• BMB Reports
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• v.34 no.2
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• pp.156-165
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• 2001

Leptin is a circulating non-glycosylated protein that is mainly produced in adipocytes. Leptin acts in the brain to regulate food intake and energy expenditure. Previously we reported our success in the isolation of a partial cDNA of the long form of the leptin receptor, OB-Rb, from rat spleen, and showed that leptin might also play a role in peripheral immune organs. In the present study, for the first time, the complete coding region of OB-Rb cDNA was cloned from rat splenocytes, and its nucleotide sequence was determined. The cDNA was then further expressed in E. coli and mammalian cells, thereby confirming the functional integrity of this receptor. Prokaryotically overexpressed OB-R protein was then used as an immunizing antigen in BALE/c mice to produce leptin receptor-specific antibodies. By using them, we confirmed the cell surface expression of OB-Rb in transfected CHO cells. It is our belief that the reagents, as produced in this study, will be of great use in further studies of the biological role of rat leptin.

• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.529-533
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• 2001

The ${\beta}$-Amylase cDNA fragment from the oomcete Saprolegnia parasitica was cloned by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers derived from conserved ${\beta}$-amylase sequences. The 5'and 3'regions of the $\beta$-amylase gene were amplified using the rapid amplification of cDNA ends (rACE) system. It consisted of an open reading frame of 1,350 bp for a protein of 450 amino acids. Comparison between the genomic and cDNA sequences revealed that the intron was not present in the coding region. The deduced amino acid sequence of the ${\beta}$-amylase gene had a 97% similarity to the ${\beta}$-amylase of Saprolegnia ferax, followed by 41% similarity to those of Arabidopsis thaliana, Hordeum vulgare, and Zea mays. The ${\beta}$-amylase gene was also expressed in Saccharomyces cerevisiae by placing it under the control of the alcohol dehydrogenase gene (ADC1) promoter.

• Molecules and Cells
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• v.20 no.1
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• pp.74-82
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• 2005

Glutaredoxins (Grxs) are thioloxidoreductases which are required for maintaining thiol/disulfide equilibrium in living cells. The Grx3 gene, which encodes one of the three monothiol Grxs in the fission yeast Schizosaccharomyces pombe, was characterized, and its transcriptional regulation studied. Genomic DNA encoding Grx3 was isolated by PCR, and a plasmid pTT3 carrying this DNA was produced. The DNA sequence has 1,267 bp, which would encode a monothiol Grx of 166 amino acids with a molecular mass of 18.3 kDa. The putative protein has 27% homology with Grx5, and contains many hydrophobic amino acid residues in its N-terminal region. S. pombe cells harboring pTT3 had increased Grx activity and enhanced survival on minimal medium plates containing aluminum (5 mM), BSO (0.05 mM), menadione (0.01 mM) or cadmium (0.2 mM). The 568 bp upstream region of Grx3 was fused into the promoterless b-galactosidase gene of the shuttle vector YEp367R to generate fusion plasmid pMJS10. Potassium chloride (KCl) and metals including aluminum and cadmium enhanced the synthesis of ${\beta}$-galactosidase from the fusion gene. The synthesis of ${\beta}$-galactosidase was also enhanced, in a Pap1-dependent manner, by fermentable carbon sources such as glucose (at low concentrations) and sucrose, but not by non-fermentable carbon sources such as ethanol and acetate. Grx3 mRNA increased in response to treatment with BSO. These observations indicate that S. pombe Grx3 is involved in the response to stress, and is regulated by stress.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.227-233
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• 2005

Fungal strain CGF was isolated from the soil of ChungNam Province, South Korea. Based on the 28S rDNA sequence analysis and the sequence of the internal transcribed spacer (ITS) region of ribosomal DNA, together with morphological and cultural characteristics, this strain was identified as Aspergillus sclerotiorum and renamed Aspergillus sclerotiorum CGF. This is the first strain of Aspergillus sclerotiorum identified in Korea. When the antifungal activity of A. sclerotiorum CGF was evaluated, among the phytopathogenic fungi, mycelial growth of only Phytophthora species was inhibited. Oermination of P. capsid zoospore was also inhibited. The bioactive compound of A. sclerotiorum CGF was highly thermo- and pH-stable.

• The Korean Journal of Mycology
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• v.46 no.1
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• pp.22-27
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• 2018

We isolated endophytic fungal strains from the leaves of Abies koreana growing in Korea. The fungal strains were identified as Coleophoma parafusiformis and Coniochaeta ligniaria, based on morphological characteristics and sequence analysis of the internal transcribed spacer region and large subunit region of ribosomal DNA. These species have not been previously reported in Korea. In this study, we report the morphological characteristics and results of phylogenetic analysis of the two novel fungal strains.

• Korean Journal of Veterinary Service
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• v.41 no.4
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• pp.257-262
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• 2018

Avian pathogenic E. coli (APEC) causes severe economic losses in the poultry farms, due to systemic infections leading to lethal colisepticemia. It causes a variety of diseases from air sac infection to systemic spread leading to septicemia. Secondary infection contains opportunistic infections due to immunosuppression disease. Collibacillosis causes the great problems in the poultry industry in Korea. Thus, it is necessary to identify and classify the characteristics of E. coli isolate of chicken origin to confirm the diversity of symptoms and whether they are transmitted among the farms. Fragment analysis is identify the difference in the number of Variable-Number Tandem-Repeats (VNTRs) for genotyping. VNTRs have repeating structure (Microsatellite, Short tandem repeats; STR, Simple sequence repeats; SSR) in the chromosome. This region can be used as a genetic marker because of its high mutation rate. And various lengths of the amplified DNA fragment cause the difference in the number of repetition of the DNA specific site. The number of repetition sequences indicates the separated size of fragments, so the each fragments can be distinguished by specific samples. The results of the sample show that there is no difference in six microsatellite loci (yjiD, aidB, molR_1, ftsZ, b1668, yibA). There are differences among the farms in relation of the number of repetitions of other six microsatellite loci (ycgW, yaiN, yiaB, mhpR, b0829, caiF). Four (ycgW, yiaB, b0829, caiF) of these six microsatellite loci show statistically significant differences (P<0.05). It means that the analysis using four microsatellite loci including ycgW, yiaB, b0829, and caiF can confirm among the farms. Five E. coli samples in one farm have same SSR repetition at all markers. But, there are significant differences from other farms at Four (ycgW, yiaB, b0829, caiF) microsatellite loci. These results emphasize again that the four microsatellite loci makes a difference in the amplified DNA fragments, enabling it to be used for E. coli genotyping.

• Genomics & Informatics
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• v.21 no.4
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• pp.52.1-52.10
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• 2023

Accurate and efficient microbial diagnosis is crucial for effective molecular diagnostics, especially in the field of human healthcare. The gold standard equipment widely employed for detecting specific microorganisms in molecular diagnosis is quantitative real-time polymerase chain reaction (qRT-PCR). However, its limitations in low metagenomic DNA yield samples necessitate exploring alternative approaches. Digital PCR, by quantifying the number of copies of the target sequence, provides absolute quantification results for the bacterial strain. In this study, we compared the diagnostic efficiency of qRT-PCR and digital PCR in detecting a particular bacterial strain (Staphylococcus aureus), focusing on skin-derived DNA samples. Experimentally, specific primer for S. aureus were designed at transcription elongation factor (greA) gene and the target amplicon were cloned and sequenced to validate efficiency of specificity to the greA gene of S. aureus. To quantify the absolute amount of microorganisms present on the skin, the variable region 5 (V5) of the 16S rRNA gene was used, and primers for S. aureus identification were used to relative their amount in the subject's skin. The findings demonstrate the absolute convenience and efficiency of digital PCR in microbial diagnostics. We suggest that the high sensitivity and precise quantification provided by digital PCR could be a promising tool for detecting specific microorganisms, especially in skin-derived DNA samples with low metagenomic DNA yields, and that further research and implementation is needed to improve medical practice and diagnosis.

• Archives of Pharmacal Research
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• v.24 no.5
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• pp.455-465
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• 2001

The highly potent cytotoxic DNA-DNA cross-linker consists of two cyclopropa[c]pyrrolo[3,4-3]indol-4(5H)-ones insoles [(+)-CPI-I] joined by a bisamido pyrrole (abbreviated to "Pyrrole"). The Pyrrole is a synthetic analog of Bizelesin, which is currently in phase II clinical trials due to its excellent in vivo antitumor activity. The Pyrrole has 10 times more potent cytotoxicity than Bizelesin and mostly form DNA-DNA interstrand cross-links through the N3 of adenines spaced 7 bp apart. The Pyrrole requires a centrally positioned GC base pair for high cross-linking reactivity (i.e., $5^1$-T$AT_2$A*-$3^1$), while Bizelesin prefers purely AT-rich sequences (i.e., $5^1$-T$AT_4$A*-$3^1$, where /(equation omitted) represents the cross-strand adenine alkylation and A* represents an adenine alkylation) (Park et al., 1996). In this study, the high-field $^1$H-NMR and rMD studies are conducted on the 1 1-mer DNA duplex adduct of the Pyrrole where the 5′(equation omitted)TAGTTA*-3′sequence is cross-linked by the drug. A severe structural perturbation is observed in the intervening sequences of cross-linking site, while a normal B-DNA structure is maintained in the region next to the drug-modified adenines. Based upon these observations, we propose that the interplay between the bisamido pyrrole unit of the drug and central C/C base pair (hydrogen-bonding interactions) is involved in the process of cross-linking reaction, and sequence specificity is the outcome of those interactions. This study suggests a mechanism for the sequence specific cross-linking reaction of the Pyrrole, and provides a further insight to develop new DNA sequence selective and distortive cross-linking agents.

• Journal of Animal Science and Technology
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• v.45 no.2
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• pp.169-182
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• 2003

To understand the structure and regulation of bovine ADRP (Adipocyte Differentiation Related Protein) gene, we have isolated the genomic clone of bovine ADRP and determined its sequence. A genomic Southern blot analysis confirmed that ADRP gene is present as a single copy in bovine genome and the ADRP gene spans 12 kb. Bovine ADRP genomic clone, HwADRPg-1, had 8 exons and 7 introns, and all splicing sites conformed to the GT/AG rule with the exon-intron boundaries located exactly. Analysis of the upstream 649 bp of the sequence of HwADRPg-1 showed that it does not contain any canonical TATAA boxes; however Sp1 binding sites and CAAT boxes are found. The promoter contained potential binding sites for AP-1, AP-2 and several putative transcription factor binding sites. The 5'-flanking region of HwADRPg-1 contained muscle specific transcription activator Myo G and C/EBP (CCAAT/ enhancer binding protein) recognizing site. These results suppose that the Myo G transcription activator regulate the transcription of bovine ADRP gene in muscular tissue and its transcriptional activity was triggered by degree of muscular development. Our results provide the necessary analysis for other flanking sequences are needed in addition to the proximal cis elements of this promoter to confer adipocyte differentiation-dependent or growth-dependent transcriptional control.

• The Korean Journal of Mycology
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• v.49 no.1
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• pp.11-19
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• 2021

In 2017, Talaromyces variabilis was isolated during a survey of fungal diversity in field soils in Korea. This isolate was described based on its morphological and molecular characteristics and it was identified molecularly using the partial 18S-ITS1-5.8S-ITS2-28S rDNA region and calmodulin (CaM)-encoding gene sequence data. Thus, this study reported morphological and multigene sequence characterization of T. variabilis.