• 생명과학회지
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• 제18권1호
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• pp.136-142
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• 2008

Rhodotorula glutinis epoxide hydrolase (EH) 유전자를 pPICZ vector에 이중발현 cassette로 재조합하여 발현시킨 재조합균주 Pichia pastoris를 제작하였으며 라세믹 styrene oxide 혼합물로부터 고순도 광학활성 (S)-styrene oxide를 제조하는데 사용하였다. 본 연구에서 사용된 R. glutinis EH 유전자는 전보에서 사용한 pPICZ B/RgEH plasmid DNA를 주형으로 하여 얻었으며 PCR 방법으로 BglII 제한자리를 돌연변이 시키고 AOX1 promoter $(P_{AOX1})$-RgEH 유전자-전사종결서열($TT_{AOX1}$)을 이중으로 가진 이중발현 cassette를 만들어 P. pastoris의 염색체 DNA에 삽입시켰다. 반응온도를 $30^{\circ}C$로 하였을 때, RgEH를 이중발현 cassette로 발현시킨 재조합균주 P. pastoris의 (R)-styrene oxide에 대한 $V_{max}$ 값은 $2.2{\mu}mol\;min^{-1} (mg\;dcw)^{-1}$으로 단일발현 cassette로 발현시킨 P. pastoris의 $0.4{\mu}mol\;min^{-1}(mg\;dcw)^{-1}$에 비하여 6배 향상되었다. 광학순도가 높은 (S)-styrene oxide를 제조하는 최적조건을 찾기 위하여 입체선택적 가수분해 속도 및 수율에 미치는 detergent 및 온도의 효과를 실험하였으며, Tween 20을 0.5% 첨가하고 $10^{\circ}C$로 반응시킨 경우 10분 반응을 통해 99.9% ee 이상의 고순도 (S)-styrene oxide를 43.4% 얻을 수 있었다.

• 생명과학회지
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• 제22권2호
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• pp.167-176
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• 2012

발효가 진행중인 멸치액젓에서 단백질분해효소를 생산하는 3종의 호염성 세균을 분리하였다. 분리된 FAM 10, FAM 114, 그리고 FAM 115는 각각 소금농도가 2~4%, 10%, 6%에서 최적 세포성장이 관찰되었으며, 18~22% 소 금농도까지 생육이 가능했다. 단백질분해효소 생산을 위한 최적 소금농도는 FAM 10은 6%, FAM 114와 FAM 115는 10%로 나타났다. FAM 10의 단백질분해효소 활성은 10%까지 첨가하면 서서히 저하되며, 14% 소금농도에서는 효소활성이 없는 반면, FAM 114와 FAM 115의 경우, 14%까지 효소활성을 나타냈으며 18%에서는 활성을 관찰할 수 없었다. 이러한 결과로부터 분리된 3종의 미생물이 중간 정도의 호염성 세균임을 증명하였다. 16S rRNA 유전자와 16S-23S 유전자 사이 공간(IGS) 서열, 생화학적 실험, 그람염색을 이용하여 비교 분석한 결과, FAM 10, FAM 114, 그리고 FAM 115는 각각 Salinivibrio sp., Halobacillus sp., 그리고 Halobacillus sp.임을 동정하였다. Salinivibrio sp. FAM 10에는 191번째 서열부터 약간 다른 2가지 종류의 16S rDNA와 16S-23S IGS내에 tRNA 유전자가 없는 형태, 이소루이신/알라닌, 글루탐산/라이신/발린을 운반하는 tRNA 유전자들을 함유하는 4가지 종류의 다른 16S-23S IGS가 존재하였다. Hablobacillus sp. FAM 114와 FAM 115는 완전히 동일한 16S rRNA 유전자 서열을 가지고 있었으며, 여러 Halobacillus sp.와 99% 서열동일성을 보여주었다. IGS의 경우, tRNA 유전자가 없는 IGS와 이소루이신/알라닌을 운반하는 tRNA 유전자들을 함유하는 3가지 16S-23S IGS가 존재하였으며, Halobacillus aidingensis와 Halobacillus sp. JM-Hb의 IGS와 99% 서열동일성을 보여주었다.

• 한국물환경학회지
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• 제24권6호
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• pp.817-822
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• 2008

In order to identify various Cryptosporidium species in environment, nested PCR-RFLP and DNA sequencing method were used. The sensitivity of nested PCR-RFLP based on 18s rRNA gene was shown to 1 oocyst. Therefore, we applied nested PCR-RFLP method to environmental samples. As a result, only 4 samples out of 8 samples confirmed as Cryptosporidium parvum by standard method of Cryptosporidium were identified as Cryptosporidium parvum by nested PCR-RFLP and DNA sequencing method. The rest of 4 samples among 8 samples were identified as Cryptosporidium muris, Cryptosporidium bailey. Therefore, in addition to standard method of Cryptosporidium, supplementary verification through nested PCR-RFLP and DNA sequencing should be needed to give more accurate information about risk of Cryptosporidium.

• Animal Systematics, Evolution and Diversity
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• 제27권1호
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• pp.47-52
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• 2011

Creeping ctenophores, Coeloplana species, were collected by SCUBA divers throughout the year (November 2006 to June 2010) from the branches and polyp masses of encrusting dendronephthyas at a depth of 20-32m off Munseom Island (Seogwipo-si, Jeju-do, Korea). A single individual of a newly recorded species in Korea, Coeloplana bocki Komai, 1920, was collected together with C. anthostella from the same location on 16 August 2009. A large number of individuals of each species were subsequently collected from the host Dendronephthya aff. dendritica on 20 June 2010. C. bocki can be distinguished from C. anthostella Song and Hwang, 2010 and C. komaii Utinomi, 1963 by its unique blue and orange colored stripes, and/or the branching and anastomosing milky-white stripes encircling the aboral sense organ towards the margin. The detailed morphology and molecular sequence information (nuclear 18S rDNA, internal transcribed spacer 1, and mitochondrial cox1 gene sequences) for C. bocki is provided, and C. bocki and C. anthostella are compared.

• Journal of Microbiology and Biotechnology
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• 제10권5호
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• pp.643-650
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• 2000

A new aniline-utilizing microorganism, strain HY1 obtained from an orchard soil, was characterized by using the BIOLOG system, an analysis of the total cellular fatty acids, and a 16S rDNA sequence. Strain HY1 was identified as a Burkholderia species, and was designated Burkholderia sp. HY1. GC and HPLC analyses revealed that Burkholderia sp. HY1 was able to degrade aniline to produce catechol, which was subsequently converted to cis,cis-muconic acid through an ortho-ring fission pathway under aerobic conditions. Strain HY1 exhibited a drastic reduction in the rate of aniline degradation when glucose was added to the aniline media. However, the addition of peptone or nitrate to the aniline media dramatically accelerated the rate of aniline degradation. A fatty acid analysis showed that strain HY1 was able to produce lipids 16:0 2OH, and 11 methyl 18:1 ${\omega}7c$ approximately 3.7-, 2.2-, and 6-fold more, respectively, when grown on aniline media than when grown on TSA. An analysison the alignment of a 1,435 bp fragment. A phylogenetic analysis of the 16S rDNA sequence based on a 1,420 bp multi-alignment sowed of the 16s rDNA sequence revealed that strain HY1 was very closely related to Burkholderia graminis with 95% similarity based that strain HY1 was placed among three major clonal types of $\beta$-Proteobacteria, including Burkholderia graminis, Burkholderia phenazinium, and Burkholderia glathei. The sequence GAT(C or G)${\b{G}}$, which is highly conserved in several locations in the 16S rDNA gene among the major clonal type strains of $\beta$-Proteobacteria, was frequently replaced with GAT(C or G)${\b{A}}$ in the 16S rDNA sequence from strain HY1.

• Journal of Microbiology
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• 제35권4호
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• pp.253-258
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• 1997

The nuclear 18S ribosomal RNA genes of seven corticioid species were sequenced. These sequences were analyzed and compared with those of 24 other species of the order Aphyllophorales and phylogenetic trees were constructed using parsimonious methods. Phylogenetic analyses showed that two species among examined members of the Corticiaceae, Resinicium bicolor and Thanatephorus praticola, are located distantly from the remaining six species. The separation of R. bicolor seems to be kphylogenetically significant because it has very unique cystidia. The independent lineage of T. practicola suggests that it is also phylogenetically distinct because it has unusual features like the homobasidium producing secondary spores and the spetal ultrastructure of pore cap. Furthermore, Auriscalpium vulgare, Bondarzewia berkeleyi, and Heterobasidion annosum from different families of the Aphyllophorales proved to be closely related to the species of the Corticiaceae. They all have amyloid spores and grouped with Aleyrodiscus amorphus, which is a member of the Corticiaceae. The amyloidity of spores seems to be an improtant character throughout the order of the Aphyllophorales.

• Journal of Microbiology
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• 제45권2호
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• pp.105-112
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• 2007

The objective of this study was to analyze the phylogenetic composition of bacterial community in the soil of an earth-cave (Niu Cave) using a culture-independent molecular approach. 16S rRNA genes were amplified directly from soil DNA with universally conserved and Bacteria-specific rRNA gene primers and cloned. The clone library was screened by restriction fragment length polymorphism (RFLP), and representative rRNA gene sequences were determined. A total of 115 bacterial sequence types were found in 190 analyzed clones. Phylogenetic sequence analyses revealed novel 16S rRNA gene sequence types and a high diversity of putative bacterial community. Members of these bacteria included Proteobacteria (42.6%), Acidobacteria (18.6%), Planctomycetes (9.0 %), Chloroflexi (Green nonsulfur bacteria, 7.5%), Bacteroidetes (2.1%), Gemmatimonadetes (2.7%), Nitrospirae (8.0%), Actinobacteria (High G+C Gram-positive bacteria, 6.4%) and candidate divisions (including the OP3, GN08, and SBR1093, 3.2%). Thirty-five clones were affiliated with bacteria that were related to nitrogen, sulfur, iron or manganese cycles. The comparison of the present data with the data obtained previously from caves based on 16S rRNA gene analysis revealed similarities in the bacterial community components, especially in the high abundance of Proteobacteria and Acidobacteria. Furthermore, this study provided the novel evidence for presence of Gemmatimonadetes, Nitrosomonadales, Oceanospirillales, and Rubrobacterales in a karstic hypogean environment.

• Parasites, Hosts and Diseases
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• 제51권3호
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• pp.269-277
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• 2013

Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.

• 대한의생명과학회지
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• 제17권2호
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• pp.151-155
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• 2011

Short tandem repeats (STRs) loci are the genetic markers used for forensic human identity test. With multiplex polymerase chain reaction (PCR) assays, STRs are examined and measured PCR product length relative to sequenced allelic ladders. In the repeat region and the flanking region of the commonly-used STR may have DNA sequence variation. A mismatch due to sequence variation in the DNA template may cause allele drop-out (i.e., a "null" or "silent" allele) when it falls within PCR primer binding sites. The STR markers were co-amplified in a single reaction by using commercial PowerPlex$^{(R)}$ 16 system and AmpFlSTR$^{(R)}$ Identifiler$^{(R)}$ PCR amplification kits. Separation of the PCR products and fluorescence detection were performed by ABI PRISM$^{(R)}$ 3100 Genetic Analyzer with capillary electrophoresis. The GeneMapper$^{TM}$ ID software were used for size calling and analysis of STR profiles. Here, this study described a forensic human identity test in which allelic drop-out occurred in the STR system D18S51. During the course of human identity test, two samples with a homozygous (16, 16 and 21, 21) genotype at D18S51 locus were discovered using the PowerPlex$^{(R)}$ 16 system. The loss of alleles was confirmed when the samples were amplified using AmpFlSTR$^{(R)}$ Identifiler$^{(R)}$ PCR amplification kit and resulted in a heterozygous (16, 20 and 20, 21) genotype at this locus each other. This discrepancy results suggest that appropriate measures should be taken for database comparisons and that allele should be further investigated by sequence analysis and be reported to the forensic community.

• 한국해양바이오학회지
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• 제2권1호
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• pp.60-67
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• 2007

For the first time in Vietnam, morphological and molecular studies of a species belonging to Bacillariophyceae collected in Northern coast of Vietnam are presented. Observations with microscope showed that this species belong to genus: Pseudo-nitzschia and seem like P. pungens. Sequence data from the partial 18S small subunit ribosomal RNA gene (18S rDNA) and the internal transcribed spacer 1 - 5.8S - internal transcribed 2 have been used to determine clearly and generate a phylogenetic framework of the obtained sequences to previously reported sequences in GenBank. These results allowed us to highlight described species of Bacillariophyceae in Northern coast of Vietnam. Furthermore, accumulation of molecular study would be helpful for the identification of scientific name of harmful algal species and further taxonomic studies in Vietnam.