• Tuberculosis and Respiratory Diseases
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• 제72권2호
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• pp.156-162
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• 2012

Background: The main goal of this study was to evaluate the diagnostic efficacy of reverse transcription-nested polymerase chain reaction (RT-nested PCR) in bronchial washing fluid with MAGE A1-6 common primers for the detection of lung cancers invisible by bronchoscopy. Methods: To determine the expression of MAGE A1-6 gene in 189 lung cancers diagnosed by conventional fluoroscopy-guided lung biopsy and 89 cancer-free controls, RT-nested PCR was performed in bronchial washing specimens. We analyzed MAGE A1-6 RT-nested PCR data according to tumor histology, stage, size, and compared them with cytological data. Results: 189 patients (111 cases in adenocarcinoma, 47 cases in squamous cell carcinoma, 22 cases in small cell lung carcinoma, and 9 cases in other cancers) and 89 benign patients were investigated. The expression of MAGE was performed by nested RT-PCR using common MAGE primer. Among 189 cancer patients, the expression rate of MAGE was 49.2%, and the positive predictive value was 89.4%. However, the expression rate of MAGE in patients with benign lesions was 12.4%. In peripheral lung cancer, the positive rate of MAGE expression was 57.4% in squamous cell carcinoma, 44.1% in adenocarcinoma and 59.1% in small cell lung cancer. Whereas the expression rate of bronchial washing cytology in peripheral lung cancer was 9.0% (p=0.011). Conclusion: MAGE RT-PCR in bronchial washing fluid gave us promising data for the detection of peripheral lung cancer. It could be a useful method for selecting diagnostic tools for peripheral lesions.

• Journal of Crop Science and Biotechnology
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• 제11권2호
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• pp.127-134
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• 2008

Tobamoviruses are the major viral pathogens of tomato and bell pepper. The preliminary results showed that Acibenzolar-Smethyl(ASM; S-methylbenzo(1,2,3) thiadiazole-7-carbothiate) pre-treatment to tomato and tobacco plants reduces the concentration of Tomato mosaic tobamovirus(ToMV) and Tobacco mosaic tobamovirus(TMV) in tomato and bell pepper seedlings, respectively. Pre-treatment of the indicator plant(Nicotiana glutinosa) with the ASM followed by challenge inoculation with tobamoviruses produced a reduced number and size of local lesions(67 and 79% protection over control to TMV and ToMV inoculation, respectively). In order to understand the mechanism of resistance the gene expression profiles of antiviral genes was examined. RT-PCR products showed higher expression of two viral resistance genes viz., alternative oxidase(AOX) and RNA dependent RNA polymerase(RdRp) in the upper leaves of the ASM-treated tomato plants challenge inoculation with ToMV. Further, the viral concentration was also quantified in the upper leaves by reverse transcription PCR using specific primer for movement protein of ToMV, as well as ELISA by using antisera against tobamoviruses. The results provided additional evidence that ASM pre-treatment reduced the viral movement to upper leaves. The results suggest that expressions of viral resistance genes in the host are the key component in the resistance against ToMV in the inducer-treated tomato plants.

• 한국발생생물학회지:발생과생식
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• 제21권3호
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• pp.327-333
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• 2017

Gonadotropins are heterodimers consisting an alpha chain ($Cg{\alpha}$) and a beta chain. Interestingly, presence of complicated $LH-{\beta}$ transcripts in rat testis was accidently found; testicular $LH-{\beta}$ transcripts were confined in seminiferous tubules to spermatids, and the translated products were localized in the elongated spermatids. We hypothesized that mouse testis has potential to produce the tissue specific $LH-{\beta}$ with similar structure to the rat testicular forms. To verify our hypothesis, we examined the adult mouse (ICR) testis using RT-PCR and immunohistochemistry. The PCR revealed the presence of the identical products in the reactions for three LH subunit types. The expected product sizes for mouse $Cg{\alpha}$ and $LH-{\beta}$ known as pituitary type were 224 bp and 503 bp, respectively. The testicular type $LH-{\beta}$ products were produced by a primer set based on the rat sequences, with unexpected size of 800 bp. Sequencing revealed that the proximal and distal parts (2-82 and 661- 773 bp, respectively) were homologous to rat testicular $LH-{\beta}$ cDNA, and middle part (83-660 bp) was a unique mouse-specific region. Both $Cg{\alpha}$ and $LH-{\beta}$ positive signals were in the round and elongated spermatids and mature sperms, and the $LH-{\beta}$ signals were more intense. In conclusion, our study demonstrated that the presence and localization of the LH subunits in mouse testis. Further studies will be needed to understand the precise structure and function of mouse testicular LH.

• Journal of Plant Biotechnology
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• 제31권1호
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• pp.19-23
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• 2004

본 연구는 붓꽃속 식물의 형태적 특성 조사와 RAPD분석을 통해 유전적 다양성과 근연성을 밝히고 유전자 도입을 위한 기초적인 자료를 얻기 위하여 실시하였다. RAPD를 이용하여 국내에서 수집한 자생붓꽃종의 DNA다형성을 조사한 결과 40개의 primer중 8개의 유용한primer가 선발되었고 이들 primer는 108개의 밴드를 나타내었다. 이들 중 다형성 밴드는 107 개였다. Jaccard (1-S$_{J}$ )법에 의한 비유사도 계수는 0.095∼0.699로 나타났고, dendrogram에서 나타난 유전적 근연성은 크게 세 개의 군으로 분류되었다. I군은 연미붓꽃, II군은 타래붓꽃, 제비붓꽃, 노랑꽃창포, 노랑무늬붓꽃이 속해 있었으며, III군은 금붓꽃, 각시붓꽃, 붓꽃, 꽃창포가 속해 있었다.

• 한국식물병리학회지
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• 제10권3호
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• pp.228-234
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• 1994

Genomic RNA was extracted from Cy strain of odontoglossum ringspot tobamovirus (ORSV-Cy) isolated from infected leaves of tobacco cv. Samsun. Size of the genomic RNA was about 6.6 kb in length. The genomic RNA was fractionated using Sephadex G-50 column chromatography into 2 fractions. They were polyadenylated at their 3'-end using E. coli poly(A) polymerase. Polyadenylated viral RNA was recovered by oligo (dT) primer adapter containing NotI restriction site and Moloney murine leukemia virus SuperScript reverse transcriptase (RNase H-). Second-strand cDNA was synthesized by using E. coli DNA ligase, E. coli DNA polymerase I and E. coli RNase H. Recombinant plasmids containing cDNAs for ORSV-Cy RNA ranged from about 800 bp to 3,000 bp. Among the selected 238 recombinants, pORCY-124 clone was the largest one covering 3'-terminal half of the viral RNA. This clone contained two restriction sites for EcoRI and XbaI and one site for AccI, AvaI, BglII, BstXI, HindIII, PstI, and TthIII 1. respectively. The clone contained partial viral replicase, a full-length movement protein and a complete coat protein genes followed by a 3' untranslated region of 414 nucleotides based on restriction mapping and nucleotide sequencing analyses. Clones pORCY-028, -068, -072, -187 and -224 were overlapped with the pORCY-124. Clones pORCY-014 and -095 covered 5' half upstream from the middle region of the viral RNA, which was estimated based on restriction mapping and partial sequence analysis. Constructed cDNA library covered more than 90% of the viral genome.

• 한국발생생물학회지:발생과생식
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• 제14권4호
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• pp.261-268
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• 2010

As in the O. mykiss electrophoretic profiles of RNA, the signals of each RNA sample from 9 individual tissues such as liver, muscle, brain, heart, pituitary gland, kidney, intestine, spleen and gill similar to positive control were obtained. The tissue distributions of the complimentary DNA (cDNA) of O. mykiss four genes were analyzed using quantitative real-time PCR with primer sets for tissue expression analysis. In this rainbow trout species, author obtained bands of various sizes, ranged from 700 bp to 1,400 bp. A dissociation curve was made at the end of each run to make sure that there was no non-specific amplification. Supplementarily, the Ct of each DNA was compared. The Ct values of vinculin with rainbow trout tissues were determined in a manner similar to those for agouti-related protein (AgRP) and melanocortin receptors (MC4R I and MC4R II). Further, obtained Cts for standard curve of each DNA were affected by specific product (vinculin, AgRP and MC4R II genes). After several experiments with four individual genes of rainbow trout, author estimated a variation ratio of the mean Ct value of the DNA extracted using the comparative CTt method was 37.27, and the standard deviation was 5.33. The correlation coefficient between the Ct values and the concentration of cDNA was -0.98, -0.99, -0.91 and -0.86, respectively (vinculin, AgRP, MC4R I and MC4R II genes). Since this correlation showed high linearity, the straight line obtained was used as a standard for the O. mykiss tissues reared in aquarium. A PCR efficiency of 100% is ideally achieved when the slopes are close to the theoretical value of -3.31. According to quantification method, the results of quantification are strongly affected by the DNA fragmentation. The size of most DNA fragments obtained from various tissues of rainbow trout used in the experiment was approximately 100 bp. According to the four slopes, an efficiency of nearly 100% was estimated for four genes detection methods. Additionally, further analysis with more individuals and primers will be required to fully establish optimization in rainbow trout.

• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.676-684
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• 1998

Baculovirus transfer and expression vectors with Hyphantria cunea nuclear polyhedrosis virus (HcNPV) were constructed. An initial transfer vector, pHcEV, constructed using HcNPV was previously reported (Park et al. 1993. J. Kor. Soc. Viral. 23: 141-151). Herein, the size of the vector was properly reduced, and a functionally perfect vector was constructed and named pHcEV-IV (6.7 kb). The vector has a 2.2-kb HcNPV DNA sequence in the 5'-flanking region of the vector's polyhedrin gene promoter. The 1.8-kb HcNPV DNA sequence, poly A signal sequence, T3 primer sequence, and 13 multicloning site sequences, in order, were ligated in front of the translation start codon of the polyhedrin gene. The cloning indicating marker lacZ gene was inserted into the pHcEV-IV, named pHcEV-IV-lacZ, and transferred into the wild-type virus. Recombinant expression virus, lacZ-HcNPV, was constructed by replacing the lacZ gene in the pHcEV-IV-lacZ with the polyhedrin gene of the wild-type virus. The recombinant virus was isolated from blue plaques that produce $\beta$-galactosidase without polyhedra. The lacZ gene insertion was confirmed by Southern hybridization analysis. The expression of the lacZ gene in Spodoptera frugiperda cells infected with the lacZ-HcNPV was examined by SDS-PAGE and colorimetric assay. One 116-kDa LacZ protein band appeared on the PAGE. The production rate of the $\beta$-galactosidase was approximately 50 international units (IU) per min per ml between 2 to 5 days postinfection (p.i.). The highest activity occurred at five days p.i. was 170 IU/min/$m\ell$. The enzyme activity first appeared about 20 h p.i. as measured by colorimetric assay.

• 한국수산과학회지
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• 제36권3호
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• pp.239-246
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• 2003

We have examined the 16S-23S rRNA intergenic spacer region (ISR) of Vibrio vulnificus KCTC 2959. ISRs were amplified by primers complementary to conserved regions of 16S and 23S rRNA genes. ISR amplicons were cloned and sequenced. Analysis of the ISR sequences showed that V. vulnificus KCTC 2959 contains five types of polymorphic ISRs. Size of ISRs ranged from 424 to 741 bp in length and the number of tRNA genes ranged from one to four. The ISRs were designated as ISR-E $(tRNA^{Glu}),\;ISR-IA\;(tRNA^{Ile}-tRNA^{Ala})$, ISR-EKV $(tRNA^{Glu}-tRNA^{Lys}-tRNA^{Val})$, ISR-IAV $(tRNA^{Ile}-tRNA^{Ala}-tRNA^{val})$ and ISR-EKAV $(tRNA^{Glu}-tRNA^{Lys}-tRNA^{Ala}-tRNA^{Val})$ based on their tRNA genes. Multiple alignment of representative sequences from different Vibrio species revealed several domains of high sequence variability. We used the sequences of variable domains to design species-specific primer for detection PCR. Specificity of the primers was examined using genomic DNA prepared from 18 different Vibrio species. The results showed that the PCR using primers designed in this study can be used to detect V. vulnificus from other Vibrio species.

• Asian-Australasian Journal of Animal Sciences
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• 제21권9호
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• pp.1233-1237
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• 2008

The genetic variability within and among three deer species in Malaysia, namely Cervus nippon (sika), Cervus timorensis (rusa) and Cervus unicolor (sambar), were evaluated using the RAPD technique. The DNA extracted from the buffy coat of 34 sika, 38 rusa and 9 sambar were analysed using ten primers that gave bands which showed good resolution. The primers generated 164 RAPD markers in total, and these ranged in size from 150 to 900 bp. The percent of polymorphism of the bands generated per primer ranged from 66.66-93.33% for rusa, 36.84-61.14% for sambar and 52.38-100% for sika. The overall percent polymorphism observed for the 164 RAPD markers was 99.39%. The results revealed five exclusive, monomorphic markers for sambar and one exclusive, monomorphic marker for sika; none was observed for rusa. However, these cannot be declared as markers for the identification of the species without analysis of more samples, populations and species. The means of within population genetic distances, based on Dice's and Jaccard's similarity indices, were similar for the rusa (0.383 and 0.542, respectively) and sika (0.397 and 0.558, respectively) populations with the sambar population being the least variable (0.194 and 0.323, respectively). The Dice based genetic distances within the species ranged from 0.194 to 0.397 and the genetic distances among the species were 0.791-0.911. The genetic distances based on Dice's and Jaccard's similarity indices between the rusa and sambar were 0.556 and 0.713, between the rusa and sika populations were 0.552 and 0.710, and between sambar and sika were 0.622 and 0.766, respectively.

• 대한의생명과학회지
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• 제17권2호
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• pp.151-155
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• 2011

Short tandem repeats (STRs) loci are the genetic markers used for forensic human identity test. With multiplex polymerase chain reaction (PCR) assays, STRs are examined and measured PCR product length relative to sequenced allelic ladders. In the repeat region and the flanking region of the commonly-used STR may have DNA sequence variation. A mismatch due to sequence variation in the DNA template may cause allele drop-out (i.e., a "null" or "silent" allele) when it falls within PCR primer binding sites. The STR markers were co-amplified in a single reaction by using commercial PowerPlex$^{(R)}$ 16 system and AmpFlSTR$^{(R)}$ Identifiler$^{(R)}$ PCR amplification kits. Separation of the PCR products and fluorescence detection were performed by ABI PRISM$^{(R)}$ 3100 Genetic Analyzer with capillary electrophoresis. The GeneMapper$^{TM}$ ID software were used for size calling and analysis of STR profiles. Here, this study described a forensic human identity test in which allelic drop-out occurred in the STR system D18S51. During the course of human identity test, two samples with a homozygous (16, 16 and 21, 21) genotype at D18S51 locus were discovered using the PowerPlex$^{(R)}$ 16 system. The loss of alleles was confirmed when the samples were amplified using AmpFlSTR$^{(R)}$ Identifiler$^{(R)}$ PCR amplification kit and resulted in a heterozygous (16, 20 and 20, 21) genotype at this locus each other. This discrepancy results suggest that appropriate measures should be taken for database comparisons and that allele should be further investigated by sequence analysis and be reported to the forensic community.