• Journal of Microbiology and Biotechnology
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• v.19 no.1
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• pp.17-22
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• 2009

The mixotrophic growth with methanol plus thiosulfate was examined in nutrient-limited mixotrophic condition for Methylobacterium goesingense CBMB5 and Methylobacterium fujisawaense CBMB37. Thiosulfate oxidation increased the growth and protein yield in mixotrophic medium that contained 150mM methanol and 20mM sodium thiosulfate, at 144 h. Respirometric study revealed that thiosulfate was the most preferable reduced inorganic sulfur source, followed by sulfite and sulfur. M. goesingense CBMB5 and M. fujisawaense CBMB37 oxidized thiosulfate directly to sulfate, and intermediate products of thiosulfate oxidation such as polythionates, sulfite, and sulfur were not detected in spent medium and they did not yield positive amplification for tested soxB primers. Enzymes of thiosulfate oxidation such as rhodanese and sulfite oxidase activities were detected in cell-free extracts of M. goesingense CBMB5, and M. fujisawaense CBMB37, and thiosulfate oxidase (tetrathionate synthase) activity was not observed. It indicated that both the organisms use the "non-S4 intermediate" sulfur oxidation pathway for thiosulfate oxidation. It is concluded from this study that M. goesingense CBMB5, and M. fujisawaense CBMB37 exhibited mixotrophic metabolism in medium containing methanol plus thiosulfate and that thiosulfate oxidation and the presence of a "Paracoccus sulfur oxidation" (PSO) pathway in methylotrophic bacteria are species dependant.

• BMB Reports
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• v.28 no.6
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• pp.509-514
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• 1995

A putative secondary structure of the mRNA for the human hepatitis B virus (HBV) X gene is proposed based on not only chemical and enzymatic determination of its single- and double-stranded regions but also selection by the computer program MFOLD for energy minimum conformation under the constraints that the experimentally determined nucleotides were forced or prohibited to base pair. An RNA of 536 nucleotides including the 461-nucleotide HBV X mRNA sequence was synthesized in vitro by the phage T7 RNA polymerase transcription. The thermally renatured transcripts were subjected to chemical modifications with dimethylsulfate and kethoxal and enzymatic hydrolysis with single strand-specific RNase T1 and double strand-specific RNase V1, separately. The sites of modification and cleavage were detected by reverse transcriptase extension of 4 different primers. Many nucleotides could be assigned with high confidence, twenty in double-stranded and thirty-seven in Single-stranded regions. These nucleotides were forced and prohibited, respectively, to base pair in running the recursive RNA folding program MFOLD. The results suggest that 6 different regions (5 within X mRNA) of 14~23 nucleotides are Single-stranded. This putative structure provides a good working model and suggests potential target sites for antisense and ribozyme inhibitors and hybridization probes for the HBV X mRNA.

• Applied Biological Chemistry
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• v.44 no.3
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• pp.153-161
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• 2001

In order to isolate genes involved in ecdysteroids biosynthesis in plants, total RNA was isolated from Achyranthes japonica Nakai, and RT-PCR was performed using degenerate primers selected based on the results of multi-alignment of four cytochrome P450 genes from plants and a putative ecdysone 20-hydroxylase gene from an insect. Fourteen partial cDNA clones showing unique base sequences were obtained, out of which six showed homologies at the levels of nucleotide and amino acid sequences to the other cytochrome P450 genes known to be involved in the ecdysteroid biosynthesis. Of the six clones, four showed relatively high homologies to a putative ecdysone 20-hydroxylase gene isolated from an insect.

• Applied Biological Chemistry
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• v.50 no.4
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• pp.276-280
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• 2007

Buckwheat often causes severe allergic reactions in sensitive people. One of the major allergenic proteins in common buckwheat (Fagopyrum esculentum) has been found to be a BW10KD protein. In this study, we developed a PCR method to detect buckwheat ingredients in food using primers corresponding to the allergenic BW10KD gene. Five pairs of oligonucleotide primers successfully enabled PCR amplification of the specific regions of the genomic BW10KD DNA from buckwheat, but no amplification from seven other cereals and beans (barley, wheat, German millet, African millet, soybean, red bean, and black bean). The proposed PCR method was applied to analyze 12 processed foods (buckwheat flour, buckwheat noodle, buckwheat jelly, wheat noodle, instant noodle, black sesame gruels, sunsik, cookie, misutkaru, and three kinds of cereal); among them, only three samples including buckwheat flour, buckwheat noodle and buckwheat jelly showed a positive reaction to the detection. This PCR method was able to detect as little as 1 ng of common buckwheat DNA. This rapid and specific PCR method would be applicable to detect allergenic buckwheat ingredients in food.

• Journal of the Korean Chemical Society
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• v.53 no.6
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• pp.742-748
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• 2009

In the meat industry, correct breed information in food labeling is required to assure meat quality. Genetic markers provide corroborating evidence to identify breed. We described the development of DNA markers to discriminate between Korean beef cattle (Hanwoo), Holstein, and mixed cow beefs. As most breeds are standardized for coat colour, the melanocortin 1 receptor (MC1R) gene, involved in the regulation of eu/pheomelanins synthesis, has been suggested as marker for breed traceability of products of animal origin. We also designed sex-determining region Y (SRY) gene specific primers for Y chromosome detection. In this study, fragments of MC1R gene and SRY gene were amplified by multiplex-PCR and subjected to digestion by MspA1I restriction endonuclease. Reaction products were analysised by denaturing high performance liquid chromatography (DHPLC). As a result, we identified 6 DHPLC peak types from MC1R gene and SRY gene analysis. DHPLC method showed more sensitive than RFLP method for DNA fragments analysis. Therefore, DHPLC method can apply to identify for Hanwoo, Holstein and mixed beef.

• Journal of Microbiology and Biotechnology
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• v.22 no.8
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• pp.1044-1053
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• 2012

The gene encoding an esterase enzyme was cloned from a metagenomic library of cow rumen bacteria. The esterase gene (est5S) was 1,026 bp in length, encoding a protein of 366 amino acid residues with a calculated molecular mass of 40,168 Da. The molecular mass of the enzyme was estimated to be 40,000 Da. The Est5S protein contains the Gly-X-Ser-X-Gly motif found in most bacterial and eukaryotic serine hydrolases. However, the Asp or Glu necessary for the catalytic triad [Ser-Asp-(Glu)-His] was not present, indicating Est5S represents a novel member of the GHSQG family of esterolytic enzymes. BlastP in the NCBI database analysis of Est5S revealed homology to hypothetical proteins and it had no homology to previous known lipases and esterases. Est5S was optimally active at pH 7.0 and $40^{\circ}C$. Among the p-nitrophenyl acylesters tested, high enzymatic activities were observed on the short-chain p-nitrophenyl acylesters, such as p-nitrophenyl acetate, etc. The conserved serine residue ($Ser_{190}$) was shown to be important for Est5S activity. The primers that amplified the est5S gene did not show any relative band with 49 species of culturable rumen bacteria. This implies that a new group esterase gene, est5S, may have come from a noncultured cow rumen bacterium.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.364-364
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• 2017

Saemangeum reclaimed land is a part of Saemangeum Development Project. Most of the persistent problems of Saemangeum reclaimed land remain to be related to soil salinity. Soil salinity is a major abiotic factor related to microbial community structure and also fungi have been reported to be more sensitive to salinity stress than bacteria. The aim of this study was conducted to investigate the effect of soil salinity levels on the microbial communities in Saemangeum reclaimed land using 454 pyrosequencing analysis. Soil samples was collected from 12 sites of in Saemangeum reclaimed land. For pyrosequencing, 27F/518R (bacteria) and ITS3/ITS4 (fungi) primers were used containing the Roche 454 pyrosequencing adaptor-key-linker (underlined) and unique barcodes (X). Pyrosequencing was performed by Chun's Lab (Seoul, Korea) using the standard shotgun sequencing reagents and a 454 GS FLX Titanium sequencing System (Roche, Inc.). In the soil samples, Proteobacteria (bacteria) and Ascomycota (fungi) shows the highest relative abundance in all the soil sample sites. Proteobacteria, Bacteroidetes, Plantomycetes, Gemmatimonadetes and Parcubacteria were shown to have significantly higher abundance in high salinity level soils than low salinity level soils, while Acidobacteria and Nitrospirae has significantly higher relative abundance in low salinity level soils. The abundance of fungal, Ascomycota has the highest relative abundance in soil samples, followed by Basidiomycota, Chlorophyta, Zygomycota and Chytridiomycota. Basidiomycota, Zygomycota, Glomeromycota and Cerozoa were show significantly higher relative abundance in low salinity level soils. The principal coordinate analysis (PCoA) and correlation analysis shown to salinity-related soil parameters such as ECe, Na+, SAR and EPS were affected to bacterial and fungal community structure. Proteobacteria, Bacteroidetes, Plantomycetes exhibited significantly positive correlation with soil salinity, while Acidobacteria exhibited significantly negative correlation. In the case of fungal community, Basidiomycota and Zygomycota were seen show significantly negative correlation with salinity related soil parameters. These results suggest that provide understanding effect of soil salinity on microbial community structure and correlation of microbial community with soil parameters in Saemangeum reclaimed land.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.36 no.3
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• pp.230-238
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• 2003

Biosurfactant-producing bacteria, showing strong crude oil degrading activity, were isolated from the caverns of National Oil Storage Basement. From the results of biochemical and molecular biological tests, the isolate was identified as Pseudomonas fluorescens PD101. It grows well on liquid media at temperature range from $20^{\circ}C\;to\;37^{\circ}C,$ but it does not produce biosurfactant when grown at $37^{\circ}C$ or at higher temperature. The biosurfactant was stable at broad pH range from 5 to 11 and under heat treatment condition of $100^{\circ}C$ for 30 min. The biosurfactant produced dark blue halo around the colony when grown on SW agar plates, which could confirm the biosurfactant as one of rhamnolipid group. The 700 bp of PCR product could be amplified from DNA of P. flurorescens PD101 by using PCR primers designed from rh1A gene of P. aeruginosa, and it showed $99\%$ of sequence homology with rh1A gene of P. aeruginosa encoding rhamnosyltransferase 1.

• Journal of Applied Biological Chemistry
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• v.52 no.1
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• pp.1-7
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• 2009

For the development of qualitative PCR detection method of genetically modified (CM) rice, rice species-specific gene, OsCc-1 (rice cytochrome c gene), was selected as suitable far use as an endogenous gene in rice. The primer pair OsCytC-5'/3'with 111 bp amplicon was used for PCR amplification of the rice endogenous gene, OsCc-1 and no amplified product was observed from 8 different crops as templates. Qualitative PCR method was carried out with stack traits of L528$\times$CryIAc1 GM rice developed in Korea. For the qualitative PCRs, some primer pairs were designed with a construct-specific and event-specific type based on T-DNA and junction sequences of T-DNA in GM rice. Actck-5'/3' amplifying between actin promoter and OsCK1 gene introduced in LS28 gave rise to an amplicon 306 bp; also, CrLB-5'/3' from CryIAcl and CKRB-5'/3'amplifying the junction region of T-DNA and genome sequence from LS28 as event-specific primers gave rise to an amplicon 142 bp and 91 bp, respectively. These primer pairs for the detection of event-specific targets not produced PCR amplicons on non-CM rice and various crops in contrast to event lines. Therefore, in this study we verified that event-specific primers were effective to specifically detect stack trait lines and demonstrated that this method presented could be provided with the detection-method data for risk assessment analysis of GM rice to be commercialized.

• Horticultural Science & Technology
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• v.29 no.3
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• pp.232-239
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• 2011

The genus Acorus is known as an indigenous medicinal plant. Genetic diversity of thirteen accessions of A. calamus and eight of A. gramineus, with an accession of Colocasia antiquorum and two of Iris pseudacorus as outgroups, were evaluated using RAPD markers for cluster analysis and principal coordinate analysis, and NIR spectroscopic profiles for principal component analysis.A total of 371 polymorphic bands were obtained by using the selected 12 random primers. The genetic distances were estimated from 0.03 to 0.31 within A. calamus and from 0.03 to 0.51 within A. gramineus. The dendrogram and three-dimensional plot separated the accessions into four distinct groups (A. calamus, A. gramineus, C. antiquorum, and I. pseudacorus). Moreover, for the diversity among genus Acorus, eleven A. calamus accessions, one A. gramineus accession, and two I. pseudacorus accessions were non-destructively analyzed from their leaves by NIR spectroscopy, which discriminated Acorus accessions like the RAPD analysis. Interestingly, thirteen accessions of A. calamus were clustered into two groups based on RAPD and NIR analyses, which indicates that there are two ecotypes of A. calamus in Korea. An accession (CZ) of A. calamus with yellow stripe on leaves was closely grouped with another (CX) at a genetic distance (GD) of 0.03, which shows that the stripe trait might be generated by chimeric mutation. The genetic distance between A. calamus and A. gramineus was revealed to be farthest from 0.80 to 0.88 GD. In genus Acorus the genetic diversity and genetic variation were identified by using RAPD marker technique and non-destructive NIRs.