plasmid size, the cloned colonies were identified by in situ colony hybridization using by plasmid size, the cloned colonies were identified by in situ colony hybridization using $[^32P]$-labeled cDNA probes and characterized the inserts with restriction endonucleases. The expression of cloned globin gene was investigated by standard radioimmunoassay using rat anti-rabbit Hb serum as primary antibody and goat antirat IgG serum as secondary antibody. The result suggested that the chimeric proteins (the part of $\\beta$-lactamase from the vector pBR322 and globin from rabbit) were supposedly produced in E. coli and the product had the antigenic determinant of rabbit hemoglobin.

• Journal of Periodontal and Implant Science
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• v.32 no.2
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• pp.281-290
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• 2002

The purpose of this study is to develop species-specific DNA probe for detection and identification of Prevotella intermedia (P. intermedia) G8-9K-3. This study procedure includes (1) whole-genomic DNA extraction of P. intermedia G8-9K-3 (2) construction of the genomic DNA library, (3) screening of strain-specific DNA probe by reverse dot hybridization, (4) confirmation of strain-specific DNA probe by Southern blot hybridization, (5) determination of nucleotide sequences of strain-specific DNA probe. Twenty-eight recombinant plasmids containing Hind III-digested DNA fragments of P. intermedia G8-9K-3 were obtained. Reverse Dot Hybridization and Southern blot analysis data showed that one of them, Pig3, could be P. intermedia G8-9K-3-specific DNA probe. This datum indicates that this Pig3 DNA probe could be useful in detection and identification of the P. intermedia G8-9K-3 strain.

• Molecules and Cells
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• v.23 no.1
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• pp.100-107
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• 2007

The moss Physcomitrella patens has two life cycles, filamentous protonema and leafy gametophore. A modified from of suppression subtractive hybridization (SSH), mirror orientation selection (MOS), was applied to screen genes differentially expressed in the P. patens protonema. Using reverse Northern blot analysis, differentially expressed clones were identified. The identified genes were involved mainly in metal binding and detoxification. One of these genes was an AP2 (APETALA2) domain-containing protein (PpACP1), which was highly up-regulated in the protonema. Alignment with other AP2/EREBPs (Ethylene Responsive Element Binding Proteins) revealed significant sequence homology of the deduced amino acid sequence in the AP2/EREBP DNA binding domain. Northern analysis under various stress conditions showed that PpACP1 was induced by ethephon, cadmium, copper, ABA, IAA, and cold. In addition, it was highly expressed in suspension-cultured protonema. We suggest that PpACP1 is involved in responses to metals, and that suspension culture enhance the expression of genes responding to metals.

• Molecular & Cellular Toxicology
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• v.2 no.1
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• pp.60-66
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• 2006

Methylmercury (MeHg), one of the heavy metal compounds, can cause severe damage to the central nervous system in humans. Many reports have shown that MeHg is poisonous to human body through contaminated foods and has released into the environment. Despite many studies on the pathogenesis of MeHg-induced central neuropathy, no useful mechanism of toxicity has been established so far. This study, using of suppression subtractive hybridization (SSH) method, was peformed to identify differentially expressed genes by MeHg in SH-SY5Y human neuroblastoma cell line. We prepared to total RNA from SH-SY5Y cells treated with solvent (DMSO) and $6.25\;{\mu}M\;(IC_{50})$ MeHg and performed forward and reverse SSH. Differentially expressed cDNA clones were screened by dot blot, sequenced and confirmed that individual clones indeed represent differentially expressed genes with real time RT-PCR. These sequences were identified by BLAST homology search to known genes or expressed sequence tags (ESTs). Analysis of these sequences may provide an insight into the biological effects of MeHg in the pathogenesis of neurodegenerative disease and a possibility to develop more efficient and exact monitoring system of heavy metals as ubiquitous environmental pollutants.

• Journal of fish pathology
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• v.19 no.3
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• pp.189-195
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• 2006

A subtracted cDNA library of a marine scuticociliate, Philasterides dicentrarchii, in response to 17β-estradiol exposure was constructed using suppression subtractive hybridization (SSH). As a result of SSH, 275 clones were isolated, and among them, only glutathione peroxidase (GPX) gene was isolated as an antioxidative enzyme responding to 17β-estradiol. The semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis revealed that the transcription of GPX gene of P. dicentrarchii was clearly increased by exposure to 17β-estradiol. The GPX transcription was also clearly increased by exposure to xenoestrogens such as bisphenol A (BPA) and genistein.

• Korean Journal of Plant Resources
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• v.19 no.3
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• pp.440-446
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• 2006

To investigate flowering related genes in winter-type oilseed rape (Brassica napus L. cv. Tammi), differentially expressed genes were isolated from leaves of the plant after low temperature treatment which is requirements for floral induction. As a result of suppression subtractive hybridization (SSH), 288 clones were randomly selected from SSH library. Using reverse Northern blot analysis, 150 of 288 clones were identified to be differentially expressed. Out of these 150 clones, 45 clones showed very high identities with the known genes. Four clones showed very high identities over 90% with metallothionein-like gene that is related to flowering-induced genes. Of these 4 clones, the cDNA clone, rfs-13, revealed high identity with meotallothionein-like protein in Arabidopsis thaliana (98%) and Brassica compestris (89%). Furthermore, gene expressed in immature flower stages was confirmed by Northern blot analysis.

• Tuberculosis and Respiratory Diseases
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• v.58 no.2
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• pp.129-134
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• 2005

Background : Ethambutol (EMB) is one of important first-line drug in the treatment of tuberculosis. Molecular techniques to detect embB gene mutations have been considered as an method to define the EMB resistance. We investigated the mutation rate within embB gene among EMB resistant strains using reverse hybridization techniques. Methods : We made 11 probes that had wild or mutated sequences containing codons 306, 406, or 497 within embB gene respectively. These probes were reverse-hybridized with PCR products amplified from embB gene which were isolated from 149 ethambutol resistant strains and 50 pan-susceptible strains. Results : Out of 149 ethambutol resistant strains, one hundred (67.1%) had mutation at least one base at codon 306, 406, or 497 in embB gene. Mutation at codon 306, 406, 497 were demonstrated in 75 (50.3%), 16 (10.7%), and 13 strains (8.7%) respectively. There were four strains that showed multi-mutation at codon 306 and codon 406 simultaneously. A high proportion (8.1%) had single mutation at codon 406. There was no mutation observed in embB gene among 50 pan-susceptible strains. Conclusion : Reverse hybridization will be useful technique for detection of gene mutation correlated to ethambutol resistance.

• The Journal of Korean Society of Virology
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• v.28 no.2
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• pp.175-182
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• 1998

We investigated the rate of hepatitis G virus infection among 50 patients who were not infected with the hepatitis C virus but showed symptoms of hepatitis. Viral RNA was extracted from the patients' sera and cDNA was synthesized and amplified by RT-PCR (reverse transcription-polymerase chain reaction) using random hexamer and 5 primers (470-20-1-77F, 470-20-1-211R, 470-20-1-211R-biotin, GV57-4512MF, GV57-4657MR). The amplified PCR products were confirmed by electrochemiluminescence (ECL), liquid hybridization (LH) and Southern blotting (SB). Among the 50 PCR products, by means of ECL, we found 4 samples to be positive and 5 samples to be indeterminate. The GV45-89M probe (5'-CYCGCTGRTITGGGGTGTACfGGAAGGC-3') was end-labelled with gamma-$^{32}P$ ATP and used for liquid hybridization with the PCR products. By using liquid hybridization, we detected specific bands from 4 positive sera and also from one indeterminate serum as determined by ECL. An 1.5% agarose gel electrophoresis of the 9 PCR products which were HGV positive or indeterminate as determined by ECL showed a 160bp band from 4 positive and one indeterminate serum. The 5 PCR products proved to be positive when SB was applied with the GV45-89M probe as well as when LH was applied. LH and SB were shown to have higher sensitivity and specificity than ECL. Two cases among 5 positive cases had relatively high SGOT, SGPT, ALP values when compared with other 48 cases. In summary, we confirmed hepatitis G virus infection in 5 cases among 50 Korean patients showing symptoms of viral hepatitis.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.7
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• pp.921-929
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• 2013

Huoyan goose is a Chinese local breed famous for its higher laying performance, but the problems of variety degeneration have emerged recently, especially a decrease in the number of eggs laid. In order to better understand the molecular mechanism that underlies egg laying in Huoyan geese, gene profiles in the pituitary gland of Huoyan geese taken during the laying period and ceased period were investigated using the suppression subtractive hybridization (SSH) method. Total RNA was extracted from pituitary glands of ceased period and laying period geese. The cDNA in the pituitary glands of ceased geese was subtracted from the cDNA in the pituitary glands of laying geese (forward subtraction); the reverse subtraction was also performed. After sequencing and annotation, a total of 30 and 24 up and down-regulated genes were obtained from the forward and reverse SSH libraries, respectively. These genes mostly related to biosynthetic process, cellular nitrogen compound metabolic process, transport, cell differentiation, cellular protein modification process, signal transduction, small molecule metabolic process. Furthermore, eleven genes were selected for further analyses by quantitative real-time PCR (qRT-PCR). The qRT-PCR results for the most part were consistent with the SSH results. Among these genes, Synaptotagmin-1 (SYT1) and Stathmin-2 (STMN2) were substantially over-expressed in laying period compared to ceased period. These results could serve as an important reference for elucidating the molecular mechanism of higher laying performance in Huoyan geese.