• Applied Biological Chemistry
• /
• v.38 no.1
• /
• pp.55-62
• /
• 1995

To understand the molecular structure of Korean garlic viruses, cDNA cloning of virus genomic RNA was attempted. Virus particles were isolated from virus-infected garlic leaves and a cDNA library was constructed from garlic virus RNA. One of these clones, S81, selected by random sequencing has been identified as a member of potexvirus group other than potyvirus and carlavirus. The clone is 873 bp long contains most of the coat protein (CP) coding region and 3'-noncoding region including poly(A) tail. A putative polyadenylation signal sequence (AAUAAA) and the hexanucleotide motif (ACUUAA), a replicational cis-acting element conserved in the 3'-noncoding region of potexvirus RNAs are noticed. The clone S81 shows about 30-40% identity in both nucleotide and amino acid sequences with CPs of potexviruses. The genome size of the virus was analysed to be 7.46 knt by Northern blot analysis, which was longer than those of other potexviruses. The open reading frame encoding CP was expressed as a fusion protein (S81CP) in Escherichia coli and the recombinant protein was purified by immobilized metal binding affinity chromatography. Polyclonal antibody was raised against S81CP in rabbit to examine the occurrence of garlic potexvirus in Korean garlic plants by immunoblot analysis. Two virus protein bands of Mr 27,000 and 29,000 from garlic leaf extract of various cultivars reacted with the antibody. It was shown that Mr 27,000 band might not be a degradation product of Mr 29,000 band, suggesting that two types of potexvirus different in size of coat protein could exist in Korean garlic plants.

• Horticultural Science & Technology
• /
• v.35 no.3
• /
• pp.344-360
• /
• 2017

Zoysiagrasses are important turf plants used for school playgrounds, parks, golf courses, and sports fields. The two most popular zoysiagrass species are Zoysia japonica and Zoysia sinica. These are widely distributed across different growing zones and are morphologically distinguishable from each other; however, it is phenotypically difficult to differentiate those that grow along the coastal line from those in beach area habitats. A combination of morphological and molecular approaches is desirable to efficiently identify these two plant cultivars. In this study, we used a rapid identification system based on DNA barcoding of the nrDNA-internal transcribed spacer (ITS) regions. The nrDNA-ITS regions of ITS1, 5.8S nrDNA, and ITS2 from Z. japonica, Z. sinica, Agrostis stolonifera, and Poa pratensis were DNA barcoded to classify these grasses according to their molecular identities. The nrDNA-ITS sequences of these species were found at 686 bp, 687 bp, 683 bp, and 681 bp, respectively. The size of ITS1 ranged from 248 to 249 bp, while ITS2 ranged from 270 to 274 bp. The 5.8S coding region ranged from 163 - 164bp. Between Z. japonica and Z. sinica, nineteen (2.8%) nucleotide sites were variable, and the G+C content of the ITS region ranged from 55.4 to 63.3%. Substitutions and insert/deletion (indel) sites in the nrDNA-ITS sequence of Z. japonica and Z. sinica were converted to cleaved amplified polymorphic sequence (CAPS) markers, and applied to the Zoysia grasses sampled to verify the presence of these markers. Among the 62 control and collected grass samples, we classified three groups: 36 Z. japonica, 22 Z. sinica, and 4 Z. japonica/Z. sinica hybrids. Morphological classification revealed only two groups; Z. japonica and Z. sinica. Our results suggest that used of the nrDNA-ITS barcode region and CAPS markers can be used to distinguish between Z. japonica and Z. sinica at the species level.

• Clinical and Experimental Pediatrics
• /
• v.45 no.2
• /
• pp.183-191
• /
• 2002

Purpose : X-linked agammaglobulinemia(XLA) is an immunodeficiency caused by abnormalities in Bruton's tyrosine kinase(Btk), and is characterized by a deficiency of peripheral blood B cells. We studied the cytoplasmic expression of Btk protein and analyzed the Btk gene in peripheral blood mononuclear cells from two siblings and one cousin with XLA, as well as additional family members. Methods : Btk protein expression was analyzed by flow cytometry. Isolation of the coding sequence of the Btk gene was performed by amplification using the reverse transcription-polymerase chain reaction(RT-PCR) technique. Sequence alterations were screened by the single-stranded conformation polymorphism(SSCP) method and characterized by standard sequencing protocols. Results : Cytoplasmic expression of Btk protein in monocytes was not detected in three patients with XLA. In addition, Btk protein analysis clearly showed cellular mosaicism in monocytes from four obligate carriers, findings further supported by SSCP. A single base pair mutation(T to C) in Btk-exon three, which encodes the PH domain, was identified in four XLA patients. A diagnostic sequencing analysis was established to detect heterozygotic pattern in 4 carrier females. Furthermore, we found significant clinical heterogeneity in individuals with the same gene mutation. Conclusion : The implicating genetic alteration provided valuable clues to the pathogenesis of XLA in Korea and the flow cytometric analysis was suggested as a useful tool for rapid detection of XLA patients and carriers. The present study has identified a genetic mutation in the Btk coding region and demonstrated heterogeneity in clinical manifestations among patients with the same mutation. A flow cytometric analysis was found to be informative in establishing a deficiency of Btk protein in both patients and carriers and is recommended as a frontline procedure in the molecular diagnosis and work-up of XLA.

• Asian-Australasian Journal of Animal Sciences
• /
• v.29 no.7
• /
• pp.925-937
• /
• 2016

In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive $F_1$ piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive $F_1$ boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive $F_1$ sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. cDNA sequencing and western blot indicated that the exogenous BMPR1B CDS was successfully expressed in host pigs. The transgenic pigs showed normal litter size performance. However, no significant differences in litter size were found between transgene-positive and negative sows. Our study provides new insight into producing cloned transgenic livestock related to reproductive traits.

• IMMUNE NETWORK
• /
• v.4 no.1
• /
• pp.23-30
• /
• 2004

Background: A human orthologue of mouse S100A6-binding protein (CacyBP), Siah-1-interacting protein (SIP) had been shown to be a component of novel ubiquitinylation pathway regulating $\beta$-catenin degradation. The role of the protein seems to be important in cell proliferation and cancer evolution but the expression pattern of SIP in actively dividing cancer tissues has not been known. For the elucidation of the role of SIP protein in carcinogenesis, it is essential to produce monoclonal antibodies specific to the protein. Methods: cDNA sequence coding for ORF region of human SIP gene was amplified and cloned into an expression vector to produce His-tag fusion protein. Recombinant SIP protein and monoclonal antibody to the protein were produced. The N-terminal specificity of anti-SIP monoclonal antibody was conformed by immunoblot analysis and enzyme linked immunosorbent assay (ELISA). To study the relation between SIP and colon carcinogenesis, the presence of SIP protein in colon carcinoma tissues was visualized by immunostaining using the monoclonal antibody produced in this study. Results: His-tag-SIP (NSIP) recombinant protein was produced and purified. A monoclonal antibody (Korea patent pending; #2003-45296) to the protein was produced and employed to analyze the expression pattern of SIP in colon carcinoma tissues. Conclusion: The data suggested that anti-SIP monoclonal antibody produced here was valuable for the diagnosis of colon carcinoma and elucidation of the mechanism of colon carcinogenesis.

• Journal of The Korean Association For Science Education
• /
• v.40 no.3
• /
• pp.347-358
• /
• 2020

He purpose of this study is to design and apply a pilot assessment tools for scientifically gifted and talented elementary school students with lower grades. The assessment tool consists of three parts: homeroom teachers' recommendation, paper and pencil test and performance tests. The tools are verified whether they are suitable for unique characteristics of young children and enable to attract active participation. For suitability check, students' performance tests were inductively analyzed and 30 behavioral patterns were shown which were included and partially exceeded the level of lower elementary students' performance expectation in NGSS. As a result, we concluded that assessment tool developed in this study will be effective in discriminating young pupils' scientific talents. Then for participation check, we compared the number of coding references as an indicator of participation. Two cases were found that students with high interest participated passively in performance tests. We found these 'passive participants' had excessive scientific experiences and extremely narrow region of interest, during the process of complex interpretation between the results of this assessment tool and in-depth interviews with homeroom teachers. We found out in this study that newly developed tools can be used in school scene after modifying and elaboration through accumulation of more case studies.

• Korean Journal of Agricultural Science
• /
• v.34 no.2
• /
• pp.161-170
• /
• 2007

To characterize $\gamma$-glutamyltranspeptidase ($\gamma$-GTP or ggt; EC 2. 3. 2. 2.) gene of Bacillus subtilis BS 62, the $\gamma$-GTP gene of BS 62 was prepared from PCR products amplified with the chromosomal DNA. The $\gamma$-GTP gene of about 2.5 kb was sequenced, and its homology was compared with the other ggt genes which were reported previously. The base sequence of the gene appeared to have an open reading frame of 1,758 bp encoding a protein of 62,175 Da. The coding region was flanked by putative ribosome binding site - AGGAGG of 7th to 12th upstream - and the stem-loof sequence was followed by transcription terminator codon. Homology of the amino acid residues sequence consisting of 587 amino acid residues was found as 98% with Bacillus subtilis gene (BSU49358), 97.4% with that of Bacillus subtilis KX 102, 37% with Pseudomonas sp. A14 (S63255) and 38% with Streptomyces avermitils (AP005028).

• Korean Journal of Plant Taxonomy
• /
• v.34 no.1
• /
• pp.43-61
• /
• 2004

Molecular taxonomic studies were conducted to evaluate interspecific relationships in Korean Viola 34 taxa including two Japanese populations using RAPD(randornly amplified polymorphic DNA), ISSR(inter simple sequence repeat) and PCR-RFLP(restriction fragment length polymorphism) analysis. Only six and four primers out of 40 arbitrary and 12 ISSR primers were screened for 34 taxa, and were revealed 70 (98.6%) and 28 (96.6%) polymorphic bands, respectively. Fifteen restriction endonucleases produced 80 restriction sites and size variations from the large single copy region of cpDNA, 16 (20%) of which were polymorphic. The separate analyses from the RAPD, ISSR and PCR-RFLP data were incongruent in the relationships among 34 taxa, but combined data was in accordance with previous infrageneric classification system based on morphological characters, especially the subsection and series level. Section Chamaemelanium placed between subsect. Patellares and Vagimtae of section Nomimium was not formed as a distinct group. Viola alb ida complex including three very closely related taxa was recognized independent group within subsect. Patellares in combined data tree. This result strongly suggested that they should be treated to series Pinmtae. RAPD analysis was very useful to clarify the interspecific relationships among the species of Korean Viola than ISSH and PCR-RFLP analyses.

• IMMUNE NETWORK
• /
• v.3 no.1
• /
• pp.16-22
• /
• 2003

Background: The S100A2 gene, also known as S100L or CaN19, encodes a protein comprised of 99-amino acids, is a member of the calcium-binding proteins of EF-hand family. According to a recent study, this gene was over-expressed in several early and malignant carcinomas compared to normal tissues. To elucidate the role of S100A2 protein in the process during carcinogenesis, production of monoclonal antibody specific to the protein is essential. Methods: First, cDNA sequence coding for ORF region of human S100A2 gene was amplified and cloned into an expression vector to produce GST fusion protein. Recombinant S100A2 protein and subsequently, monoclonal antibody to the protein were produced. The specificity of anti-S100A2 monoclonal antibody was confirmed by immunoblot analysis of cross reactivity to other recombinant proteins of S100A family (GST-S100A1, GST-S100A4 and GST-S100A6). To confirm the relation of S100A2 to cervical carcinogenesis, S100A2 protein in early cervical carcinoma tissue was immunostained using the monoclonal antibody. Results: GST-S100A2 recombinant protein was purified by affinity chromatography and then fusion protein was cleaved and S100A2 protein was isolated. The monoclonal antibody (KK0723; Korean patent pending #2001-30294) to the protein was produced and the antibody did not react with other members of EF-hand family proteins such as S100A1, S100A4 and S100A6. Conclusion: These data suggest that anti-S100A2 monoclonal antibody produced in this study can be very useful for the early detection of cervical carcinoma and elucidation of mechanism during the early cervical carcinogenesis.

• Horticultural Science & Technology
• /
• v.29 no.5
• /
• pp.461-466
• /
• 2011

In genetic and molecular breeding studies of plants, researchers need to design various kinds of primers based on their research purposes. So far many kinds of web- or script-based non-commercial programs for primer design are available. Because most of them do not include user interface for multipurpose usage including gene structure prediction and direct target selection on sequences, it has been a laborious work to design primers targeting on the exon or intron regions of interesting genes. Here we report a primer designing graphic user interface program, Pickprimer, that includes gene structure prediction and primer design modules by combining source codes of the Spidey and Primer3 programs. This program provides simple graphic user interface to input sequences and design primers. Genomic sequence and mRNA or coding sequence of genes can be copy and pasted or input as fasta or text files. Based on alignment of the input sequences using the Spidey module, a putative gene structure is graphically visualized along with exon-intron sequences of color codes. Primer design can be easily performed by dragging mouse on the displayed sequences or input primer targeting position with desirable values of primers. The output of designed primers with detailed information is provided by the Primer3 module. PCR evaluation of 24 selected primer sets successfully amplified single amplicons from six Brassica rapa cultivars. The Pickprimer will be a convenient tool for genetic and molecular breeding studies of plants.