• Korean Journal of Environmental Biology
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• v.21 no.1
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• pp.77-85
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• 2003

We have examined the 16S-23S rRNA intergenic spacer regions (ISR) of Vibrio fluvialis. ISRs were PCR amplified, cloned into a plasmid vector and then sequenced. As results of ISR nucleotide sequence analysis, total of 6 clones were isolated depending on the size. The clones were different in both the number and the composition of the tRNA genes, and were designated ISR-A, ISR-E, ISR-El, ISR-lA, ISR-EKV, ISR-EKAV. ISR-A contains $tRNA^{Ala}$; ISR-lA, $tRNA^{Ile}$-$tRNA^{Ala}$; ISR-EKV, $tRNA^{GIu}$-$tRNA^{Lys}$-$tRNA^{Val}$;ISE-EKAV, $tRNA^{GIu}$-$tRNA^{Lys}$-$tRNA^{Ala}$-$tRNA^{Val}$; ISR -E and E1, $tRNA^{GIu}$ clusters. ISR-EKV was shown to be a minor type out of the six ISR types and showed a very limited homology between ISR-EKV from V, fluvialis and ISRa from other Vibrio species. Therefore ISR-EKV sequence was used to design species-specific primers to detect V, fiuvialis from other Vibrio species by PCR reaction. The specificity of the primers was examined using genomic DNA of other Vibrios as templates for PCR reaction. The result showed that PCR can be a useful method to detect V. fluvialis among Vibrio species in a single PCR reaction.

• Korean Journal Plant Pathology
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• v.11 no.3
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• pp.271-277
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• 1995

The RNA nucleotide sequences of the 3/-noncoding regions (3'-NCRs) of two Korean strains of turnip mosaic virus (TuMV), Ca and cqs, have been determined from their cDNA clones that encompassed the 3'-terminal regions of the viral genomic RNAs. The 3'-NCRs of both strains were 209 nucleotides long, terminated with GAC residues and poly (A) tails. The potential polyadenylational signal motif, UAUGU, was located 140 nucleotides upstream from the poly (A) tail in each of the virus. A highly conserved hexanucleotide sequence [A G U G A/U G/C], which was common in the 3'-NCRs of the potyvirus RNAs, was also found at the regions of 119 bases upstream from the 3'-end. Comparison of the 3'-NCRs of the two Korean isolates with those of four strains from Canada, China and Japan showed significantly identical genotypes (94.3∼99.5%). The secondary structure of three loops with long stems was found within the 3'-NCRs by sequence analysis. The substituted bases in the region among the six TuMV strains did not alter their secondary structures. Length of the 3'-NCRs of the know 11 potyviral RNAs and TuMV RNAs was different from one another and their nucleotide sequences showed 55.7% to 24.0% of homology. The 3'-NCR, therefore, is considered to be useful for phylogenetic studies in potyviruses.

• Archives of Pharmacal Research
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• v.19 no.6
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• pp.497-501
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• 1996

A gene coding for triosephosphate isomerase (TPI) from a rat skeletal muscle cDNA library was cloned and its nucleotide sequence was determined. The 1, 348-bp cDNA clone contains 24 bp $5^I$ noncoding region, the entire 750 bp coding region corresponding to a protein of 249 amino acids, $547bp 3^I$ noncoding region and part of a poly(A) tail. It also contains a polyadenylation signal, AATAAA, starting from 17 bp upstream of the poly(A) tail. The calculated molecular weight of rat TPI is 27.8 kDa and the net charge is +4. The deduced amino acid sequence from rat TPI CDNA sequence has 93% and 94% homology with that of mouse and human clones, respectively. The amino acids at the residue of Asn12, Lys14, His96, Glu 166, His96, His101, Ala177, Tyr165, Glu13O, Tyr2O9, and Ser212 in catalytic site are completely identical, confirming that the functional residues in TPI proteins are highly conserved throughout evolution. The most profound characteristic of rat TPI enzyme, compared with other TPIs, is that there are five cysteine substitutions at the residue of 21, 27, 159, 195 and 204. A Glu123 instead of Gly was found in rabbit, rhesus, mouse and human sequences. Through the method of RT-PCR, the mRNA transcription level of TPI gene was found to be different among various tissues and was highest in muscle.

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.115-128
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• 1997

Three cDNA fragments located within NS5 region of HCV were synthesized by RT using viral RNA extracted from blood sample of Korean patient as a template. The cDNAs were amplified by PCR, cloned into the T-vector, and the nucleotide sequences were determined. Comparative analysis of the nucleotide and amino acid sequence of NS5 cDNAs showed that it is closely related with HCV type 1b. The cloned NS5 cDNA showed 91-94% homology at the nucleotide sequence level and 96-98% homology at the amino acid sequence level with several strains of the HCV type 1b. The NS5 cDNAs were subcloned into E. coli expression vectors to construct pRSETA5-1, pTHAN5-1, pRSETC5-2, pRSETBB1, pRESTCB1 and pRSETB-H3. Expression of the NS5 proteins was achieved by inducing the promoter with isopropyl-thio-${\beta}$-D-galactoside (IPTG) and confirmed by SDS-polyacrylamide gel electrophoresis. The NS5 proteins were immunoreactive against sera from Korean hepatitis C patients in Western blot analysis. Among the recombinant NS5 proteins, pRSETAS-1 plasmid derived protein, coded from aa2022 to aa2521 of HCV polyprotein, showed the strongest immunoreactivity against sera from Korean hepatitis C patients in immunoblot analysis. These results suggest that NS5 proteins would be useful as an antigen for detection of antibody against HCV in the blood samples.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.3
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• pp.329-334
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• 2006

Adipocyte-specific secretory factor (ADSF)/resistin, a hormone, is a small cysteine-rich protein secreted from adipose tissue and has been implicated in modulating adipogenesis in humans and rodents. The objective of this study was to clone a gene encoding ADSF/resistin and to characterize its function in Korean Native Cattle (Hanwoo). The coding sequence was 330 base pairs and it encoded a protein of 109 amino acids. An NCBI BLAST-search revealed the cloned cDNA fragment shared significant homology (82%) with the cDNA encoding the human ADSF/resistin. The nucleotide sequence homology of the Hanwoo sequence was 73% and 64% for the rat and mouse, respectively. A 654 bp ADSF/resistin gene promoter was cloned and putative binding sites of transcription factors were identified. Tissue distribution of ADSF mRNA was examined in liver, skeletal muscles (tenderloin, biceps femoris), subcutaneous fat, and perirenal fat by RT-PCR. ADSF mRNAs were detected in fat tissues but not in liver and muscles, suggesting that ADSF/resistin expression may be induced during adipogenesis. Although, the physiological function of ADSF/resistin in the cow remains to be determined, these data indicate ADSF is related to the adipocyte phenotype and may have a possibly regulatory role in adipocyte function.

• The Journal of Korean Society of Virology
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• v.28 no.3
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• pp.233-245
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• 1998

The gene encoding F protein of CBP-1 strain, a heat-stable Newcastle disease virus (NDV) isolated from the diseased pheasants in Korea, was characterized by reverse transcription-polymerase chain reaction (RT-PCR), nucleotide and amino acid sequences. Virus RNA was prepared from the chorioallatoic fluid infected with NDV CBP-1 virus and cDNA was amplified by RT-PCR, cloned and sequenced to analyze. The PCR was sensitive as to detect the virus titer above $2^5$ hemagglutination unit. 1.7kb (1,707bp) size of the cDNA was amplified and cloned into BamHI site of pVL1393 Baculo transfer vector. The nucleotide sequences for F protein were determined by dye terminator cyclic sequencing using four pairs of primers, and 553 amino acid sequences were predicted. In comparison of the nucleotide sequence of F gene of CBP-1 with those of other NDV strains, the homology revealed 88.8%, 98.5% and 98.7% with Kyojungwon (KJW), Texas GB and Beaudette C strains, respectively. As the deduced 553 amino acid sequences of F protein of CBP-1 were compared with those of other NDV strains, the homology appeared 89.9%, 98.7% and 98.9% with KJW, Texas GB and Beaudette C strains, respectively. The putative protease cleavage site (112-116) was R-R-Q-K-R, indicating that CBP-1 strain is velogenic type. The amino acid sequences include 6 sites of N-asparagine-linked glycosylation and 13 cysteine residues. These data indicate that the genotype of CBP-1 strain is more closely associated with the strains of Texas GB and Beaudette C than KJW strain.

• Journal of Microbiology and Biotechnology
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• v.22 no.12
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• pp.1621-1628
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• 2012

The agar-degrading bacterium GNUM-1 was isolated from the brown algal species Sargassum serratifolium, which was obtained from the West Sea of Korea, by using the selective artificial seawater agar plate. The cells were Gram-negative, $0.5-0.6{\mu}m$ wide and $2.0-2.5{\mu}m$ long curved rods with a single polar flagellum, forming nonpigmented, circular, smooth colonies. Cells grew at $20^{\circ}C-37^{\circ}C$, between pH 5.0 and 9.0, and at 1-10% (w/v) NaCl. The DNA G+C content of the GNUM-1 strain was 45.5 mol%. The 16S rRNA sequence of the GNUM-1 was very similar to those of Alteromonas stellipolaris LMG 21861 (99.86% sequence homology) and Alteromonas addita $R10SW13^T$(99.64% sequence homology), which led us to assign it to the genus Alteromonas. It showed positive activities for agarase, amylase, gelatinase, alkaline phosphatase, esterase (C8), lipase (C14), leucine arylamidase, valine arylamidase, ${\alpha}$-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, ${\alpha}$-galactosidase, ${\beta}$-galactosidase, ${\beta}$-glucosidase, catalase, and urease. It can utilize citrate, malic acid, and trisodium citrate. The major fatty acids were summed feature 3 (21.5%, comprising $C_{16:1}{\omega}7c/iso-C_{15:0}$ 2-OH) and C16:0 (15.04%). On the basis of the variations in many biochemical characteristics, GNUM-1 was considered as unique and thus was named Alteromonas sp. GNUM-1. It produced the highest agarase activity in modified ASW medium containing 0.4% sucrose, but lower activity in rich media despite superior growth, implying that agarase production is tightly regulated and repressed in a rich nutrient condition. The 30 kDa protein with agarase activity was identified by zymography, and this report serves as the very first account of such a protein in the genus Alteromonas.

• Plant Biotechnology Reports
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• v.2 no.1
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• pp.75-85
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• 2008

Prune dwarf virus (PDV) is an Ilarvirus systemically infecting almond trees and other Prunus species and spreading through pollen, among other means. We have studied strategies based on coat protein (cp) gene to block PDV replication in host plant cells. A Portuguese isolate of PDV was obtained from infected almond leaves and used to produce the cDNA of the cp gene. Various constructs were prepared based on this sequence, aiming for the transgenic expression of the original or modified PDV coat protein (cpPDVSense and cpPDVMutated) or for the expression of cpPDV RNA (cpPDVAntisense and cpPDVwithout start codon). All constructs were tested in a PDV host model, Nicotiana benthamiana, and extensive molecular characterization and controlled infections were performed on transformants and their progenies. Transgenic plants expressing the coat protein RNA were able to block the proliferation of a PDV isolate sharing only 91% homology with the isolate used for cpPDV cloning, as evaluated by DAS-ELISA on newly developed leaves. With cp expression, the blockage of PDV proliferation in newly developed leaves was only achieved with the construct cpPDV Mutated, where the coat protein has a substitution in the 14th aa residue, with arginine replaced by alanine. This result points to a possible role of the mutated amino acid in the virus ability to replicate and proliferate. This work reveals the possibility of achieving protection against PDV through either coat protein RNA or mutated cp sequence.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.5
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• pp.216-222
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• 2020

Tumor necrosis factor receptor associated factor 4 (TRAF4) is found to be overexpressed in human breast cancer. It plays a role in cancer metastasis, production of reactive oxygen species, and cell polarity at membranes. The characteristics and functions of TRAF4 in pigs have not yet been identified. As the first step of research, the mRNA sequence of TRAF4 in porcine cells has been determined. To obtain the full-length sequence, rapid amplification of cDNA ends (RACE) has been carried out. Upon cloning, 2,030 bp of nucleotides were found to encode 470 amino acids, and 8 and 12 amino acids were different from those of the human and mouse TRAF4, respectively. The coding region of porcine TRAF4 was shown to be 93% and 90% homologous to human and mouse TRAF4, respectively. qPCR was conducted to determine the relative expression level of TRAF4. TRAF4 expression in pK15 was enhanced by cell-cell contacts. The mRNA levels of CLDN4, OCLN, and TJP1 at 60% and 80% confluency were significantly higher than at 40% confluency. Further, TRAF4 and tight junction-related genes were down-regulated upon treatment with TRAF4 siRNA. Thus, TRAF4 may affect the function of tight junctions in pig.

• Development and Reproduction
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• v.10 no.3
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• pp.177-184
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• 2006

Retinol-binding protein(RBP) plays an important role in the specific transport of retinol to target cells through the blood stream in higher vertebrates. In order to clarify the effects of 4-nonylphenol(4-NP) on RBP mRNA expression in the rockfish, Sebastes schlegeli which is common in coastal waters of Korea and commercially important species, the cDNA library was constructed from the liver, and a partial fragment of the RBP gene was cloned. The deduced amino acid sequence from the RBP mRNA showed a high homology to the amino acid sequence from Sparus aurata(80%), Oncorhynchus mykiss(72%) or Anguilla anguilla(78%). Effects of 4-NP on RBP and vitellogenin(VTG) mRNA expression level in rockfish were examined by the northern blot analysis. In female and male rockfish injected with 4-NP(10 mg/kg BW, lower dose), there was no changes in the levels of VTG mRNA expression in the liver. The RBP mRNA levels, however, decreased at 48 hours after the injection in male. In the rockfish injected with 4-NP(25 mg/kg BW, higher dose), the level of VTG mRNA expression increased after 24 hours, regardless of sex. The level of RBP mRNA expression decreased at 48 hours after the injection in both sexes. These data indicate that estrogenic mimics such as 4-NP exhibit a contrasting effect on RBP and VTG gene expression in rockfish.