• Journal of Veterinary Clinics
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• v.36 no.4
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• pp.190-195
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• 2019

We aimed to identify genomic variations as well as the marker genes related with chronic mitral valve disease (CMVD) in Canis lupus familiaris using whole genome resequencing, which provides valuable resources for further study. Two ten-year old female Canis lupus familiaris English cocker spaniels were used for this study, one control and one who had been diagnosed as CMVD. For the whole genome resequencing, muscles from the left ventricular wall were collected from each dog. With the HiSeq DNA Shotgun library and $HiSeq^{TM}$ 2000 platform, whole genome resequencing was performed. From the results, we identified 5 million and 6 million variants in gene expression in the control and CMVD-diagnosed subject, respectively. We then selected the top 1,000 genes from the SNP, INS, and DEL mutation and 675 genes among them were overlapped for every mutation between the control and CMVD-diagnosed patient. Interestingly, in both groups, the intron variant (91.16 and 91.18%) and upstream variant (3.10 and 3.08%) are most highly related. Among the overlapped 675 genes, gene ontology for intracellular signal transduction is highly counted in INS, and DEL, and SNPs (35, 33, 31, respectively). In this study, we found that the COL and CDH gene families could be key molecules in identifying the difference in gene expression between control and CMVD-diagnosed dogs. We believe further studies will prove the importance of variants in key molecule expression and that these data will serve as a valuable foundation stone the study of canine CMVD.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.65.2-65
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• 2003

To protect the plant against several soil-borne pathogens, we are currently constructing disease-resistant transgenic root stock for the growth of cucurbitaceae vegetable plants, watermelon and gourd. We made a watermelon cDNA library from Cladosporium cucumerinum-Infected leaves for substractive hybriazation and differential screening. We isolated the several pathogen inducible cDNA clones, such as caffeoyl-CoA-methyltransferase, LAA induced protein, receptor-like kinase homolog, hydroxyproline-rich glycoprotein, catalase, calmodulin binding protein, mitochondrial ATPase beta subunit, methyl tRNA synthetase and WRKY transcription factors. We previously obtained CaMADS in pepper and galactinol synthase ( CsGolS) in cucumber that were confirmed to be related with disease-resistance. CaMADS and CsGolS2 were transformed into the inbred line 'GO701-2' gourd, the inbred line '6-2-2' watermelon and the Kong-dye watermelon by Agrobacterium tumerfaciens LBA4404. Plant growth regulators (zeatin, BAP and IAA) were used for shoot regeneration and root induction for optimal condition. Putative transgenic plants were selected in medium containing 100mg/L kanamycin and integration of the CaMADS and CsGO/S2 into the genomic DNA were demonstrated by the PCR analysis. We isolated major soil-borne pathogens, such as Monosporascus cannonballus, Didymella bryoniae, Cladosporium cuvumerinum from the cultivation area of watermelon or root stock, and successfully established artificial inoculation method for each pathogen. This work was supported by a grant from BioGreen 21 program, Rural Development Administration, Republic of Korea.

• Animal Bioscience
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• v.37 no.2
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• pp.184-192
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• 2024

Objective: This study aims to investigate the genetic structure and characteristics of the Angus cattle population in Hungary. The survey was performed with the assistance of the Hungarian Hereford, Angus, Galloway Association (HHAGA). Methods: Genetic parameters of 1,369 animals from 16 Angus herds were analyzed using the genotyping results of 12 microsatellite markers with the aid of PowerMarker, Genalex, GDA-NT2021, and STRUCTURE software. Genotyping of DNA was performed using an automated genetic analyzer. Based on pairwise identity by state values of animals, the Python networkx 2.3 library was used for network analysis of the breed and to identify the central animals. Results: The observed numbers of alleles on the 12 loci under investigation ranged from 11 to 18. The average effective number of alleles was 3.201. The overall expected heterozygosity was 0.659 and the observed heterozygosity was 0.710. Four groups were detected among the 16 Angus herds. The breeders' information validated the grouping results and facilitated the comparison of birth weight, age at first calving, number of calves born and productive lifespan data between the four groups, revealing significant differences. We identified the central animals/herd of the Angus population in Hungary. The match of our group descriptions with the phenotypic data provided by the breeders further underscores the value of cooperation between breeders and researchers. Conclusion: The observation that significant differences in the measured traits occurred among the identified groups paves the way to further enhancement of breeding efficiency. Our findings have the potential to aid the development of new breeding strategies and help breeders keep the Angus populations in Hungary under genetic supervision. Based on our results the efficient use of an upcoming genomic selection can, in some cases, significantly improve birth weight, age at first calving, number of calves born and the productive lifespan of animals.

• Journal of Life Science
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• v.20 no.3
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• pp.411-415
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• 2010

Anisakis simplex is one of the parasitic nematodes, and has a complex life cycle in crustaceans, fish, squid or whale. When people eat under-processed or raw fish, it causes anisakidosis and also plays a critical role in inducing serious allergic reactions in humans. However, no web-based database on A. simplex at the level of DNA or protein has been so far reported. In this context, we constructed a web-based database for Anisakis research. To build up the web-based database for Anisakis research, we proceeded with the following measures: First, sequences of order Ascaridida were downloaded and translated into the multifasta format which was stored as database for stand-alone BLAST. Second, all of the nucleotide and EST sequences were clustered and assembled. And EST sequences were translated into amino acid sequences for Nuclear Localization Signal prediction. In addition, we added the vector, E. coli, and repeat sequences into the database to confirm a potential contamination. The web-based database gave us several advantages. Only data that agrees with the nucleotide sequences directly related with the order Ascaridida can be found and retrieved when searching BLAST. It is also very convenient to confirm contamination when making the cDNA or genomic library from Anisakis. Furthermore, BLAST results on the Anisakis sequence information can be quickly accessed. Taken together, the Web-based database on A. simplex will be valuable in developing species specific PCR markers and in studying SNP in A. simplex-related researches in the future.

• Journal of Plant Biotechnology
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• v.33 no.3
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• pp.153-160
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• 2006

Brassica rape is an important species used as a vegetable, oil, and fodder worldwide. It is related phylogenically to Arabidopsis thaliana, which has already been fully sequenced as a model plant. The 'Multinational Brassica Genome Project (MBGP)'was launched by the international Brassica community with the aim of sequencing the whole genome of B. rapa in 2003 on account of its value and the fact that it has the smallest genome among the diploid Brassica. The genome study was carried out not only to know the structure of genome but also to understand the function and the evolution of the genes comprehensively. There are two mapping populations, over 1,000 molecular markers and a genetic map, 2 BAC libraries, physical map, a 22 cDHA libraries as suitable genomic materials for examining the genome of B. rapa ssp. pekinensis Chinese cabbage. As the first step for whole genome analysis, 220,000 BAC-end sequences of the KBrH and KBrB BAC library are achieved by cooperation of six countries. The results of BAC-end sequence analysis will provide a clue in understanding the structure of the genome of Brassica rapa by analyzing the gene sequence, annotation and abundant repetitive DHA. The second stage involves sequencing of the genetically mapped seed BACs and identifying the overlapping BACs for complete genome sequencing. Currently, the second stage is comprises of process genetic anchoring using communal populations and maps to identify more than 1,000 seed BACs based on a BAC-to-BAC strategy. For the initial sequencing, 629 seed BACs corresponding to the minimum tiling path onto Arabidopsis genome were selected and fully sequenced. These BACs are now anchoring to the genetic map using the development of SSR markers. This information will be useful for identifying near BAC clones with the seed BAC on a genome map. From the BAC sequences, it is revealed that the Brassica rapa genome has extensive triplication of the DNA segment coupled with variable gene losses and rearrangements within the segments. This article introduces the current status and prospective of Korea Brassica Genome Project and the bioinformatics tools possessed in each national team. In the near future, data of the genome will contribute to improving Brassicas for their economic use as well as in understanding the evolutional process.

• Journal of Life Science
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• v.19 no.5
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• pp.566-572
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• 2009

A recombinant plasmid, pDH3, was obtained from the genomic library of Serattia marcescens KCTC 2172, and several recombinant subclones constructed from pDH3. The nucleotide sequence of a 5,137 bp segment, pPH4, was determined and three open reading frames were detected. The three ORFs encoded the phosphate specific transport (pst) operon, which was pstC, pstA, and pstB, with the same direction of transcription. Comparison of the pst operon of S. marcescens with that of other organisms revealed that the genes for pstS and phoU were missing. A potential CRP bonding site and pho box sequence was found in the upstream of the putative promoter at the regulatory region. Analysis of the nucleotide sequence showed that homology in amino acid sequences between the PstC protein and Yersinia sp., Vibrio sp., and Pseudomonas sp. were 49, 37 and 33%, respectively. The PstA protein and Yersinia sp., Vibrio sp., and Pseudomonas sp. showed homologies of 64, 51, and 47%, respectively. PstB protein and Methanocaldococcus sp., E. coli, and Mycoplasma sp. showed homologies of 60, 50, and 48%, respectively. The pst genes could be expressed in vivo and positively regulated by cAMP-CRP. The E. coli strain harboring plasmid pPH7, with pst genes, increased with the transport of phosphate.

• Journal of Sericultural and Entomological Science
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• v.53 no.1
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• pp.44-49
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• 2015

We characterized tissue specific-expressed genes in the posterior silk gland of Bombyx mori using by the Annealing Control Primer based differential display-PCR manner. In this study, we isolated 34 differentially expressed PCR amplicons, which one of these was identified as a novel transcript named as ACP-16 (366 bp), its expression was observed only in the posterior silk glands by Northern blot analysis. To determine promoter region of the ACP-16, we isolated and analyzed a phage DNA having 1.7 kb-long genome DNA including the open reading flame and 5'- upstream untranslated region of the ACP-16 gene from a genomic DNA library. We have estimated a promoter region of the ACP-16 gene by a web promoter prediction engine, which locates -750 ~ -165 from translation initiation site (ATG, +1). ACP-16 gene is necessary to more studies about critical biological role in order to apply the silkworm's transgenic system.

• Journal of Sericultural and Entomological Science
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• v.52 no.1
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• pp.39-44
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• 2014

We isolated highly-expressed genes in the posterior silk glands of silkworm on a previously study, which one of these was identified as RNA binding protein-1 homologue (RBP-1) gene. In this study, we investigated gene expressional characteristics of the RBP-1 depending on silkworm development stages and several tissues of the larvae, respectively. Northern blot hybridization analysis showed that the RBP-1 gene was expressed high in larval and pupal periods, and highly expressed than endogenous internal control gene (BmA3) on all tested larval tissues. In addition, we isolated and analyzed a phage DNA having 1,660 bp-long promoter region of the RBP-1 gene from a genomic DNA library. To study the RBP-1 gene promoter activity, RBP-1 (-740/+ 30) was amplified by PCR and subcloned into a pGL3 basic vector to generate pGL-RBP1. A luciferase report vector carrying RBP-1 gene promoter (770 bp) was tested by luciferase assay in Sf9 cells. In the result, the RBP-1 gene promoter was more efficient than constitutive promoter (BmA3) by approximately ten percent.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.58-66
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• 2007

Isolation, expression, and characterization of a novel $endo-{\beta}-1,3(4)-D-glucanase$ with high specific activity and homology to Bacillus lichenases is described. One clone was screened from a genomic library of Paenibacillus sp. F-40, using lichenan-containing plates. The nucleotide sequence of the clone contains an ORF consisting of 717 nucleotides, encoding a ${\beta}-glucanase$ protein of 238 amino acids and 26 residues of a putative signal peptide at its N-terminus. The amino acid sequence showed the highest similarity of 87% to other ${\beta}-1,3-1,4-glucanases$ of Bacillus. The gene fragment Bg1 containing the mature glucanase protein was expressed in Pichia pastoris at high expression level in a 3-1 high-cell-density fermenter. The purified recombinant enzyme Bg1 showed activity against barley ${\beta}-glucan$, lichenan, and laminarin. The gene encodes an $endo-{\beta}-1,3(4)-D-glucanase$ (E. C. 3.2.1.6). When lichenan was used as substrate, the optimal pH was 6.5, and the optimal temperature was $60^{\circ}C$. The $K_m,\;V_{max},\;and\;k_{cat}$ values for lichenan are 2.96mg/ml, $6,951{\mu}mol/min{\cdot}mg,\;and\;3,131s^{-1}$, respectively. For barley ${\beta}-glucan$ the values are 3.73mg/ml, $8,939{\mu}mol/min{\cdot}mg,\;and\;4,026s^{-1}$, respectively. The recombinant Bg1 had resistance to pepsin and trypsin. Other features of recombinant Bg1 including temperature and pH stability, and sensitivity to some metal ions and chemical reagents were also characterized.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.880-884
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• 2001

Rumen microbiology research has undergone several evolutionary steps: the isolation and nutritional characterization of readily cultivated microbes; followed by the cloning and sequence analysis of individual genes relevant to key digestive processes; through to the use of small subunit ribosomal RNA (SSU rRNA) sequences for a cultivation-independent examination of microbial diversity. Our knowledge of rumen microbiology has expanded as a result, but the translation of this information into productive alterations of ruminal function has been rather limited. For instance, the cloning and characterization of cellulase genes in Escherichia coli has yielded some valuable information about this complex enzyme system in ruminal bacteria. SSU rRNA analyses have also confirmed that a considerable amount of the microbial diversity in the rumen is not represented in existing culture collections. However, we still have little idea of whether the key, and potentially rate-limiting, gene products and (or) microbial interactions have been identified. Technologies allowing high throughput nucleotide and protein sequence analysis have led to the emergence of two new fields of investigation, genomics and proteomics. Both disciplines can be further subdivided into functional and comparative lines of investigation. The massive accumulation of microbial DNA and protein sequence data, including complete genome sequences, is revolutionizing the way we examine microbial physiology and diversity. We describe here some examples of our use of genomics- and proteomics-based methods, to analyze the cellulase system of Ruminococcus flavefaciens FD-1 and explore the genome of Ruminococcus albus 8. At Illinois, we are using bacterial artificial chromosome (BAC) vectors to create libraries containing large (>75 kbases), contiguous segments of DNA from R. flavefaciens FD-1. Considering that every bacterium is not a candidate for whole genome sequencing, BAC libraries offer an attractive, alternative method to perform physical and functional analyses of a bacterium's genome. Our first plan is to use these BAC clones to determine whether or not cellulases and accessory genes in R. flavefaciens exist in clusters of orthologous genes (COGs). Proteomics is also being used to complement the BAC library/DNA sequencing approach. Proteins differentially expressed in response to carbon source are being identified by 2-D SDS-PAGE, followed by in-gel-digests and peptide mass mapping by MALDI-TOF Mass Spectrometry, as well as peptide sequencing by Edman degradation. At Ohio State, we have used a combination of functional proteomics, mutational analysis and differential display RT-PCR to obtain evidence suggesting that in addition to a cellulosome-like mechanism, R. albus 8 possesses other mechanisms for adhesion to plant surfaces. Genome walking on either side of these differentially expressed transcripts has also resulted in two interesting observations: i) a relatively large number of genes with no matches in the current databases and; ii) the identification of genes with a high level of sequence identity to those identified, until now, in the archaebacteria. Genomics and proteomics will also accelerate our understanding of microbial interactions, and allow a greater degree of in situ analyses in the future. The challenge is to utilize genomics and proteomics to improve our fundamental understanding of microbial physiology, diversity and ecology, and overcome constraints to ruminal function.