• The Journal of Korean Society of Virology
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• v.29 no.4
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• pp.221-233
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• 1999

Prospect Hill Virus (PHV) is the well known serotype of hantavirus, a newly established genus in family Bunyaviridae. Extensive studies have upheld the original view of PHV genetics with three genes such as nucleocapsid (N) protein, envelope proteins (G1, G2) and RNA dependent RNA polymerase. In this study, we report the existence of additional gene that is encoded in an overlapping reading frame of the N protein gene within S genome segment of PHV. This gene is expected to encode a nonstructural small (NSs) protein and it seems to be only found in PHV infected cell. The presence and synthesis of NSs protein could be demonstrated in the cell infected with PHV using anti-peptide sera specific to the predicted amino acid sequence deduced from the second open reading frame. Ribosomal synthesis of this protein appears to occur at AUG codon at the 83rd base of S genome segment, downstream of N protein initiation codon. This protein is small in size (10.4 KDa) and highly basic in nature. The expression strategy of NSs protein appears that a signal mRNA is used to translate both N and NSs protein in PHV infected cell. 10 KDa protein in virus infected cell lysates can bind to mimic dsRNA. This fact strongly suggests that NSs protein may be involved in virus replication on late phase of viral life cycle.

• Korean Journal of Microbiology
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• v.38 no.1
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• pp.45-49
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• 2002

Bradyrhizobium japonicum is a soil bacterium with a unique ability to infect the roots of leguminous plants and establish a nitrogen-fixing symbiosis, which has been used as a microbial manure. In this study, we examined the stress response after pretreatment of cells with cold temperature. When pre-treated with cold temperature ($4^{\circ}C$) for 16 hr, B. japonicum increased the viability in subsequent stress-conditions such as alcohol, $H_2O_2$, heat, and dehydration. For cold adpatation, cultured B. japonicum was exposed to $4^{\circ}C$. Upon subsequent exposure to various conditions, the number of adapted cells pretreated by cold adaptation was 10-1000 fold higher than that of non-adaptated ones. It appeared de novo protein synthesis occurred during adaptation, because a protein synthesis inhibitor, chloramphenicol abolished the increased stress tolerance. By using a degenerate PCR primer set, a csp homolog was amplified from B. japonicum genome and sequenced. The deduced partial amino acid sequence of the putative Csp (Cold shock protein) shares a significant similarity with known Csp proteins of other bacteria.

• BMB Reports
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• v.44 no.6
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• pp.387-392
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• 2011

To investigate the molecular scavenging capabilities of the larvae of Hermetia illucens, two serine proteases (SPs) were cloned and characterized. Multiple sequence alignments and phylogenetic tree analysis of the deduced amino acid sequences of Hi-SP1 and Hi-SP2 were suggested that Hi-SP1 may be a chymotrypsin- and Hi-SP2 may be a trypsin-like protease. Hi-SP1 and Hi-SP2 3-D homology models revealed that a catalytic triad, three disulfide bonds, and a substrate-binding pocket were highly conserved, as would be expected of a SP. E. coli expressed Hi-SP1 and Hi-SP2 showed chymotrypsin or trypsin activities, respectively. Hi-SP2 mRNAs were consistently expressed during larval development. In contrast, the expression of Hi-SP1 mRNA fluctuated between feeding and molting stages and disappeared at the pupal stages. These expression pattern differences suggest that Hi-SP1 may be a larval specific chymotrypsin-like protease involved with food digestion, while Hi-SP2 may be a trypsin-like protease with diverse functions at different stages.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.761-768
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• 2007

Serratia proteamaculans HY-3 isolated from the digestive tract of a spider produces an extracellular protease named arazyme, with an estimated molecular mass of 51.5 kDa. The purified enzyme was characterized as having high activities at wide pH and temperature ranges. We further characterized biochemical features of the enzymatic reactions under various reaction conditions. The protease efficiently hydrolyzed a broad range of protein substrates including albumin, keratin, and collagen. The dependence of enzymatic activities on the presence of metal ions such as calcium and zinc indicated that the enzyme is a metalloprotease, together with the previous observation that the proteolytic activity of the enzyme was not inhibited by aspartate, cysteine, or serine protease inhibitors, but strongly inhibited by 1,10-phenanthroline and EDTA. The araA gene encoding the exoprotease was isolated as a 5.6 kb BamHI fragment after PCR amplification using degenerate primers and subsequent Southern hybridization. The nucleotide sequence revealed that the deduced amino acid sequences shared extensive similarity with those of the serralysin family of metalloproteases from other enteric bacteria. A gene(inh) encoding a putative protease inhibitor was also identified immediately adjacent to the araA structural gene.

• The Plant Pathology Journal
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• v.31 no.4
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• pp.371-378
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• 2015

Surveys of yellowing viruses in plastic tunnels and in open field crops of melon (Cucumis melo cultivar catalupo), oriental melon (C. melo cultivar oriental melon), and cucumber (C. sativus) were carried out in two melon-growing areas in 2014, Korea. Severe yellowing symptoms on older leaves of melon and chlorotic spots on younger leaves of melon were observed in the plastic tunnels. The symptoms were widespread and included initial chlorotic lesions followed by yellowing of whole leaves and thickening of older leaves. RT-PCR analysis using total RNA extracted from diseased leaves did not show any synthesized products for four cucurbit-infecting viruses; Beet pseudo-yellows virus, Cucumber mosaic virus, Cucurbit yellows stunting disorder virus, and Melon necrotic spot virus. Virus identification using RT-PCR showed Cucurbit aphid-borne yellows Virus (CABYV) was largely distributed in melon, oriental melon and cucumber. This result was verified by aphid (Aphis gossypii) transmission of CABYV. The complete coat protein (CP) gene amplified from melon was cloned and sequenced. The CP gene nucleotide and the deduced amino acid sequence comparisons as well as phylogenetic tree analysis of CABYV CPs showed that the CABYV isolates were undivided into subgroups. Although the low incidence of CABYV in infections to cucurbit crops in this survey, CABYV may become an important treat for cucurbit crops in many different regions in Korea, suggesting that CABYV should be taken into account in disease control of cucurbit crops in Korea.

• Journal of Life Science
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• v.11 no.2
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• pp.111-115
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• 2001

This study was designed to clone the SNF2/SW12 helicase-related genes from the fission yeast Schizosaccha-romyces pombe and thereafter to elucidate the common functions of the proteins in this family. The $hrp^{2+}$gene was cloned by polymerase chain reaction amplification using degenerative primers from conserved SNF2 motifs within the ERCC6 gene, which encodes a protein involved in DNA excision repair. Like other SNF2/SW12 family proteins, the deduced amino acid sequence of Hrp2 contains DNA-dependent ATPase/7 helicase domains as well as the chromodomain and the DNA binding domain. This configuration is similar to that of mCHD1 (mouse chromo-ATPase/helicase-DNA-dinding protein 1), suggesting that Hrp2 is a S. pombe homolog of mCHD1, which is thought to function in altering the chromatin structure to control the gene expression. To characterize the function of Hrp2, 4 Uracil-Hrp2 fusion protein, it was purified near homogeneity by affinity chromatography on $Ni^{2+}$-NTA agarose, DEAE-Sepharose ion exchange arid Sephacryl S-200 gel filtration chromatographies. The purified fusion protein exhibited DNA-dependent ATPase activity, which was stimulated by both double-stranded and single-stranded DNA. To determine the steady-state level of $hrp^{2+}$ transcripts during growth, cells were cultured in medium and collected at every 2hr to prepare total RNAs. The northern blot analysis showed that the level of $hrp^{2+}$ transcripts reached its maximum before the cells entered the exponential growth phase and then decreased gradually, This result implies that Hrp2 may be required at early stages of cell growth.h.

• Korean Journal of Ichthyology
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• v.19 no.4
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• pp.266-273
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• 2007

Heat shock proteins (HSPs) are a family of highly conserved proteins playing an important role in the functioning of unstressed and stressed cells. The HSP70 family, the most widely studied of the hsps, is constitutively expressed (hsc70) in unstressed cells and is also induced in response to stressors (hsp70), especially those affecting the protein machinery. The HSP/HSC70 proteins act as molecular chaperones and are crucial for protein functioning, including folding, intracellular localization, regulation, secretion, and protein degradation. Here, we report the identification and characterization of the putative amino acid sequence deduced from one cDNA clone identified as heat shock protein 70. The alignment showed that the putative sequence is 100% identical to the heat shock protein 70 cognate (HSC 70) of olive flounder. The 5'-flanking region sequence (approximately 1 kb) ahead of the hsc70 gene was cloned by genome walking and a putative core promoter region and transcription elements were identified. We characterized the promoter of the olive flounder hsc70 gene by examining the ability of 5'-upstream fragments to drive expression of green fluorescent protein (GFP) in live embryos.

• International Journal of Industrial Entomology and Biomaterials
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• v.18 no.1
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• pp.18-27
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• 2009

B. thuringiensis strain LY-99 belonging to subsp. alesti (H3a3c), was isolated from Chinese tobacco warehouse and showed significantly high toxicity to Plutella xylostella. For the identification of the cry1-type genes from B. thuringiensis LY-99, an extended multiplex PCRrestriction fragment length polymorphism (PCRRFLP) method was established by using two pairs of universal primers based on the conserved regions of the cry1-type genes to amplify around 2.4 kb cry1-type gene fragments. Then the DNA fragment was cloned into pGEM-T Easy vector and digested with EcoRI and EcoRV enzymes. Through this method, a known cry1-type gene was successfully identified from the reference strain, B. thuringiensis subsp. alesti. In addition, the RFLP patterns revealed that B. thuringiensis LY-99 included a novel cry1A-type gene in addition to cry1Aa, cry1Ac, cry1Be and cry1Ea genes. The novel cry1A-type gene was designated cry1Ah2 (Genbank accession No DQ269474). An inverse PCR method was used to amplify the flank regions of cry1Ah2 gene. Finally, 3143 bp HindIII fragment from B. thuringiensis LY-99 plasmid DNA including 5' region and partial ORF was amplified, and sequence analysis revealed that cry1Ah2 gene from LY-99 showed 89.31% of maximum sequence similarity with cry1Ac1 crystal protein gene. In addition, the deduced amino acid sequence of Cry1Ah2 protein shared 87.80% of maximum identity with that of Cry1Ac2. This protein therefore belongs to a new class of B. thuringiensis crystal proteins.

• Korean Journal of Poultry Science
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• v.38 no.2
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• pp.89-96
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• 2011

The outbreak of Newcastle disease occurred for the first time at a commercial chicken farm near Ulaanbaatar, Mongolia in August 2010. Newcastle disease virus (NDV) obtained from infected chickens in Mongolia was characterized by biological and molecular biological approches. Mongolian NDV isolate killed all of chicken embryos within 60 h in the mean death time assay, indicating virulent for chicken. A genomic region of 695 nts between nts 1055 of the M gene and 508 of the F gene was amplified by RT-PCR and sequenced. The deduced amino acid sequence of the F protein cleavage site was $^{112}RRQKRF^{117}$, which is a typical sequence of velogenic strains of NDV and is agreement with the result of the MDT assay. The sequence of the partial F gene (nts 47 to 435) was used for genotyping by phylogenetic analysis. The phylogenetic analysis showed that the Mongolian isolate was of genotype VII within class II of NDV. Further phylogenetic analysis on the genotype VII strains revealed that the isolates placed in a genetic sublineage of VIId and most closely related with velogenic strains of NDV circulating in Far-east Asian region especially China, suggesting the introduction of velogenic NDV into Mongolia from neighboring countries.

• BMB Reports
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• v.40 no.5
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• pp.715-722
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• 2007

A new full-length cDNA encoding hyoscyamine $6\beta$-hydroxylase (designated as aah6h, GenBank Accession No. EF187826), which catalyzes the last committed step in the scopolamine biosynthetic pathway, was isolated from young roots of Anisodus acutangulus by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of aah6h was 1380 bp and contained a 1035 bp open reading frame (ORF) encoding a deduced protein of 344 amino acid residues. The deduced protein had an isoelectric point (pI) of 5.09 and a calculated molecular mass of about 38.7 kDa. Sequence analyses showed that AaH6H had high homology with other H6Hs isolated from some scopolamine-producing plants such as Hyoscyamus niger, Datura metel and Atropa belladonna etc. Bioinformatics analyses results indicated AaH6H belongs to 2-oxoglutarate-dependent dioxygenase superfamily. Phylogenetic tree analysis showed that AaH6H had closest relationship with H6H from A. tanguticus. Southern hybridization analysis of the genomic DNA revealed that aah6h belonged to a multi-copy gene family. Tissue expression pattern analysis firstly founded that aah6h expressed in all the tested tissues including roots, stems and leaves and indicated that aah6h was a constitutive-expression gene, which was the first reported tissue-independent h6h gene compared to other known h6h genes.