• Journal of Life Science
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• v.26 no.7
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• pp.855-867
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• 2016

The horse is relatively earlier domesticated animal species. Domesticated horses have been selected for their ability of racing, robustness, and disease-resistance. As a result, the thoroughbred horse genome has been condensed many genotypes related to exercise ability. In recent years, with the advent of NGS technologies, many studies were concentrated on finding superior genetic species in the horse genome in terms of genomics. Consequently, GWAS (Genome-wide Association study) is applied to horse genome, then genetic marker is revealed for superior racing ability. In addition, RNA-Seq is utilized as a method for analyze of whole transcript profiling in specific samples. By using this approach, specific gene expression patterns and transcript sequences can be revealed in various samples such as each individual, before and after exercise state, and each tissue. DNA methylation, a strong factor that regulate gene expression without the change of DNA sequence, have got a lot of attention. In horse genome, exercise- or individual-specific DNA methylation patterns were detected, and could be useful to develop selective marker of superior horses. MicroRNAs inhibit gene expression, and transposable elements accounted for half of the mammalian genome. These two elements are the crucial factors in functional genomics, and could be applied to the selection of superior horses. As the functional genomics and epigenomics advance, then these technologies introduced in this paper were applied to select superior horses. In this paper, the studies for selection of superior horses through genetic technologies, and development possibilities of these studies were discussed.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.909-915
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• 2005

We investigated microRNA (miRNA) biogenesis of Epstein-Barr virus (EBV) which is the first virus shown to produce viral miRNAs. As expected, expression of all the reported EBV miRNAs were detected by Northen blot in an EBV-infected B cell line, B95-8; BHRF1-1, BHIU1-2, BHRF1-3, BART1, and BART2. The putative EBV pri-miRWAs and pre-miRNAs predicted from the known mature EBV miRNA sequences were detected by RT-PCR in B95-8 cells. Many animal miRNA genes exist as clusters of 2-7 genes and they are expressed polycistronically. As the EBV miRNAs are clustered in two regions of the EBV genome, we examined whether these clustered EBV miRNA genes are also expressed polycistronically. A long polycistronic transcript with the expected size (1602 bp) corresponding to the BHRF1-1~BHRF1-2~BHRF1-3 was amplified. However, any polycistronic transcript containing both BART1 and BART2 was detectable in B95-8. These results suggest that EBV miRNAs may be processed in a similar way with animal miRNAs and that some of the clustered EBV miRNAs can be transcribed polycistronically.

• Molecules and Cells
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• v.23 no.1
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• pp.80-87
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• 2007

Beet curly top virus (BCTV) and Beet severe curly top virus (BSCTV), members of curtoviruses, encode seven open reading frames (ORFs) within a ~3 kb genome. One of these viral ORFs, C1, is known to play an important role in the early stage of viral infection in plants during initiation of viral DNA replication. We used promoter:: reporter (${\beta}$-glucuronidase) gene fusions in transgenic Arabidopsis to identify the putative promoter region of BCTV ORF C1. Unlike other geminiviruses, the intergenic region of BCTV was not sufficient to promote C1 expression in transgenic plants. When sequences extending into the coding region of C1 were tested, strong expression of the reporter protein was observed in vascular tissues of transgenic plants. This expression was not dependent on the presence of the intergenic regions or proximal 5' portions of the C1 coding region. Transgenic plants expressing a reporter gene under control of the putative complete C1 promoter were inoculated with virus to determine if any viral transcript affected C1 expression. Virus inoculated plants did not show any altered pattern or change in of reporter gene expression level. These results suggest that (1) important transcriptional activator elements for C1 expression reside in the 3' portion of C1 coding area itself, (2) C1 protein does not auto-regulate its own expression and (3) C1 expression of two curtoviruses is controlled differently compared to other geminiviruses.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.131-137
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• 2001

Previous work has identified that only the catabolite responsive element A (creA; previously called cre-2) out of two potential cre sequences (cre-1: nucleotide +160 to +173 and cre-2: +173 to +186), recognized within the coding region of the xylanaseA gene (xynA) of Bacillus stearothermophilus No.236, was actually, was actually involved in the carbon catabolite repression(CCR) of xynA expression in B. subtilis. However, the level of CCR of xynA expression in the original B.stearothermophilus No.236 strain (70-fold repression). Therefore, to search for an additional cre element in the promoter region, the upstream region of the xynA gene was subcloned by chromosome walking, and as a result, another potential cre element (nucleotide -124∼-137; designated creB) was recognized in this region. The cre-like sequence revealed a high homology to the cre consensus sequence. The xylanase activity of B. subtilis MW15 bearing pWPBR14 (containing creA and creB) cultured in a medium containing xylose as the sole carbon source was about 7.7 times higher than that observed for the same culture containing glucose. B. subtilis MW15 bearing pWPBR23 (containing only creA) produced an activity about 2.4 times higher. This pattern of CCR was confirmed using derivatives of xynA::aprA fusion plasmids. Furthermore, a measurement of the amounts of the xynA transcript showed a similar pattern as that for the production of xylanase. In addition, the synthesis of xylanase in B. subtilis QB7115 [a catabolite control protein A (ccpA) mutant strain] carrying pWPBR14 was almost completely relieved from glucose repression. Together, these results lead to a conclusion that the CCR of the expression of the xynA gene is mediated by CcpA binding at creA and creB sites in B. subtilis.

• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.988-992
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• 2006

The ptsHI operon from Leuconostoc mesenteroides ssp. mesenteroides SY1 (L. mesenteroides SY1), a strain isolated from kimchi, was cloned and characterized. The ptsH open reading frame (ORF) was 273 bp in size, which can encode a protein of 90 amino acid residues with a molecular weight of 9,212 Da. The pfsI ORF was 1,719 bp in size, which was capable of encoding a protein of 572 amino acids with a molecular mass of 62,549 Da. ptsH and pfsI genes were transcribed as a single transcript of 2.0 kb in size regardless of carbon sources, supporting the operon structure. Although the deduced amino acid sequences of the HPr and EI were highly homologous with those of other Gram-positive bacteria, an additional amino acid (glutamine at the $3^{rd}$ amino acid) was present in HPr from L. mesenteroides SY1. Phosphorylation sites of HPr included the histidine residue ($16^{th}$) and serine residue ($47^{th}$). Mutant HPrs, in which each phosphorylation site was mutated into alanine, were obtained, and phosphorylation with HPr and mutated HPrs showed that HPr was phosphorylated at the serine residue ($47^{th}$) by HPr kinaseiphosphorylase (HPr K/P).

• BMB Reports
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• v.38 no.5
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• pp.571-576
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• 2005

Cytokines are proteins produced by many different cells of the immune system and play a significant role in initiating and regulating the inflammatory process. In this research, an important cytokine, interleukin-10 (IL-10) gene, has been identified and characterized from zebrafish (Danio rerio) genome database. Zebrafish IL-10 is located within a 2690 bp fragment and contains five exons and four introns, sharing the same organization with mammalian IL-10 genes. An open reading frame of 543 bp was found to encode a putative 180 amino acid protein with a signal peptide of 22 amino acids, which shares 29.7-80.9% homology with amino acid sequences of other known IL-10. The signature motif of IL-10 is also conserved in zebrafish IL-10. The predicted transcript was finally confirmed by sequencing of cDNA clones. Multi-tissue reverse transcriptase PCR (RT-PCR) was performed to examine the tissue distribution and expression regulation of this gene in seven organs of normal and lipopolysaccharide (LPS) stimulation zebrafish. The results demonstrated that this gene was expressed slightly in normal kidney, gill and gut, no expression was detected in other four tissues. The expression was clearly upregulated after LPS stimulation. Using the ideal zebrafish model, further study of IL-10 characterization and function may provide insight on the understanding of the innate immune system.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.4
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• pp.489-498
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• 2018

Objective: Foot and mouth disease (FMD) and porcine reproductive and respiratory syndrome (PRRS) are major diseases that interrupt porcine production. Because they are viral diseases, vaccinations are of only limited effectiveness in preventing outbreaks. To establish an alternative multi-resistant strategy against FMD virus (FMDV) and PRRS virus (PRRSV), the present study introduced two genetic modification techniques to porcine cells. Methods: First, cluster of differentiation 163 (CD163), the PRRSV viral receptor, was edited with the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 technique. The CD163 gene sequences of edited cells and control cells differed. Second, short hairpin RNA (shRNAs) were integrated into the cells. The shRNAs, targeting the 3D gene of FMDV and the open reading frame 7 (ORF7) gene of PRRSV, were transferred into fibroblasts. We also developed an in vitro shRNA verification method with a target gene expression vector. Results: shRNA activity was confirmed in vitro with vectors that expressed the 3D and ORF7 genes in the cells. Cells containing shRNAs showed lower transcript levels than cells with only the expression vectors. The shRNAs were integrated into CD163-edited cells to combine the two techniques, and the viral genes were suppressed in these cells. Conclusion: We established a multi-resistant strategy against viral diseases and an in vitro shRNA verification method.

• Journal of Microbiology
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• v.34 no.1
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• pp.68-73
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• 1996

An nprX gene of Bacillus subtilis NS15-4 encoding a neutral protease was cloned and its molecular characteristics were analyzed. The complete nucleotide sequence indicated that there is an open reading frame (0RF) possibly encoding 521 amino acid polypeptide. The ORF used all codons expected two cysteine and a proline having a codon bias index (CBI) of 0.09 in Escherichia coli. There were homologous sequences to the consensus sequence of -35 and -10 regions of E. coli promoters and to a Shine-Dalgarno (SD) sequence located 25 bp downstream of a mojor transcription initiation site. Moreover, there were also five minor transcription initiation sites at 6. 7. 8. 14 and 15 nt downstream of the major site. Northern blot analysis revealed the presence of about 1.8 kb mRNA transcript in E. coli having the nprX gene. The nucleotide sequence was identified in GenBank to be a gene for a neutral protease of B. sutilis with six nucleotide difference in the ORF region. The flanking regions of the NprX ORF showed much more differences form those of other neutral protease genes except the nprE gene of B. subtilis, which has the most homology to the nprX gene, and of which the flanking regions were identical to those of the nprX gene.

• BMB Reports
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• v.35 no.2
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• pp.213-219
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• 2002

Malonyl-CoA decarboxylase (E.C.4.1.1.9) catalyzes the conversion of malonyl-CoA to acetyl-CoA. Although the metabolic role of this enzyme has not been fully defined, it has been reported that its deficiency is associated with mild mental retardation, seizures, hypotonia, cadiomyopathy, developmental delay, vomiting, hypoglycemia, metabolic acidosis, and malonic aciduria. Here, we isolated a cDNA clone for malonyl CoA decarboxylase from a rat brain cDNA library, expressed it in E. coli, and characterized its biochemical properties. The full-length cDNA contained a single open-reading frame that encoded 491 amino acid residues with a calculated molecular weight of 54, 762 Da. Its deduced amino acid sequence revealed a 65.6% identity to that from the goose uropigial gland. The sequence of the first 38 amino acids represents a putative mitochondrial targeting sequence, and the last 3 amino acid sequences (SKL) represent peroxisomal targeting ones. The expression of malonyl CoA decarboxylase was observed over a wide range of tissues as a single transcript of 2.0 kb in size. The recombinant protein that was expressed in E. coli was used to characterize the biochemical properties, which showed a typical Michaelis-Menten substrate saturation pattern. The $K_m$ and $V_{max}$ were calculated to be $68\;{\mu}M$ and $42.6\;{\mu}mol/min/mg$, respectively.

• International Journal of Industrial Entomology and Biomaterials
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• v.23 no.2
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• pp.231-238
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• 2011

Attacin is an insect antibacterial protein that plays an important role in immune response to injury and infection. In this report, we have isolated and characterized of cDNA encoding for the attacin from the immunized larvae of swallowtail butterfly, $Papilio$ $xuthus$. A full length cDNA of $P.$ $xuthus$ attacin was obtained by employing annealing control primer (ACP)-based differential display PCR and 5' RACE. The complete $P.$ $xuthus$ attacin cDNA was comprised of 949 bp encoding a 250 amino acid precursor. It contains a putative 18 amino acid signal peptide sequence, a 42 amino acid propeptide sequence, and a 190 amino acid mature protein with a theoretical molecular mass of 19904.01 and a pI of 9.13. The putative mature protein of $P.$ $xuthus$ attacin showed 48-52% and 24-30% identity in amino acid sequences with that of lepidopteran and dipteran insects, respectively. Semiquantitive RT-PCR results revealed that the transcript of $P.$ $xuthus$ attacin gene was up-regulated at significant levels after injection with bacterial lipopolysaccharide (LPS). We sub-cloned cDNA fragment encoding mature $P.$ $xuthus$ attacin into the expression vector, highly expressed in $E.$ $coli$ BL21 cells, and its antibacterial activity was analyzed. Recombinant $P.$ $xuthus$ attacin evidenced considerably antibacterial activity against Gram-negative bacteria, $E.$ $coli$ ML 35 and $Klebsiella$ $pneumonia$.