• Journal of Microbiology and Biotechnology
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• v.20 no.9
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• pp.1283-1287
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• 2010

In eukaryotes, eRF3 participates in translation termination and belongs to the superfamily of GTPases. In this work, the dissociation constants for nucleosides bound to Euplotes octocarinatus eRF3 in the presence and absence of eRF1a were determined using fluorescence spectra methods. Furthermore, a GTP hydrolyzing assay of eRF3 was carried out using an HPLC method, and the kinetic parameters for GTP hydrolysis by eRF3 were determined. Consistent with data from humans, the results showed that eRF1a promoted the binding of GTP to eRF3 and the GTP hydrolyzing activity of eRF3. However, in contrast to the lack of GTP binding in the absence of eRF1 in human eRF3, the E. octocarinatus eRF3 was able to bind GTP by itself. The nucleotide binding affinity of the E. octocarinatus eRF3 also differed from the human data. A structure model and amino acid sequence alignment of potential G domains indicated that these differences may be due to valine 317 and glutamate 452 displacing the conserved glycine and lysine involved in GTP binding.

• BMB Reports
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• v.40 no.3
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• pp.426-431
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• 2007

To investigate the possible mechanisms of high-altitude native animals in adapting to high altitude, we cloned hemoglobin alpha-chain (alpha-chain Hb) gene from Pantholops hodgsonii, an animal species that indigenously lives at elevations of 3700-5500 m on the Qinghai-Tibetan plateau. Using reverse transcription polymerase chain reaction (RT-PCR) technique, the alpha-chain Hb gene was amplified from total RNA in the liver of the Pantholops hodgsonii. TA cloning technique was used and the PCR product was cloned into pGEM-T vector. The DNA sequence of the gene was highly homologous with sheep (99.1%), goat (98.6%), cattle (95.6%) and human (86.5%). The alpha-chain Hb gene encoded a 142-amino acid protein that could be identified with the homology of alpha-chain Hb protein in sheep (98%), goat (96%), cattle (91%) and human (87%). However, 18 alternations were detected when compared with the alpha-chain Hb gene in human, and 2 in sheep. Moreover, the alterations of a117 GluAsp and $\alpha$132 AsnSer in important regions were noted in human and sheep, respectively. Phylogenetic analysis suggested that the structure of alpha-chain Hb was highly similar to that in sheep. This study provided essential information for elucidating the possible roles of hemoglobin in adapting to extremely high altitude in Pantholops hodgsonii.

• Korean Journal of Plant Tissue Culture
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• v.27 no.5
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• pp.407-409
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• 2000

Outer envelope membrane proteins of chloroplasts encoded by the nuclear genome are transported without the N-terminal transit peptide. Here, we investigated the targeting mechanism of AtOEP7, an Arabidopsis homolog of small outer envelope membrane proteins in vivo. AtOEP7 was expressed transiently in protoplasts or stably in transgenic plants as fusion proteins with GFP. In both cases AtOEP7:GFP was targeted to the outer envelope membrane when assayed under a fluorescent microscope or by Western blot analysis. Except the transmembrane domain, deletions of the N- or C-terminal regions of AtOEP7 did not affect targeting although a region closed to the C-terminal side of the transmembrane domain affected the targeting efficiency. Targeting experiments with various hybrid transmembrane mutants revealed that the amino acid sequence of the transmembrane domain determines the targeting specificity The targeting mechanism was further studied using a fusion protein, AtOEP7：NLS：GFP, that had a nuclear localization signal. AtOEP7：NLS：GFP was efficiently targeted to the chloroplast envelope despite the presence of the nuclear localization signal. Taken together, these results suggest that the transmembrane domain of AtOEP7 functions as the sole determinant of targeting specificity and that AtOEP7 may be associated with a cytosolic component during translocation to the chloroplast envelope membrane.

• Korean Journal of Environmental Biology
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• v.34 no.2
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• pp.97-106
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• 2016

$Na^+/K^+$-ATPase is a membrane protein and plays a key role in osmotic regulation in living organisms. In the present study, a cDNA sequence encoding the $Na^+/K^+$-ATPase alpha subunit from the monogonont rotifer, Brachionus koreanus was cloned by rapid amplification of cDNA ends technique. To investigate the role of this enzyme in osmotic stress, enzymatic activities of $Na^+/K^+$-ATPase were measured after exposure to different salinities for 48 h. The full-length Bk $Na^+/K^+$-ATPase cDNA was 3069 bp-long, encoding a 1022-amino acid polypeptide. Bk $Na^+/K^+$-ATPase possesses eight membrane spanning regions and five conserved domains. Phylogenetic analysis showed that Bk $Na^+/K^+$-ATPase had high identity with those of other species, and was closely clustered with other Brachionus sp. These findings indicate that this protein was conserved both structurally and functionally. B. koreanus $Na^+/K^+$-ATPase activity was stimulated in both hyposaline (6 psu) and hypersaline (32 psu) conditions, suggesting that this protein may play a role in osmoregulation. This study would provide better understanding of the physiology of B. koreanus and this enzyme may be useful as a molecular marker for evaluation of osmotic stress in aquatic environment.

• Korean Journal of Plant Tissue Culture
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• v.22 no.3
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• pp.149-155
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• 1995

The coat protein (CP) gene was cloned from RNA genome of the Cucumber Mosaic Virus strain ABI (CMV-ABI) isolated in Korea. The comparisons of the nucleotide sequence of the cloned CP gene and its deduced amino acid sequences with other CP genes revealed that the CMV-ABI belongs to subgroup I (type I), CMV-ABI developed the typical mosaic symptom in infected plants. Tobacco plants (Samsun and NC82) were transformed by leaf-disc transformation via Agrobacterium, temefaciens LB4404 harboring pVCP, witch CMV-ABI CP gene was inserted into the pBI121, and a number of mature transgenic tobacco plants were developed. Southern and PCR analysis of genomic DNA from the transgenic plants showed that the CP gene was integrated into the genomes of the most of the transgenic plant. Result of the segregation patterns of resistance in T1 seedlings of the plants to kanamycin showed that the transgenic plants containing l,2 and 3 copies of CP gene were50%, 39% and 11% of the total transgenic plants, respectively.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.11
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• pp.1591-1598
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• 2004

To isolate the $\beta$-galactosidase producing thermophilic bacteria, samples of mud and water were collected from hot springs of avolcanic area near Golden Springs in New Zealand. Among eleven isolated strains, the strain of KNOUC112 produced the highest amounts of $\beta$-galactosidase at 40 h incubation time (0.013 unit). This strain was aerobic, asporogenic bacilli, immobile, gram negative, catalase positive, oxidase positive, and pigment producing. Optimum growth was at 70-72$^{\circ}C$, pH 7.0-7.2, and it could grow in the presence of 3% NaCl. The main fatty acids of cell components were iso-15:0 (30.26%), and iso-17:0 (31.31%). Based on morphological and biochemical properties and fatty acid composition, the strain could be identified as genus Thermus, and finally as Thermus thermophilus by phylogenetic analysis based on 16S rRNA sequence. So the strain is designated as Thermus thermophilus KNOUC112. A gene from Thermus thermophilus KNOUC112 encoding $\beta$-galactosidase was amplified by PCR using redundancy primers prepared based on the structure of $\beta$-galactosidase gene of Thermus sp. A4 and Thermus sp. strain T2, cloned and expressed in E. coli JM109 DE3. The gene of Thermus thermophilus KNOUC112 $\beta$-galactosidase(KNOUC112$\beta$-gal) consisted of a 1,938 bp open reading frame, encoding a protein of 73 kDa that was composed of 645 amino acids. KNOUC112$\beta$-gal was expressed as dimer and trimer in E. coli JM109 (DE3) via pET-5b.

• Fisheries and Aquatic Sciences
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• v.14 no.4
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• pp.347-354
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• 2011

Aeromonas salmonicida is an important fish pathogen commonly associated with furunculosis in salmonids. Typical A. salmonicida strains have the surface virulence A-layer protein, a major virulence determinant encoded by the vapA gene. In this study, 880 chum salmon Oncorhynchus keta were collected from the east coast of Korea during 2006-2011, including 560 wild adults and 320 artificially hatched fry pools, and the presence of typical A. salmonicida was examined by PCR using the typical A. salmonicida-specific vapA gene primers. The results demonstrated that 34.5% of the samples (304/880 samples) were PCR positive, implying that a typical A. salmonicida infection is highly prevalent among chum salmon in Korea. Twenty typical A. salmonicida isolates were recovered based on their brown pigmentation on Trypticase Soy Agar (TSA) plates, which indicates the existence of the A-layer protein. Further biochemical analyses with the four randomly selected typical A. salmonicida isolates revealed some variations in their amino acid decarboxylation and carbohydrate fermentation activity. A phylogenetic analysis based on the entire vapA gene sequence suggested that the A. salmonicida isolates from chum salmon were clustered with those isolated from Atlantic salmon in Europe. Further study is needed to resolve such an interesting relationship in detail.

• Journal of Microbiology and Biotechnology
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• v.17 no.9
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• pp.1498-1503
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• 2007

To identify novel crystal proteins, Bacillus thuringiensis 2385-1 was isolated from Korean soil samples and characterized. The H-serotype of 2385-1 was identical to that of subsp. kenyae (H4a4c), and its crystal toxin was bipyramidal-shaped. However, 2385-1 showed a much higher toxicity towards Plutella xylostella and Spodoptera exigua larvae than subsp. kenyae. In addition, the crystal protein profile and plasmid DNA pattern of 2385-1 differed from those of subsp. kenyae. To verify the crystal protein gene types of 2385-1, a PCR-RFLP analysis was performed, and the results revealed that 2385-1 contained two novel cry1-type crystal protein genes, cryl-5 and cry1-12, in addition to the crylJal gene. The deduced amino acid sequences of cryl-5 and cry1-12 showed a 97.9% and 75.7% sequence similarity with the CrylAb and CrylJa crystal proteins, respectively. Among the novel crystal proteins, Cry1-5 showed a high toxicity towards P. xylostella and S. exigua larvae. In conclusion, B. thuringiensis 2385-1 is a new isolate in terms of its gene types, and should be a promising source for an insecticide to control lepidopteran larvae.

• Journal of Microbiology and Biotechnology
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• v.23 no.6
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• pp.759-765
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• 2013

The gene encoding squalene synthase (SQS) of the lipid-producing heterotrophic microalga Aurantiochytrium sp. KRS101 was cloned and characterized. The krsSQS gene is 1,551 bp in length and has two exons and one intron. The open reading frame of the gene is 1,164 bp in length, yielding a polypeptide of 387 predicted amino acid residues with a molecular mass of 42.7 kDa. The deduced krsSQS sequence shares at least four conserved regions known to be required for SQS enzymatic activity in other species. The protein, tagged with $His_6$, was expressed into soluble form in Escherichia coli. The purified protein catalyzed the conversion of farnesyl diphosphate to squalene in the presence of NADPH and $Mg^{2+}$. This is the first report on the characterization of an SQS from a Thraustochytrid microalga.

• The Journal of Korean Society of Virology
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• v.29 no.2
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• pp.65-74
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• 1999

Reovirus was found to inhabit both the respiratory and the enteric tract of human and animals. The genome of reovirus comprises 10 segments of double-stranded RNA, total size 24 kbp. Nine strains of reovirus were isolated from human and field mice in Korea. Aseptically collected sera from human and lung tissues from field mice were used for virus isolation. For serotype determination, hemagglutination inhibition test was used, and three strains were confirmed to type 2 and six strains to type 3. To determine the genomic diversity and molecular phylogeny of reoviruses isolated in Korea, part of S4 genomic segment of reovirus was enzymatically amplified and directly sequenced. In nucleotide level, Apo98-35 strain showed 15.4%, 19.3%, and 14.4% differences compared to type 1 (T1L, Lang), type 2 (T2J), and type 3 reference strains, respectively. In amino acid level, Apo98-35 strain showed 10.5%, 13.7%, and 9.5% differences compared to type 1, type 2, and type 3 reference strains, respectively. Using the maximum parsimony method based on 285 bp spaning region of the S4 genomic segment, phylogenetic analysis indicated that Apo98-35 from Korea formed different phylogenetic branch. Our data obtained by sequence and phylogenetic analyses of reoviruses are consistent with the distinct geographically dependent evolution of reoviruses in Korea.