• Journal of Plant Biotechnology
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• v.33 no.2
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• pp.123-131
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• 2006

Chinese cabbage (Brassica rapa ssp. pekinesis) is one of the most important vegetable crops in korea and other East Asian countries. Cytosolic fructose-1,6-bisphospha-tase (cytFBPase) is a key enzyme in sucrose biosyn-thesis, which controls the sucrose levels as well as the productivity at plants. The Chinese cabbage cytFBPase gene, BrFBPase, encodes the 340 amino acid polypep-tide, giving a theoretical molecular weight of 37.2 kD and a isolectric point of 5.4. BrFBPase showed high sequence identity with Brassica homologs and its functional domains, such as 12,6P$_2$ binding site or active site and F6P binding site, were highly conserved in diverse sources of organisms. Although the genome of Chinese cabbage seemed to be triplicated, BrFBPase appears to be a single copy gene. The expression of BrFBPase was examined at transcript and protein levels under various conditions. BrFBPase expression was observed only in photosynthetic source tissue, not in sink tissue. The expression was slightly higher during the day than at night, and it showed a diurnal cycle with circadian rhythmicity. Short-term exposure to low temperature inhibited the expression of the BrFBPase, while long-term exposure increased the expression, supporting that sugar levels are high in late autumn when temperature are low.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.44-44
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• 2003

$BK_{Ca}$ channels were suggested to contain one or more domains of the ‘regulator of K+ conductance’(RCK) in their cytosolic carboxyl termini (Jiang et al.2001). It was also shown that the RCK domain in mammalian $BK_{Ca}$ channels might sense the intracellular $Ca^{2+}$ with a low affinity (Xia et al. 2002). We aligned the amino acid sequence of the $\alpha$-subunit of rat $BK_{Ca}$ channels (rSlo) with known RCK domains and identified a second region exhibiting about 50％ homology. This putative domain, RCK2, contains the characteristic amino acids conserved in other RCK domains. We wondered whether this second domain is involved in the domain-domain interaction and the gating response to intracellular $Ca^{2+}$ for rSlo channel, as revealed in the structure of RCK domain of E. coli channel (Jiang et al.2001). In order to examine the possibility, site-directed mutations were introduced into the RCK2 domain of rSlo channel and the mutant channels were expressed in Xenopus oocytes for functional studies. One of such mutation, G772D, in the putative nucleotide-binding domain resulted in the enhanced $Ca^{2+}$ sensitivity and the channel gating of rSlo channel. These results suggest that this region of $BK_{Ca}$ channels is important for the channel gating and may form an independent domain in the cytosolic region of $BK_{Ca}$ channels. In order to obtain the mechanistic insights of these results, G772 residue was randomly mutagenized by site-directed mutagenesis and total 17 different mutant channels were constructed. We are currently investigating these mutant channels by electrophysiological techniques.ical techniques.

• Korean Journal of Plant Taxonomy
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• v.38 no.1
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• pp.51-61
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• 2008

Leaves are indeterminate organs and possess a lot of genes which is involved in establishing leaf polarities. These polarities are regulated relatively early during leaf development and defined relative to the factors intrinsic to the primordia and interactions with the shoot apical meristem (SAM). Recently, several genes that control the polarity of lateral organs have been identified. Our genetic study of deformed root and leaf1 (drl1) mutant, which produces narrow, filament‐like leaves and defective meristems, revealed that DRL1 is involved in the regulation of SAM activity and leaf polarity. The DRL1 gene was found to encode a novel protein showing homology to Elongator‐associate protein (EAP) of yeast KTI12. The amino acid sequence of DRL1 is universally conserved in prokaryotes and eukaryotes. DRL1 and the plant DRL1 homologs clearly formed a monophyletic clade, suggesting the evolutionary conservation of DRL1 homologs was maintained in the genomes of all land plants.

• Bulletin of the Korean Chemical Society
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• v.30 no.6
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• pp.1360-1364
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• 2009

The bacterium Sphingomonas chungbukensis DJ77 produces the extracellular polysaccharide gellan in high yield. Gellan produced by this bacterium is widely used as a gelling agent, and the enzyme UDP-glucose pyrophosphorylase (UGP) is thought to play a key role in the gellan biosynthetic pathway. The UGP gene has been successfully cloned and over-expressed in E. coli. The expressed enzyme was purified with a molecular weight of approximately 32 kDa, as determined by a SDS-polyacrylamide gel, but the enzyme appears as ca. 63 kDa on a native gel, suggesting that the enzyme is present in a homodimer. Kinetic analysis of UDP-glucose for UGP indicates $K_m$ = 1.14 mM and $V_{max}$ = 10.09 mM/min/mg at pH 8.0, which was determined to be the optimal pH for UGP catalytic activity. Amino acid sequence alignment against other bacteria suggests that the UGP contains two conserved domains: An activator binding site and a glucose-1-phosphate binding site. Site-directed mutagenesis of Lys194, located within the glucose-1-phosphate binding site, indicates that substitution of the charge-reversible residue Asp for Lys194 dramatically impairs the UGP activity, supporting the hypothesis that Lys194 plays a critical role in the catalysis.

• 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.

• Archives of Pharmacal Research
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• v.29 no.5
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• pp.384-393
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• 2006

Protein carboxylmethylation methylates the free carboxyl groups in various substrate proteins by protein carboxyl O-methyltransferase (PCMT) and is one of the post-translational modifications. There have been many studies on protein carboxylmethylation. However, the precise functional role in mammalian systems is unclear. In this study, immunoglobulin, a specific form of $\gamma-globulin$, which is a well-known substrate for PCMT, was chosen to investigate the regulatory roles of protein carboxylmethylation in the immune system. It was found that the anti-BSA antibody could be carboxylmethylated via spleen PCMT to a level similar to $\gamma-globulin$. This carboxylmethylation increased the hydrophobicity of the anti-BSA antibody up to 11.4%, and enhanced the antigen-binding activity of this antibody up to 24.6%. In particular, the Fc region showed a higher methyl accepting capacity with 80% of the whole structure level. According to the amino acid sequence alignment, indeed, 7 aspartic acids and 5 glutamic acids, as potential carboxylmethylation sites, were found to be conserved in the Fc portion in the human, mouse and rabbit. The carboxylmethylation of the anti-BSA antibody was reversibly demethylated under a higher pH and long incubation time. Therefore, these results suggest that protein carboxylmethylation may reversibly regulate the antibody-mediated immunological events via the Fc region.

• Journal of Microbiology
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• v.38 no.2
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• pp.66-73
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• 2000

To investigate the genetic relationship among 12 species belonging to the Fusarium section Martiella, Dlaminia, Gibbosum, Arthrosporiella, Liseola and Elegans, the internal transcribed spacer(ITS) regions of ribosomal DNA (rDNA) were amplified with primer pITS1 and pITS4 using the polymerase chain reaction(PCR). After the amplified products were digested with 7 restriction enzymes, restriction fragment length polymorphism (RFLP) patterns were analyzed. The partial nucleotide sequences of the ITS region were determined and compared. Little variation was observed in the size of the amplified product having sizes of 550bp or 570bp. Based on the RFLP analysis, the 12 species studied were divided into 5 RFLP types. In particular, strains belonging to the section Martiella were separated into three RFLP types. Interestingly, the RFLP type of F. solani f. sp. piperis was identical with that of isolates belonging to the section Elegans. In the dendrogram derived from RFLP analysis of the ITS region, the Fusarium spp. examined were divided into two major groups. In general, section Martiella excluding F. solani f. sp. piperis showed relatively low similarity with the other section. The dendrogram based on the sequencing analysis of the ITS2 region also gave the same results as that of the RFLP analysis. As expected, 5.8S, a coding region, was highly conserved, whereas the ITS2 region was more variable and informative. The difference in the ITS2 region between the length of F. solani and its formae speciales excluding F. solani f. sp. piperis and that of other species was caused by the insertion/deletion of nucleotides in positions 143-148 and 179-192.

• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1221-1226
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• 2008

The soft rot bacterium Pectobacterium wasabiae is an economically important pathogen of many crops. A new phytase gene, appA, was cloned from P. wasabiae by degenerate PCR and TAIL-PCR. The open reading frame of appA consisted of 1,302 bp encoding 433 amino acid residues, including 27 residues of a putative signal peptide. The mature protein had a molecular mass of 45 kDa and a theoretical pI of 5.5. The amino acid sequence contained the conserved active site residues RHGXRXP and HDTN of typical histidine acid phosphatases, and showed the highest identity of 48.5% to PhyM from Pseudomonas syringae. The gene fragment encoding the mature phytase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant phytase had a specific activity of 1,072$\pm$47 U/mg for phytate substrate. The optimum pH and temperature for the purified phytase were pH 5.0 and 50$^{\circ}C$, respectively. The $K_m$ value was 0.17 mM, with a $V_{max}$ of 1,714 $\mu$mol/min/mg. This is the first report of the identification and isolation of phytase from Pectobacterium.

• Plant Biotechnology Reports
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• v.3 no.3
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• pp.213-223
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• 2009

Lipid transfer proteins (LTPs) are widely distributed in the plant kingdom, but their functions remain elusive. The proteins AlLTP2-4 were isolated from three related Allium plants: garlic (A. sativum L.), Welsh onion (A. fistulosum L.), and Nanking shallot (A. ascalonicum L.). These novel proteins comprise a new class of LTPs associated with the Ace-AMP1 from onion (A. cepa L.). The AlLTP genes encode proteins harboring 132 common amino acids and also share a high level of sequence identity. Protein characteristics and phylogenetic analysis suggest that LTPs could be classified into five distinct groups. The AlLTPs were clustered into the most distantly related plant LTP subfamily and appeared to be restricted to the Allium species. In particular, the number of amino acids existing between the fourth and fifth Cys residue was suggested as a conserved motif facilitating the categorization of all the LTP-related proteins in the family. Unlike other LTPs, AlLTPs harboring both the putative C-terminal propeptide and N-terminal signal peptide were predicted to be localized to cytoplasmic vacuoles. When a chimeric GFP protein fused with both N-terminal and C-terminal AlLTP2 signal peptides was expressed in rice cells, the fluorescence signal was detected in the endomembrane compartments, thereby confirming that AlLTPs are an unprecedented intracellular type of LTP. Collectively, our present data demonstrate that AlLTPs are a novel type of LTP associated with the Allium species.

• Molecules and Cells
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• v.28 no.5
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• pp.463-472
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• 2009

Although the possible cellular roles of several ubiquitin-specific proteases (UBPs) were identified in Arabidopsis, almost nothing is known about UBP homologs in rice, a monocot model plant. In this report, we searched the rice genome database (http://signal.salk.edu/cgi-bin/RiceGE) and identified 21 putative UBP family members (OsUBPs) in the rice genome. These OsUBP genes each contain a ubiquitin carboxyl-terminal hydrolase (UCH) domain with highly conserved Cys and His boxes and were subdivided into 9 groups based on their sequence identities and domain structures. RT-PCR analysis indicated that rice OsUBP genes are expressed at varying degrees in different rice tissues. We isolated a full-length cDNA clone for OsUBP6, which possesses not only a UCH domain, but also an N-terminal ubiquitin motif. Bacterially expressed OsUBP6 was capable of dismantling K48-linked tetra-ubiquitin chains in vitro. Quantitative real-time RT-PCR indicated that OsUBP6 is constitutively expressed in different tissues of rice plants. An in vivo targeting experiment showed that OsUBP6 is predominantly localized to the nucleus in onion epidermal cells. We also examined how knock-out of OsUBP6 affects developmental growth of rice plants. Although homozygous T3 osubp6 T-DNA insertion mutant seedlings displayed slower growth relative to wild type seedlings, mature mutant plants appeared to be normal. These results raise the possibility that loss of OsUBP6 is functionally compensated for by an as-yet unknown OsUBP homolog during later stages of development in rice plants.