• BMB Reports
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• v.39 no.1
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• pp.68-75
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• 2006

In higher plants, P450s participate in the biosynthesis of many important secondary metabolites. Here we reported for the first time the isolation of a new cytochrome P450 cDNA that expressed in a stem-specific manner from Camptotheca acuminata (designated as CaSS), a native medicinal plant species in China, using RACE-PCR. The full-length cDNA of CaSS was 1735 bp long containing a 1530 bp open reading frame (ORF) encoding a polypeptide of 509 amino acids. Bioinformatic analysis revealed that CASS contained a heme-binding domain PFGXGRRXCX and showed homology to other plant cytochrome P450 monooxygenases and hydroxylases. Southern blotting analysis revealed that there was only one copy of the CaSS present in the genome of Camptotheca acuminata. Northern blotting analysis revealed that CaSS expressed, in a tissue-specific manner, highly in stem and lowly in root, leaf and flower. Our study suggests that CaSS is likely to be involved in the phenylpropanoid pathway.

• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.545-554
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• 2013

Plasmid isolation of kimchi-derived Weissella cibaria KLC140 revealed six different plasmids. The smallest plasmid, pKW2124, was DNA sequenced and characterized, showing 2,126 bp with a GC content of 36.39% and five putative open reading frames (ORFs). In silico analysis of these ORFs showed ORF1 encodes a putative replication protein similar to rolling circular replication proteins from other lactic acid bacteria. However, a single-stranded intermediate was not detected when S1 nuclease was treated, suggesting it may follow theta replication. Interestingly, the replication initiation site of this plasmid is 100% identical to other plasmids from lactic acid bacteria, suggesting it may function for replication initiation. To construct a surface layer expression vector, pTSLGFP, slpA encoding the surface layer protein from Lactobacillus acidophilus was PCR amplified and fused with the gfp gene, forming a SLGFP fused gene. The plasmid pKW2124 was cloned into the XbaI site of pUC19, forming an Weissella-E. coli shuttle vector pKUW22. NheI-linearized pTSLGFP was ligated into pKUWCAT containing pKUW22 and the chloramphenicol acetyltransferase gene from pEK104, resulting in an 8.6 kb pKWCSLGFP surface layer expression vector. After transformation of this vector into W. cibaria KLC140, a GFP fluorescence signal was detected on the surface of the transformant, substantiating production of SLGFP fused protein and its secretion. This is the first report for construction of a Weissella surface layer expression vector, which may be useful for surface layer production of beneficial proteins in Weissella.

• Journal of Life Science
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• v.20 no.8
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• pp.1193-1200
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• 2010

The cDNA cloning and developmental profiles of the mRNA for A. pernyi arylphorin was determined. The complete A. pernyi arylphorin cDNA sequence comprised 2,234 bp (without the poly $A^+$ tail), including an open reading frame of 2,112 bp beginning with a methionine ATG at bp34. The A. pernyi arylphorin contained 704 amino acids which are highly enriched in aromatic amino acids, phenylalanine and tyrosine. The calculated molecular mass of the A. pernyi arylphorin from the ORF was 83,439 Da. The deduced amino acid sequence of A. pernyi arylphorin showed 78, 71, 62 and 64% identity with those of H. cecropia, M. sexta $\alpha$ subunit, M. sexta $\beta$ subunit and B. mori storage protein. In Northern blot analysis, the A. pernyi arylphorin mRNA only in the fat body of the 5th instar larvae was responsible for gene expression of the protein, and the synthetic activity of the mRNA was detected strongly in the early larvae, but not in the middle or late-stage larvae. In addition, a very weak signal in mRNA activity was detected in pupal stages, but this was considered to be inactive mRNA after reviewing the results of the labeling experiment of this protein.

• Journal of Life Science
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• v.27 no.11
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• pp.1276-1289
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• 2017

This study isolated and purified the antimicrobial peptide McSSP-31 from an acidified hemocyte extract of a Mytilus coruscus. The antimicrobial peptide was purified by using a $C_{18}$ reversed-phase high-performance liquid chromatography (HPLC). The peptide was determined to be 3330.549 Da by matrix assisted-laser desorption ionization time-of-flight mass spectrophotometer (MALDI-TOF/MS). The N-terminus of a 14 amino-acid sequence was identified as P-S-P-T-R-R-S-T-S-R-S-K-S-R by Edman degradation method. The acquired sequence showed a 93% similarity with the sperm-specific protein Phi-1, which is from M. californianus. The identified open-reading frame (ORF) of peptide was 306 bp encoding 101 amino acids, which was analyzed by rapid amplification of cDNA ends (RACE), cloning and sequencing analysis. We compared the full sequence with other known proteins that reveal the sperm-specific protein Phi-1 (93.5%) of M. californianus. Synthesized antimicrobial peptide (McSSP-31) showed antibacterial activity against gram-positive bacteria including B. subtilis, S. mutans, S. aureus and gram-negative bacteria including E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa and fungi, C. albicans. Also, synthesized peptide showed strong antibacterial activity against antibiotic-resistant strains, including S. aureus. The cytotoxicity of the peptide was determined by using the HUVEC human cell line. The peptide did not exhibit any significant cytotoxic effects on the normal human cell line, and it had very low hemolytic activity with flounder hemoglobin. The results demonstrated that peptide purified from the hemocyte of a M. coruscus exhibits antibacterial activity against various bacteria and has the potential to be an alternative antibiotic agent.

• Journal of Life Science
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• v.19 no.5
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• pp.566-572
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• 2009

A recombinant plasmid, pDH3, was obtained from the genomic library of Serattia marcescens KCTC 2172, and several recombinant subclones constructed from pDH3. The nucleotide sequence of a 5,137 bp segment, pPH4, was determined and three open reading frames were detected. The three ORFs encoded the phosphate specific transport (pst) operon, which was pstC, pstA, and pstB, with the same direction of transcription. Comparison of the pst operon of S. marcescens with that of other organisms revealed that the genes for pstS and phoU were missing. A potential CRP bonding site and pho box sequence was found in the upstream of the putative promoter at the regulatory region. Analysis of the nucleotide sequence showed that homology in amino acid sequences between the PstC protein and Yersinia sp., Vibrio sp., and Pseudomonas sp. were 49, 37 and 33%, respectively. The PstA protein and Yersinia sp., Vibrio sp., and Pseudomonas sp. showed homologies of 64, 51, and 47%, respectively. PstB protein and Methanocaldococcus sp., E. coli, and Mycoplasma sp. showed homologies of 60, 50, and 48%, respectively. The pst genes could be expressed in vivo and positively regulated by cAMP-CRP. The E. coli strain harboring plasmid pPH7, with pst genes, increased with the transport of phosphate.

• The Korean Journal of Malacology
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• v.30 no.4
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• pp.399-408
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• 2014

Glutathione S-transferases (GSTs) are a superfamily of detoxification enzymes that primarily catalyze the nucleophilic addition of reduced glutathione to both endogenous and exogenous electrophiles. In this study, we isolated and characterized a full-length of alpha class GST cDNA from the abalone (Haliotis discus hannai). The abalone GST cDNA encodes a 223-amino acid polypeptide with a calculated molecular mass of 25.8 kDa and isoelectric point of 5.69. Multiple alignments and phylogenetic analysis with the deduced abalone GST protein revealed that it belongs to the alpha class GSTs and showed strong homology with disk abalone (Haliotis discus discus) putative alpha class GST. Abalone GST mRNA was ubiquitously detected in all tested tissues. GST mRNA expression was comparatively high in the mantle, gill, liver, and digestive duct, however, lowest in the hemocytes. Expression level of abalone GST mRNA in the mantle, gill, liver, and digestive duct was 182.7-fold, 114.8-fold, 4675.8-fold, 406.1-fold higher than in the hemocytes, respectively. Expression level of abalone GST mRNA in the liver was peaked at 6 h post-infection with Vibrio parahemolyticus and decreased at 12 h post-infection. While the expression level of abalone GST mRNA in the hemocytes was drastically increased at 3 h post-infection with Vibrio parahemolyticus. These results suggest that abalone GST is conserved through evolution and may play roles similar to its mammalian counterparts.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.1
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• pp.116-121
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• 2010

Long-chain polyunsaturated fatty acids (LC-PUFA) promote the development of brain and vision of the fetus, relieve inflammation, inhibit oral dysplasia of rumor cell, decrease the incidence of cardiovascular disease and regulate arrhythmia. ${\Delta}-6$ desaturase is the rate-limited enzyme in the desaturation process. This study reports the cloning, characterization and tissue expression of a ${\Delta}-6$ desaturase gene in the chicken. PCR primers were designed based on the predicted sequence of chicken ${\Delta}-6$ desaturase (accession number: XM421053) and used to isolate a cDNA fragment of 1,323 bp from chicken liver. Based on the 1,323 bp fragment an EST (BI390105) was obtained by BLAST. The EST and 5'nd of the 1,323 bp fragment were partially overlapped. Gene specific primers derived from the EST were used for amplification of the 5'nd. Another gene-specific primer derived from the 1,323 bp fragment was used for amplification of the 3'nd by 3'ACE. Then the three overlapping cDNA sequences obtained were assembled with DNAMAN software and a full-length ${\Delta}-6$ desaturase of 2,153 bp was obtained. The full-length cDNA contained an ORF of 1,335 bp with a 5'ntranslated region of 147 nucleotides followed by an ATG initiation codon. Stop codon TGA was at position 1,481-1,483 bp. The deduced amino acids shared an homology above 77% with bovine, mice, orangutan, rat and human. The protein sequence had three histidine-rich regions HDFGH (HisI region), HFQHH (HisII region) and HH (HisIII region), a cytochrome $b_{5}$-like domain containing a heme-binding motif and two transmembrane domains. Sequence analysis of the chicken genomic DNA revealed that the coding sequence of chicken ${\Delta}-6$ desaturase included 12 exons and 11 introns. Semi-quantitative RT-PCR showed that the ${\Delta}-6$ desaturase expression levels were in turn liver, spleen, pancreas, lung, breast muscle, heart, and abdominal fat. The expression of ${\Delta}-6$ desaturase in liver was significantly higher than that in breast muscle (p<0.01). The expression of ${\Delta}-6$ desaturase in lung was significantly higher than that in abdominal fat (p<0.01). This is the first clone of chicken ${\Delta}-6$ desaturase.

• Journal of the Korean Wood Science and Technology
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• v.34 no.3
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• pp.56-63
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• 2006

In order to evaluate the mechanism of cellulose hydrolysis, the complementary DNA encoding Glycoside Hydrolase Family (GHF)74 was cloned from Phanerochaete chrysosporium. Depending on the presence of Cellulose Binding Module (CBM), it can be classified as GHF74A or GHF74B. The GHF74A gene from P. chrysosporium (PcGHF74A) consists of 2163 bp encoding a protein of 721 amino acid residues. The PcGHF74A showed homology of 70~77% compared with the GHF74 from other filamentous fungi. The PcGHF74B, which contains CBM and is a member of family 1, was transcribed to various transcripts depending on the nature of carbon sources and their concentration. To study the possible presence of splice variants in GHF74B transcripts in P. chrysospoium, we carried out RT-PCR analysis using primers that designed based on the annotation data and sequenced data. Our result indicated that PcGHF74B was transcribed to several splicing variants in various culture conditions. Especially in the culture of 2% cellulose, three transcript products were observed. First transcript was presumed to be a full length ORF that contained 11th intron with stop codon at position 2562 bp. The second one consisted of 12 exons and 11 introns with stop codon at position 1187 bp with 7th exon. The shortest transcript consisted of 10 exons and 9 introns with stop codon at 910 bp in the 7th exon. These premature stop codon might prevent the synthesis of fully active GHF74 or contribute for the production of protein with distinct function depending on the ambient carbon sources.

• Journal of Microbiology and Biotechnology
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• v.23 no.11
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• pp.1519-1528
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• 2013

Bacillus pumilus SG2, a halotolerant strain, expresses two major chitinases designated ChiS and ChiL that were induced by chitin and secreted into the supernatant. The present work aimed to obtain a mutant with higher chitinolytic activity through mutagenesis of Bacillus pumilus SG2 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis and screening on chitin agar and subsequent formation of halos, the mutated strains were examined for degradation of chitin under different conditions. A mutant designated AV2-9 was selected owing to its higher chitinase activity. To search for possible mutations in the whole operon including ChiS and ChiL, the entire chitinase operon, including the intergenic region, promoter, and two areas corresponding to the ChiS and ChiL ORF, was suquenced. Nucleotide sequence analysis of the complete chitinase operon from the SG2 and AV2-9 strains showed the presence of a mutation in the catalytic domain (GH18) of chitinase (ChiL). The results demonstrated that a single base change had occurred in the ChiL sequence in AV2-9. The wild-type chitinase, ChiL, and the mutant (designated ChiLm) were cloned, expressed, and purified in E. coli. Both enzymes showed similar profiles of activity at different ranges of pH, NaCl concentration, and temperature, but the mutant enzyme showed approximately 30% higher catalytic activity under all the conditions tested. The results obtained in this study showed that the thermal stability of chitinase increased in the mutant strain. Bioinformatics analysis was performed to predict changes in the stability of proteins caused by mutation.

• Research in Plant Disease
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• v.22 no.4
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• pp.289-292
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• 2016

We surveyed the occurrence of Japanese yam mosaic virus (JYMV) on Yam in Gyeongsangbukdo pronvince from 2013 to 2015. The symptoms of JYMV were yellow stripes and chlorosis in yam leaves and the infection rate was ranged from 33.6% to 40.8%. We determined nucleotide sequence encoding the polyprotein of JYMV isolate BRI from yam leaves using next-generation sequencing (NGS) method. The partial nucleotide portion (7,736 nucleotides) of the genomic RNA of the JYMV isolate BRI has been sequenced (accession No. KU309315). The region sequenced includes a single open reading frame (ORF) encoding a polyprotein composed of 2,497 amino acids containing the coat protein (CP) and 3' untranslated region (UTR). The genomic organization of this isolate shows almost the same to that of other members of JYMV. The JYMV isolate BRI showed 77% to 79% nucleotide identity with the Japanese and Chinese strains and isolates. This is the first report of the genome nucleotide sequence of JYMV from Dioscorea opposita in Korea.