• Preventive Nutrition and Food Science
• /
• v.9 no.4
• /
• pp.324-329
• /
• 2004

In order to investigate the molecular mechanism of $\gamma$-aminobutyric acid (GABA) production in lactic acid bacteria, we cloned a glutamate decarboxylase (GAD) gene from Lactobacillus plantarum using polymerase chain reaction (PCR). One PCR product DNA was obtained and inserted into a TA cloning vector with a T7 promoter. The recombinant plasmid was used to transform E. coli. The insertion of the product was con­firmed by EcoRI digestion of the plasmid purified from the transformed E. coli. Nucleotide sequence analysis showed that the insert is a full-length Lactobacillus plantarum GAD and that the sequence is $100\%$ and $72\%$ identical to the regions of Lactobacillus plantarum GAD and Lactococcus lactis GAD sequences deposited in GenBank, accession nos: NP786643 and NP267446, respectively. The amino acid sequence deduced from the cloned Lactobacillus plantarum GAD gene showed $100\%$ and $68\%$ identities to the GAD sequences deduced from the genes of the NP786643 and NP267446, respectively. To express the GAD protein in E. coli, an expression vector with the GAD gene (pkk/GAD) was constructed and used to transform the UT481 E. coli strain and the expression was confirmed by analyzing the enzyme activity. The Lactobacillus plantarum GAD gene obtained may facilitate the study of the molecular mechanisms regulating GABA metabolism in lactic acid bacteria.

• Horticulture, Environment, and Biotechnology : HEB
• /
• v.59 no.6
• /
• pp.857-864
• /
• 2018

Previous studies have not detected transcripts of the gene encoding anthocyanidin synthase (ANS) in white pomegranates (Punica granatum L.) and suggest that a large-sized insertion in the coding region of the ANS gene might be the causal mutation. To elucidate the identity of the putative insertion, 3887-bp 5' and 3392-bp 3' partial sequences of the insertion site were obtained by genome walking and a gene coding for an expansin-like protein was identified in these genome-walked sequences. An identical protein (GenBank accession OWM71963) isolated from pomegranate was identified from BLAST search. Based on information of OWM71963, a 5.8-Mb scaffold sequence with genes coding for the expansin-like protein and ANS were identified. The scaffold sequence assembled from a red pomegranate cultivar also contained all genome-walked sequences. Analysis of positions and orientations of these genes and genome-walked sequences revealed that the 27,786-bp region, including the 88-bp 5' partial sequences of the ANS gene, might be translocated into an approximately 22-kb upstream region in an inverted orientation. Borders of the translocated region were confirmed by PCR amplification and sequencing. Based on the translocation mutation, a simple PCR codominant marker was developed for efficient genotyping of the ANS gene. This molecular marker could serve as a useful tool for selecting desirable plants at young seedling stages in pomegranate breeding programs.

• Animal cells and systems
• /
• v.15 no.2
• /
• pp.161-168
• /
• 2011

Siberian chipmunks, Tamias sibiricus, are one of several popular companion animals found in the pet shops of South Korea. At present, however, there have been no studies done in South Korea examining their origin even though they could be potential carriers of zoonotic diseases, and are a species of concern for efficient conservation and management strategies. Sequences of the mitochondrial cytochrome b gene (1140 bp) were determined to investigate the origin of Siberian chipmunks sold in four South Korean pet shops through comparison with sequence data from animals of known locality. Nine Siberian chipmunks were collected from pet shops in South Korea, which resulted in nine haplotypes. One (AR) of these coincided with the haplotype previously described. Phylogenetic and network analyses using 53 haplotypes including 45 haplotypes from GenBank showed three phylogenetic groups in South Korea, almost concordant to locality, designated as northern, central, and southern parts as described in a previous study. Of the nine individuals examined from the pet shops, eight were clustered into the northern phylogroup but one (cgrb9153) was grouped with the southern phylogroup, implying that at least the Siberian chipmunks examined in this study did not originate from other countries. It is likely that most individuals sold in the pet shops of Seoul were caught in the wild in Gyeonggi-do and Gangwon-do, or are maternal descendants of captive-bred individuals originating from the northern part of South Korea. It is recommended that conservation and management units of Korean chipmunks should be examined in further detail.

• Journal of Ginseng Research
• /
• v.45 no.1
• /
• pp.48-57
• /
• 2021

Background: Ginsenoside Rh2 is well known for many pharmacological activities, such as anticancer, antidiabetes, antiinflammatory, and antiobesity properties. Glycosyltransferases (GTs) are ubiquitous enzymes present in nature and are widely used for the synthesis of oligosaccharides, polysaccharides, glycoconjugates, and novel derivatives. We aimed to synthesize new ginsenosides from Rh2 using the recombinant GT enzyme and investigate its cytotoxicity with diverse cell lines. Methods: We have used a GT gene with 1,224-bp gene sequence cloned from Lactobacillus rhamnosus (LRGT) and then expressed in Escherichia coli BL21 (DE3). The recombinant GT protein was purified and demonstrated to transform Rh2 into two novel ginsenosides, and they were characterized by nuclear magnetic resonance (NMR) techniques and evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide assay. Results: Two novel ginsenosides with an additional glucopyranosyl (6→1) and two additional glucopyranosyl (6→1) linked with the C-3 position of the substrate Rh2 were synthesized, respectively. Cell viability assay in the lung cancer (A549) cell line showed that glucosyl ginsenoside Rh2 inhibited cell viability more potently than ginsenoside Rg3 and Rh2 at a concentration of 10 μM. Furthermore, glucosyl ginsenoside Rh2 did not exhibit any cytotoxic effect in murine macrophage cells (RAW264.7), mouse embryo fibroblasts cells (3T3-L1), and skin cells (B16BL6) at a concentration of 10 μM compared with ginsenoside Rh2 and Rg3. Conclusion: This is the first report on the synthesis of two novel ginsenosides, namely, glucosyl ginsenoside Rh2 and diglucosyl ginsenoside Rh2 from Rh2 by using recombinant GT isolated from L. rhamnosus. Moreover, diglucosyl ginsenoside Rh2 might be a new candidate for treatment of inflammation, obesity, and skin whiting, and especially for anticancer.

• Journal of Microbiology and Biotechnology
• /
• v.7 no.4
• /
• pp.223-228
• /
• 1997

A DNA fragment containing the gene for cell wall hydrolase of alkalophilic Bacillus subtilis BL-29 was cloned into E. coli JM109 using pUC18 as a vector. A recombinant plasmid, designated pCWL45B, was contained in the fragment originating from the alkalophilic B. subtilis BL-29 chromosomal DNA by Southern hybridization analysis. The nucleotide sequence of a 1.6-kb HindIII fragment containing a cell wall hydrolase-encoding gene was determined. The nucleotide sequence revealed an open reading frame (ORF) of 900 bp with a concensus ribosome-binding site located 6 nucleotide upstream from the ATG start codon. The primary amino acid sequence deduced from the nucleotide sequence revealed a putative protein of 299 amino acid residues with an M.W. of 33, 206. Based on comparison of the amino acid sequence of the ORF with amino acid sequences in the GenBank data, it showed significant homology to the sequence of cell wall amidase of the PBSX bacteriophage of B. subtilis.

• Parasites, Hosts and Diseases
• /
• v.53 no.1
• /
• pp.105-108
• /
• 2015

Most of the diphyllobothriid tapeworms isolated from human samples in the Republic of Korea (=Korea) have been identified as Diphyllobothrium nihonkaiense by genetic analysis. This paper reports confirmation of D. nihonkaiense infections in 4 additional human samples obtained between 1995 and 2014, which were analyzed at the Department of Parasitology, Hallym University College of Medicine, Korea. Analysis of the mitochondrial cytochrome c oxidase 1 (cox1) gene revealed a 98.5-99.5% similarity with a reference D. nihonkaiense sequence in GenBank. The present report adds 4 cases of D. nihonkaiense infections to the literature, indicating that the dominant diphyllobothriid tapeworm species in Korea is D. nihonkaiense but not D. latum.

• Proceedings of the Korea Inteligent Information System Society Conference
• /
• 2002.11a
• /
• pp.480-488
• /
• 2002

본 논문에서는 하나의 시스템 안에서 효율적인 유전자 데이터의 관리와 다양한 서열 분석작업이 가능한 기능 유전체학을 지원하는 서열 분석 및 관리 시스템인 GWB(Gene WorkBench)를 설계하고 구현하였다. GWB는 로컬 데이터베이스 관리뿐만 아니라 GenBank, EMBL, SWISSPROT와 같은 외부 공공 데이터베이스에 대한 접근 기능도 제공하며, 권한을 가진 내부 이용자와 그렇지 못한 외부 이용자들을 구분하여 일부 유용한 기능들은 외부 사용자들도 이용할 수 있도록 설계되었다. 또 GWB는 유전자에 관한 문헌정보 검색과 관련 유전자 탐색 기능 등 일부 유전자 기능 연구를 지원하는 기능을 제공하고 있다.

• Korean Journal of Medicinal Crop Science
• /
• v.17 no.1
• /
• pp.26-32
• /
• 2009

The region containing 18S rRNA gene, ITS 1 and part of the 5.8S rRNA gene of the Atractylodes japonica Koidz was amplified by PCR and the product cloned in a pBluescript SK II plasmid. DNA sequence of the cloned DNA was determined and submitted to the GenBank (accession number EU678363). Phylogenetic analysis of the ITS 1 DNA showed close similarity with the other plant species of the family Compositae. The extract of the plant materials of five different members of the family Compositae was analyzed by HPLC to detect atractylon. Extract of the A. japonica Koidz showed presence of significant amount of atractylon. However, noticeable amount of atractylon was not detected by the same analyses from the extracts of the other plants belonging to the family Compositae including Artemisia capillaris, Chrysantemum zawadskii, Eclipta prostrata or Taraxacum platycarpum.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.8
• /
• pp.1324-1327
• /
• 2015

The nucleotide sequence of the TRP1 gene encoding phosphoribosyl anthranilate isomerase in yeast Saccharomycopsis fibuligera was determined by degenerate polymerase chain reaction and genome walking. Sequence analysis revealed the presence of an uninterrupted open-reading frame of 759 bp, including the stop codon, encoding a 252 amino acid residue. The deduced amino acid sequence of Trp1 in S. fibuligera was 43.5% homologous to that of Komagataella pastoris. The cloned TRP1 gene (SfTRP1) complemented the trp1 mutation in Saccharomyces cerevisiae, suggesting that it encodes a functional TRP1 in S. fibuligera. A new auxotrophic marker to engineer starch-degrading yeast S. fibuligera is now available. The GenBank Accession No. for SfTRP1 is KR078268.

• Korean Journal of Microbiology
• /
• v.29 no.4
• /
• pp.203-208
• /
• 1991

The dapA-complementing gene (L-2, 3-dihydrodipicolinate synthetase: DHDP synthetase, dapA) has been cloned by using a cosmid genomic bank of Corynebacterium glutamicum JS231 that is a lysine overproducer, AEC (s-(2-aminoethyl)-L-cysteine) resistant mutant. By enzymatic deletion analysis, the DNA region complementing the escherichia coli dapA host could be confined to 4.5kb SalI-generated DNA fragment. This DNA fragment was inserted into the C. glutamicum/E. coli shuttle vector pECCG117 to construct pDHDP5812. The specific activity of DHDP synthetase detected in C. glutamicum JS231/pDHDP5812 was increased about 10 fold above that of C. glutamicum JS231. The addition of leucine during growth did not repress the expressin of dapA, and the enzyme activity was not inhibited by lysine.