• Journal of Plant Biotechnology
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• v.33 no.4
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• pp.309-313
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• 2006

Discovery of single nucleotide polymorphisms (SNPs), including small insertions and deletions, is one of the hot topics in genetic research. The most common type of sequence variant consists of single base differences or small insertions and deletions at specific nucleotide positions. Significance of SNPs in rice is increasing for genetic research, positional cloning and molecular breeding. $F_2$ 170 lines and $F_3$ 194 lines derived from Sangjuchalbyeo/HR13721-53-3-1-3-3-2-2 Were used for Searching SNP markers related to bacterial blight resistance. Sangjuchalbyeo is susceptible to bacterial blight, but HR13721-53-3-1-3-3-2-2 has Xa1 gene resistant to bacterial blight. Individual lines were inoculated with $K_1$ race of bacterial blight and resistant or susceptible was evaluated after 3 weeks from inoculation. The genotypes of population were analysed by PCR-RFLP for SNP marker developing. The segregation of $F_2\;and\;F_3$ population showed almost 3:1, 1:1 ratio, respectively. Analysis of genotype using SNP marker is capable of confirming resistance for $K_1$ race and genotype through amplifying the gene using 16PFXal primer and digested the PCR product with Eco RV. There were close relation between resistance test for $K_1$ race and SNP marker genotype. Especially, DNA analysis using SNP marker is capable of judging homozygote/heterozygote in $F_2$ population compared with resistant test for Kl race. So, it seems to improve the selection efficiency in disease resistant breeding.

• Journal of Plant Biotechnology
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• v.32 no.3
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• pp.167-173
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• 2005

Three different cDNAS for cinnamate 4-hydroxylase (C4H) which are involved in the second step of the general phenylpropanoid pathway were isolated and designated as pc4h1 (1,755 bp), pc4h2 (1,655 bp), and pc4h3 (1,316 bp), respectively. The nucleotide sequence analysis revealed that both pc4h1 and pc4h2 clones encode polypeptides of 505 amino acids frame but pc4h3 clone was truncated at the 5'-end of coding region. The alignment of the deduced amino acid sequences showed that PC4H1 and PC4H2 are highly homologous (95.8% identical) with each other and contain three conserved domains which are typical in cytochrome P450 monooxygenase: proline-rich region, threonine-containing binding pocket for the oxygen molecule, and heme binding region. In addition, result of the phylogenic tree analysis revealed that both pepper C4Hs belong to Class 1. pc4h2 transcription was strongly induced in wounded fruit (400%) and root (200%) relative to its very low basal level but not in leaf or stem tissue. In case of pc4h1, the basal level of transcription was higher than pc4h2 but induction by wounding was lower in fruit and root while leaf and stem tissues did not respond to wounding. The basal level of pc4h3 transcripts was not, if any, detectable and response to wounding was not observed.

• Journal of Plant Biotechnology
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• v.42 no.1
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• pp.25-33
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• 2015

A PyrcpPR-10 gene with differentially expressed was isolated by using the suppression subtractive hybridization assay between '93-3-98' (highly resistant against scab caused by Venturia nashicola) and 'Sweat Skin'(highly susceptible) and analyzed the expression pattern according to organs and cultivars. The full length of PyrcpPR-10 was cloned as 743bp with 480bp's ORP, and was determined to encode a protein of 159 amino acid residues. On analyzing PyrcpPR-10 gene sequence compared with resistant and susceptible cultivars, 'Hwangsilri' (resistant), 'Gamcheonbae' (moderately resistant), 'Wonhwang' (moderately susceptible), 'Niitaka' (highly susceptible), and 'Sweat Skin' (highly susceptible) had identical gene sequence but 'Bartlett' (highly resistant) showed partly different sequences. The deduced amino acid sequence showed 64 ~ 98% homology and had the GXGGXG motif to known amino acid of other plants PR-10 by the BLAST X analysis. Among several organs or tissues, petal was showed highest expression level of PyrcpPR-10 gene followed by leaf, floral axis, bud, and bark. The expression level of PyrcpPR-10 gene was dramatically increased at 24 hr after inoculation in all cultivars and also up-regulated in accordance with resistant degree of cultivars. While resistant cultivars ('Bartlett', '93-3-98', and 'Hwangsilri') induced relatively high expression level of PyrcpPR-10 gene, susceptible cultivars ('Niitaka', and 'Sweat Skin') showed low expression level. PyrcpPR-10 gene is assumed that it is directly connected with defense mechanisms to pear scab.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.12 no.2
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• pp.140-149
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• 2009

Purpose: Helicobacter pylori infection is probably acquired in childhood and persists as an asymptomatic infection for decades in most individuals. It is unclear why only a minority of those infected develop a clinical manifestation, even in childhood, such as peptic ulcer disease. H. pylori infection activates local immune responses and causes lymphocyte infiltration in the gastric mucosa. We have previously reported that both T and B cells in the lamina propria play important roles in the local immune response of H. pylori-infected children. The aim of this study was to investigate the association between H. pylori genotypes and gastric mucosal lymphocytes. Methods: Twenty-five H. pylori-infected children (10 with peptic ulcer disease and 15 with gastritis) were enrolled in this study. We investigated the genotypes (cagA, cagE, vacA, and babA2) and evaluated the association with clinical manifestations, histopathology, and gastric mucosal lymphocytes. Results: The prevalence of cagA, cagE, vacA s1m1, and babA2 was 80%, 60%, 84%, and 88%, respectively. The most prevalent (68%) combination of cagA, vacA, and babA2 genotypes was cagA+/vacA s1m1+/babA2+. H. pylori genotypes were not associated with clinical manifestations, histopathology, or gastric mucosal lymphocytes. Conclusion: There was no association between the cagA, cagE, vacA, or babA2 status and gastric mucosal lymphocytes. The role of the host immune response in relation to H. pylori genotypes and disease potential in children needs further studies.

• Pediatric Infection and Vaccine
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• v.5 no.1
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• pp.79-87
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• 1998

Purpose : The P1 protein of Mycoplasma pneumoniae mediates the attachment of the pathogen to its host cell and elicits a strong humoral immune response during infection with this organism. Mycoplasma pneumoniae strains can be classified into two groups(I and II) by PCR method of P1 cytadhesin gene. In this study, we evaluated the prevalence, epidemiological and clinical characteristics of each group. Methods : From 155 patients with Mycoplasma pneumoniae, who admitted to the Department of Pediatrics, Sung-Ae and Kwangmyung Sung-Ae Hospital between November 1996 and October 1997, we collected their throat swabs or nasopharyngeal aspirates for DNA extraction and serum for indirect hemagglutination test of Mycoplasma pneumoniae. The group specific PCR amplification were performed using specific oligonucleotide primers designed for P1 gene genotyping. Results : Group I(137 patients, 88.4%) occurred frequently than group II(18 patients, 11.6%). In both group, the most prevalent season was winter in 1996(Nov. to Dec.) and fall in 1997(Aug. to Oct.) The prevalent age was four to six years old. The number of male was more than female in both group; Group 1(1.2:1), Goup 2(1.6:1). No significant relationship were found between two groups in duration of fever and hospital days(P>0.05). The rate of high antibody titers(>1:5120) was lower in group I(6/137, 4.4%) than group II(2/18, 11.1%). Conclusion : Group I was much more prevalent than group II during 1996~1997 in Korea. There was no difference between two groups in epidemiological and clinical parameters except the rate of high antibody titers. Further follow-up survey will be needed for the epidemiologic and clinical studies of Mycoplasma pneumoniae in Korea.

• Pediatric Infection and Vaccine
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• v.11 no.1
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• pp.59-72
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• 2004

Purpose : Rotaviruses are the major cause of gastroenteritis in infants and young children worldwide. It is important to get the epidemiologic data of rotavirus genotype for the application of rotavirus vaccine. So we tried to investigate the distribution of rotavirus genotypes with RT-PCR. Methods : A total of 120 rotavirus latex agglutinin test positive stool samples were collected continually from 120 children from Sep. 2000 to Apr. 2001. Rotavirus P(VP4), G (VP7) genotypes were determined by RT-PCR. Results : The genotype was identified in 116 stool samples of total 120 samples(96%). The incidence of G genotype was as follow; G1 17(14.2%), G2 74(61.7%), G4 1(0.8%), G9 1(0.8%). There were four cases of multiple genotypes; G1/G2, G1/G4, G1/G9, G8/G9 and genotype of G3, G8 were not found. Twenty three(19.2%) samples were nontypeable. The incidence of P was as follow; P[4] 77(64.2%), P[6] 22(18.3%), P4/P6 12(10%), P[4]/P[8] 1(0.8%) p[8] 1(0.8%). Seven(5.9%) samples were nontypeable. Conclusion : Various combinations of G and P genotypes were observed. Most rotavirus strains were P[4]G2 62(51.74%), followed by P[6]G2 7(5.8%), and P[6]G1 7(5.8%), P[4/P[6] G1 4(3%), P[4]/P[6]G2 4(3%), P[4]G1 3(2.5%), P[8]G2 1(0.8%), P[4]G4 1(0.8%) in Kyoungsangnamdo, Korea during 2000~2001.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 1995.06b
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• pp.77-96
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• 1995

To obtain basic informations for the improvement of seed potato production in Korea, some etiological properties of potato virus Y(PVY) distributed in the major seed potato production area(Daekwanryeong) were characterized, and the nucleotide and amino acid sequences of the coat protein gene of the PVY strains isolated were analyzed. PVY strains in Daekwonryeong, an alpine area, were identified to be two strains, PVYo and PVYN by symptoms of indicator plants, and their distribution in potato fields was similar. Major symptom on potato varieties by PVY was grouped as either mosaic alone or mosaic accompanied with veinal necrosis in the lower leaves. The symptom occurrence of the two symptoms was similar with Irish Cobbler, but Superior showed a higher rate of mosaic symptom than the other. The PVY strain which was isolated from potato cv. Superior showing typical mosaic symptoms produced symptoms of PVY-O on the indicator plants of Chenopodium amaranticolor, Nicotiana tabacum cv. Xanthi nc and Physalis floridana, but no symptom o Capsicum annum cv. Ace. Moreover, results from the enzyme-linked immunosorbent assay with monoclonal and polyclonal antibodies showed that the isolated PVY reacts strongly with PYV-O antibodies but does not react specifically with PVY-T antibodies. The purified virus particles were flexious with a size of 730$\times$11nm. On the basis of the above characteristics, the strain was identified to be a PVY-O and named as of PVY-K strain. The flight of vector aphids was observed in late May, however, the first occurrence of infected plants was in mid June with the bait plants surrounded with PVY-infected potato plants and early July with the bait plants surrounded with PVY-free potato plants. PVY infection rates by counting symptoms on bait plants (White Burley) were 1.1% with the field surrounded with PVY-free potato plants and 13.7% the fields surrounded with PVY-infected potato plants, showing the effect of infection pressure. The propagated PVY-K strain on tobacco(N. sylvestris) was purified, and the RNA of the virus was extracted by the method of phenol extraction. The size of PVY-K RNA was measured to be 9, 500 nucleotides on agarose gel electrophoresis. The double-stranded cDNAs of PVY-K coat protein(CP) gene derived by the method of polymerase chain reaction were transformed into the competent cells of E. coli JM 109, and 2 clones(pYK6 and pYK17) among 11 clones were confirmed to contain the full-length cDNA. Purified plasmids from pYK17 were cut with Sph I and Xba I were deleted with exonuclease III and were used for sequencing analysis. The PVY-K CP gene was comprised of 801 nucleotides when counted from the clevage site of CAG(Gln)-GCA(Ala) to the stop codon of TGA and encoded 267 amino acids. The molecular weight of the encoded polypeptides was calculated to be 34, 630 daltons. The base composition of the CP gene was 33.3% of adenine, 25.2% of guanine, 20.1% of cytosine and 21.4% of uracil. The polypeptide encoded by PVY-K CP gene was comprised of 22 alanines, 20 threonines, 19 glutamic acids and 18 glycines in order. The homology of nucleotide sequence of PVY-K CP gene with those of PVY-O(Japan), PVY-T(Japan), PVY-TH(Japan), PVYN(the Netherlands), and PVYN(France) was represented as 97.3%, 88.9%, 89.3%, 89.6% and 98.5%, respectively. The amino acid sequence homology of the polypeptide encoded by PVY-K CP gene with those encoded by viruses was represented as 97.4%, 92.5%, 92.9%, 92.9%, and 98.5%, respectively.

• Journal of Life Science
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• v.15 no.2 s.69
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• pp.253-260
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• 2005

Mitotic centromere-associated kinesin (MCAK), which is a member of the Kin I (internal motor domain) subfamily of kinesin-related proteins, is known to play a role in mitotic segregation of chromosome during M phase of the cell cycle. In the present study, we have produced a rat polyclonal antibody using human MCAK (HsMCAK) expressed in E. coli as the antigen. The antibody specifically recognized the HsMCAK protein (81 kDa), and could detect its nuclear localization in human Jurkat T cells and 293T cells by Western blot analysis. The specific stage of the cell cycle was obtained through blocking by either hydroxyl urea or nocodazole and subsequent releasing from each blocking for 2, 4, and 7 h. While the protein level of HsMCAK reached a maximum level in the S phase with slight decline in the $G_{2}-M$ phase, the electrophoretic mobility shift from $p81^{MCAK}\;to\;p84^{MCAK}$ began to be induced in the late S phase and reached a maximum level in the $G_{2}/M $ phase, and then it disappeared as the cells enter into the $G_{1}$ phase. Immunocytochemical analysis revealed that HsMCAK protein localized to centrosome and nucleus at the interphase, whereas it appeared to localize to the spindle pole, centromere of the condensed mitotic DNA, spindle fiber, or midbody, depending on the specific stage of the M phase. These results demonstrate that a rat polyclonal antibody raised against recombinant HsMCAK expressed in E. coli specifically detects human MCAK, and indicate that the electrophoretic mobility shift from $p81^{MCAK}\;to\;p84^{MCAK}$, which may be associated with its differential intracellular localization during the cell cycle, fluctuates with a maximum level of the shift at the $G_{2}-M$ phase.

• Korean Journal of Veterinary Research
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• v.44 no.2
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• pp.241-249
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• 2004

As a part of epidemiologic investigation of tsutsugamushi disease, the wild rodents which were captured in Gyeongnam area were diagnosed with indirect immunofluorescent antibody assay (IFA) and Polymerase Chain Reaction (PCR) to find if they have an antibody against Orientia tsutsugamushi. The conclusion was drawn as followings. (1) The captured 58 wild rodents showed that the subspecies distribution of Apodemus agrarius was 86.2%, Microtus fortis was 8.6% and Crocidura lasiura was 5.2%. (2) The antibody positive rate of O. tsutsugamushi Gilliam, Karp, Karto and Boryong by IFA method was 32.0% in Apodemus agrarius among 50 wild rodents and 40.0% in Microtus fortis among 5 wild rodents, respectively. It was negative in the case of all the 3 Crocidura lasiura. (3) The antibody titers on Apodemus agrarius, Microtus fortis and Crocidura lasiura against Gilliam, Karp, Karto and Boryong were measured between 1:20 and 1:640. The antibody titer against each antigen was in the order Boryong>Gilliam>Karp. (4) O. tsutsugamushi was detected from the blood, spleen and kidney from the artificially infected mice by IFA and PCR. IFA showed the positive response between 3 and 18 days after inoculation. On the other hand, positive response was found from all the samples by PCR. (5) From PCR of the genomic DNA extracted from the blood, spleen and kidney samples of the captured wild rodents, Boryong-specific amplification product with size of 210 bp, which is particular in Boryong, was detected from spleen and kidney samples, but not detected in the blood. (6) Boryong-specific amplification product was detected from spleen and kidney samples which were obtained at 3, 6, 12, 18 and 24 days after the infection with Boryong. But, it wasn't detected from the uninfected samples. (7) From PCR of spleen and kidney samples of the captured wild rodents, it was found that positive rate of O. tsutsugamushi in Apodemus agrarius and Microtus fortis were 25.0% (4/16) and 20.0% (1/5), respectively. From the above results, it can be concluded that Apodemus agrarius resided in Gyeongnam area carried O. tsutsugamushi and PCR method might be a simple, precise, rapid and useful diagnostic tool than IFA for the diagnosis of O. tsutsugamushi.

• The Korean Journal of Nuclear Medicine
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• v.28 no.2
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• pp.220-226
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• 1994

This study was performed to evaluate the clinical applicability of the reverse transcription polymerase chain reaction (RT-PCR ) kit of HCV-RNA using biotinylated and radioiodinated primers. Study subjects were 118 patients with positive anti-HCV. HCV-RNA in patient's serum was extracted by guanidium thiocyanate method. After first amplification, the product was reamplified by primers labelled with biotin and I-125. The final amplification product was defected by counting the radioactivity after incubation in avidin coated tubes. In 51 samples, the test was repeated for evaluation of reproducibility. This new method was also compared with conventional RT-PCR methods in 34 samples from patients with chronic liver disease. The results were as follows ; 1) HCV-RNA was positive in 85(97%)of 88 patients with chronic liver disease, and in 23 (73%) of 30 patients with normal liver function. 2) In comparison with conventional method, HCV-RNA was detected in 32(94%) of 34 patients with new method, whereas in 27(79%) of the same group with conventional method. 3) Repeated test with new method in 52 samples demonstrated 82% of concordant result. In conclusion, new method with biotinylated and radioiodinated primers was more sensitive than conventional method. However, great care must be taken for quality control because there were considerable interassay variation and possiblity of false positivity and false negativity.