• 미생물학회지
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• 제41권3호
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• pp.195-200
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• 2005

Comamonas sp. strain DJ-12의 pmcABCDEFT 유전자군은 protocatechuate (PCA)의 분해과정에 관여하는 PCA 4,5-dioxygenase, 4-carboxy-2hydroxymuconic semialdehyde (CHMS) dehydrogenase, 2-pyrone04,5-dicarboxylate(PDC) hydrolase, 4-oxalomesaconate (OMA) hydratase, 그리고 4-oxalocitramalate (OCM) aldolase 등의 효소들을 생산하는 유전자들과 transporter의 역학을 하는 유전자로 각각 확인되었다. 이 유전자군은 Comamonas sp. strain DJ-12의 chromosomal DNA로부터 얻은 PCR 산물들을 T-vector에 ligation하여 재조합 플라스미드 pMT1, pMT2, pMT3, pMT4, pMT5, pMT6, pMT7, pMT8, pMT9, pMT10을 제조하였다. 이들 재조합 플라스미드의 염기서열을 분석한 결과 PCA 4,5-dioxygenase 유전자는 alpha(pmcA)와 beta(pmcB) 두 개의 subunit으로 구성 되어있으며, 각각 450 bp와 870 bp이었다. CHMS dehydrogenase 유전자(pmcC)는 960 bp, PDC hydrolase 유전자(pmcD)는 918 bp이였으며, OMA hydratase 유전자(pmcE)는 1029 bp, OCM aldolase 유전자 (pmcF)는 689 bp, 그리고 transporter 유전자(pmcT)는 1,398 bp이였다. 이들 pmc 유전자들은 pmcT-pmcE-pmcF-pmcD-pmcA-pmcB-pmcC의 순서로 배열되어 있었다. Comamonas sp. strain DJ-12의 pmcABCDEFT 유전자산물의 아미노산 서열을 분석한 결과, Comamonas testosteroni BR6020 및 Psedomonas ochraceae NG.J1와 $94{\~}98\%$의 높은 유사성을 보였고, 그 유전자들의 배열 순서도 동일하였다. 그러나 Sphingomonas paucimobilis SYK-6, Sphingomonas sp. LB126, 그리고 Arthrobacter keyser 12B와는 아미노산 서열이 $52{\~}74\%$의 유사성을 보였고, 그 유전자의 배열 구조도 상이하였다.

• Reproductive and Developmental Biology
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• 제38권3호
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• pp.123-127
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• 2014

This study was conducted for SNPs in the 5'-regions of estrogen receptor-${\alpha}$ (ESR-${\alpha}$), and association with calving interval (CI), service per conception (SPC) and 305 days milk yield in Hanwoo and Holstein dairy cattle. The genetic improvement was incurred low reproduction performance. The objective of this study was to investigate connections between single nucleotide polymorphisms (SNP) of Estrogen receptor-${\alpha}$ (ESR-${\alpha}$) with reproduction performance (calving interval, service per conception, and 305 d milk yield) in Hanwoo and Holstein dairy cattle. Hanwoo and Holstein blood samples were collected from 183 and 124 dam of breeding farms and DNA was extracted. Primer design was based on NCBI GenBank (Accession No. AY340579). The PCR-RFLP method with Bgl I was used to genotype the cattle. The result showed two variants of the ESR-${\alpha}$ gene. The Bgl I cut the 492 bp amplification product into 322 bp and 170 bp fragments for allele G, while allele A remained uncut, resulting in two restriction fragments for homozygote G/G and three fragments for heterozygote A/G. We found two of different genotypes in these breeds, A/G and G/G. In Hanwoo, the A/G genotype frequency was 0.13, and G/G was 0.87. The CI of A/G was $382.18{\pm}10.03$ days, and G/G was $381.69{\pm}5.22$ days. The SPC of A/G was $1.62{\pm}0.16$, and G/G was $1.32{\pm}0.04$. While CI showed no significance difference, SPC exhibited significant difference (p<0.05). In Holstein cattle, the frequency of genotype A/G was 0.02 and G/G was 0.98. The 305 days milk yield of A/G was $7,253.00{\pm}936.00kg$ and of G/G was $8,747.51{\pm}204.88kg$, showing no significant difference.

• Asian-Australasian Journal of Animal Sciences
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• 제21권12호
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• pp.1689-1695
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• 2008

Leptin, the hormone product of the obese gene, is secreted predominately from white adipose tissue and regulates feed intake, energy metabolism and body composition. It has been considered a candidate gene for performance, carcass and meat quality traits in beef cattle. The objective of this study was to identify SNPs in the promoter region of the leptin gene and to evaluate the possible association of the SNP genotypes with carcass and meat quality traits in Korean cattle. We identified a total of 25 SNPs in the promoter region (1,208-3,049 bp upstream from the transcription start site) of the leptin gene, eleven (g.1508C>G, g.1540G>A, g.1545G>A, g.1551C>T, g.1746T>G, g.1798ins(G), g.1932del(T), g.1933del(T), g.1934del(T), g.1993C>T and g.2033C>T) of which have not been reported previously. Their sequences were deposited in GenBank database with accession number DQ202319. Genotyping of the SNPs located at positions g.2418C>G and g.2423G>A within the promoter region was performed by direct sequencing and PCR-SSCP method to investigate the effects of SNP genotypes on carcass and meat quality traits in Korean cattle. The SNP and SSCP genotypes from the two mutations of the leptin promoter were shown to be associated with the BF trait. The average BF value of animals with heterozygous SNP genotype was significantly greater than that of animals with the homozygous SNP genotypes for the g.2418C>G and g.2423G>A SNPs (p<0.05). Analysis of the combined genotype effect in both SNPs showed that animals with the AC SSCP genotype had higher BF value than animals with BB or AA SSCP genotypes (p<0.05). These results suggest that SNP of the leptin promoter region may be useful markers for selection of economic traits in Korean cattle.

• The Korean Journal of Physiology and Pharmacology
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• 제20권2호
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• pp.201-211
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• 2016

Although the antioxidant and cardioprotective effects of NecroX-5 on various in vitro and in vivo models have been demonstrated, the action of this compound on the mitochondrial oxidative phosphorylation system remains unclear. Here we verify the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity during hypoxia-reoxygenation (HR). Necrox-5 treatment ($10{\mu}M$) and non-treatment were employed on isolated rat hearts during hypoxia/reoxygenation treatment using an ex vivo Langendorff system. Proteomic analysis was performed using liquid chromatography-mass spectrometry (LC-MS) and non-labeling peptide count protein quantification. Real-time PCR, western blot, citrate synthases and mitochondrial complex activity assays were then performed to assess heart function. Treatment with NecroX-5 during hypoxia significantly preserved electron transport chain proteins involved in oxidative phosphorylation and metabolic functions. NecroX-5 also improved mitochondrial complex I, II, and V function. Additionally, markedly higher peroxisome proliferator-activated receptor-gamma coactivator-$1{\alpha}$ ($PGC1{\alpha}$) expression levels were observed in NecroX-5-treated rat hearts. These novel results provide convincing evidence for the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity and in preserving $PGC1{\alpha}$ during cardiac HR injuries.

• 한국잠사곤충학회지
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• 제51권2호
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• pp.137-141
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• 2013

ITS 1, 2, 5.8S ribosomal RNA gene 염기서열 분석과 주사전자현미경 구조 분석을 통해 3종의 페루산 곤충병원성진균들의 동정을 수행하고자 하였다. 이를 위해 두개의 ITS 부위와 5.8S rRNA gene 부위를 포함하는 PCR product를 증폭하여 염기서열 분석을 수행하였으며 분석된 염기서열을 이용하여 NCBI의 BLAST를 이용하여 가장 높은 상동성을 보이는 종들의 ITS1-5.8S-ITS2 염기서열 정보와 비교분석을 위한 근연종들의 염기서열 정보를 다운로드하여 neighbor joining 분석을 수행하였다. 이를 통해 5.8S rRNA 유전자 염기서열은 속 수준에서도 거의 차이를 보여주지 않을 정도로 매우 안정적으로 보존되어 있음을 확인할 수 있었으며 종간 구분이 모호한 결과를 보여주었다. 그와 반대로 ITS 부위의 염기서열은 종에 매우 특이적임을 확인할 수 있었으며, 비교분석에 사용된 Beauveria bassiana strain 간의 차이는 확인할 수 없었다. ITS 염기서열 분석결과를 뒷받침하고자 곤충병원성 진균류의 동정을 위한 분류 key로 사용되는 미세구조 관찰을 위해 주사전자현미경 관찰과 광학현미경 관찰을 통해 B. bassiana 및 Lecanicillium attenuatum의 전형적 구조를 관찰할 수 있었다.

• Asian Pacific Journal of Cancer Prevention
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• 제15권17호
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• pp.7169-7174
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• 2014

Background: NPAS2 is a product of the circadian clock gene. It acts as a putative tumor suppressor by playing an important role in DNA damage responses, cell cycle control and apoptosis. Chronic lymphocytic leukemia (CLL) appears to be an apoptosis related disorder and alteration in the NPAS2 gene might therefore be directly involved in the etiology of CLL. Here, the Ala394Thr polymorphism (rs2305160:G>A) in the NPAS2 gene was genotyped and melatonin concentrations were measured in a total of seventy-four individuals, including thirty-seven CLL cases and an equal number of age- and sex-matched healthy controls in order to examine the effect of NPAS2 polymorphism and melatonin concentrations on CLL risk in a Pakistani population. Materials and Methods: Genotyping of rs2305160:G>A polymorphism at NPAS2 locus was carried out by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). Melatonin concentrations were determined by enzyme linked immunosorbent assay (ELISA). Statistical analysis was performed using Statistical Package for Social Sciences software. Results: Our results demonstrated no association of the variant Thr genotypes (Ala/Thr and Thr/Thr) with risk of CLL. Similarly, no association of rs2305160 with CLL was observed in either females or males after stratification of study population on a gender basis. Moreover, when the subjects with CLL were further stratified into shift-workers and non-shift-workers, no association of rs2305160 with CLL was seen in either case. However, significantly low serum melatonin levels were observed in CLL patients as compared to healthy subjects (p<0.05). Also, lower melatonin levels were seen in shift-workers as compared to non-shift-workers (p<0.05). There was no significant difference (p>0.05) in the melatonin levels across NPAS2 genotypes in all subjects, subjects with CLL who were either shift workers or non-shift-workers. General Linear Model (GLM) univariate analysis revealed no significant association (p>0.05) of the rs2305160 polymorphism of the NPAS2 gene with melatonin levels in any of the groups. Conclusions: While low melatonin levels and shift-work can be considered as one of the risk factors for CLL, the NPAS2 rs2305160 polymorphism does not appear to have any association with risk of CLL in our Pakistani population.

• 미생물학회지
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• 제40권3호
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• pp.183-188
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• 2004

TNT 분해 세균 Stenotrophomonas sp. OK-5는 세 개의 다른 NAD(P)H-nitroreductase의 활성 fractions (NTR fractions I, II, III)을 갖고 있는 것으로 확인된 바 있다. 본 연구에서는 NTR fractions I, II, III에 대한 생리학적 특성과 분자생물학적 특성을 규명하고자 하였다. TNT에 대한 균주 OK-5의 NTR fractions I, II, 그리고 III의 활성은 억제 물질인 $\beta$-mercaptoethanol의 첨가 시에 효소의 활성 이 모두 억제되는 것으로 확인되었다. TNT와 그 유사 기질을 이용하여 균주 OK-5에서 분리된 NTR의 기질 특이성을 조사한 결과, nitrobenzene, 그리고 RDX에 대 해서는 비교적 활성 이 높게 나타났으나 2,6-DNT와 2,4-DNT에서는 낮은 활성을 나타내는 것으로 확인되었다. 균주 OK-5에서 정제된 NTR fraction I의 N-말단 아미노산 서 열은 $^1MSDLLNADAVVQLFRTARDS^20$로 분석되었고, Xanthomonas campestris의 NTR과 X. axonopodis의 NTR에서 각각 70%와 65%로 비교적 높은 유사성을 가지는 것으로 나타났다. 균주 OK-5의 NTR fraction I의 효소를 암호화하는 SmOK5nrI 유전자의 염기서열을 확인하고 분석된 유전자로부터 유추되는 아미노산 서열을 각각 비교한 결과 X. campestris의 NTR과 81%, X. axonopodis의 NTR과 75%,그리고 Streptomyces avermitilis의 NTR과 30%의 유사성이 있는 것으로 조사되었으나, Pseudomonas putida KT2440의 NTR (pnrB)과는 16%로 낮은 유사성이 있는 것으로 확인되었다.

• 한국미생물·생명공학회지
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• 제31권2호
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• pp.103-110
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• 2003

그람 양성세균에서 PyrR단백질에 의하여 피리미딘의 생합성이 조절된다는 발견을 바탕으로 하여, Synechocystis sp.PCC6803과 Haemophilus influenzae의 PyrR orthologue 유전자를 Bacillus subtilis에서 형질전환 시켜 피리미딘 생합성의 조절 유무를 조사하였다. Synechocystis sp.PCC6803과 H. influenzae의 PyrR orthologue유전자를 pUC19과 T-vector에 클로닝 한후 pKH1, pKH2, pHPSK1, pHPSK2으로 각각 명명하였다. 이것을 다시 Escherichia. coli와 B. subtiius용 shuttle vector인 pHPS9에 클로닝 하여 pKH3, pKH4, pHPSK3, pHPSK4로 각각 명명하였다. B. subtilis DB104Δ PyrR에 pKH3, pKH4, pHPSK3, pHPSK4을 형질전환후 ATCase 활성을 측정결과 pHPSK3을 가진 균주만 피리미딘에 의한 조절작용이 일어난다는 사실을 통하여, H. influenzae의 PyrR orthologue 유전자의 선도 부분에 조절에 관여하는 미지의 부분이 있음을 예측할 수 있었다. 서로 다른 유래의 PyrR orthologue단백질을 정제하기 위하여 pET14b에 클로닝후 pKH5, pHPSK5으로 각각 명명하였다. SDS-PAGE로 분석한 결과 각각 약 18 kDa과 21 kDa의 분자량을 나타내었다. 정제된 PyrR orthologue 단백질의 UPRTase 활성을 측정한 결과 H. infuenzae의 PyrR orthologue 단백질은 UPRTase 활성을 나타내었으며 다양한 pH에서 측정한 결과 pH 5에서 가장 높은 활성을 나타내었다. 반면에, Synechocystis sp. PCC6803의 PyrR orhologue 단백질은 UPRTase 활성을 나타내지 않았다. 여러 가지 균주의 PyrR 아미노산 서열을 비교한 계통수 분석은 PyrR 단백질의 조절기작과 어느 정도 연관됨을 시사해 주었다.

• 한국수정란이식학회지
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• 제32권3호
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• pp.73-79
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• 2017

Transplantation is considered to be a very useful approach to improve human welfare and to prolong life-span. Heterologous organ transplantation using pig organs which are similar to human beings and easy to make mass-production has known as one of the alternatives. To ensure potential usage of the pig organ for transplantation application, it is essentially required to generate transgenic pig modifying immuno-related genes. Previously, we reported production of heterozygous ${\alpha}1,3$-galactosyltransferase (GalT) knock-out and human membrane cofactor protein (MCP) expressing pig ($GalT^{-MCP/+}$), which is enforced for suppression of hyperacute and acute immunological rejection. In this study, we reported generation of homozygous pig ($GalT^{-MCP/-MCP}$) by crossbreeding $GalT^{-MCP/+}$ pigs. Two female founders gave birth to six of $GalT^{-MCP/-MCP}$, and seven $GalT^{-MCP/+}$ pigs. We performed quantitative real-time PCR, western blot, and flow cytometry analyses to confirm GalT and MCP expression. We showed that fibroblasts of the $GalT^{-MCP/-MCP}$ pig do not express GalT and its product Gal antigen, while efficiently express MCP. We also showed no expression of GalT, otherwise expression of MCP at heart, kidney, liver and pancreas of transgenic pig. Taken together, we suggest that the $GalT^{-MCP/-MCP}$ pig is a useful candidate to apply xenotransplantation study.

• 대한방사성의약품학회지
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• 제1권2호
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• pp.118-122
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• 2015

Suicidal gene therapy is based on the transduction of tumor cells with "suicide" genes encoding for prodrug-activating enzymes that render target cells susceptible to prodrug treatment. Suicidal gene therapy results in the death of tumor with the expression of gene encoding enzyme that converts non-toxic prodrug into cytotoxic product. Cytochrome P450 4B1 (CYP4B1) activates 4-ipomeanol (4-IPO) or 2-aminoanthracene (2-AA) to cytotoxic furane epoxide and unsaturated dialdehyde intermediate.In this study, therapeutic effects of suicidal gene therapy with rabbit CYP4B1/2-AA or 4-IPO system were evaluated in HT-29 (human colon cancer cell). pcDNA-CYP4B1 vector was transfected into HT-29 by lipofection and stable transfectant was selected by treatment of hygromycin ($500{\mu}g/mL$) for 3 weeks. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed for confirmation of CYP4B1 expression in CYP4B1 gene transduced cell. The cytotoxic effects of CYP4B1 transduced cell were determined using dye-exclusion assay after treatment of 2-AA or 4-IPO for 96 hrs. Dye-exclusion assay showed that $IC_{50}$ of HT-29 and CYP4B1 transduced HT-29 was 0.01 mM and 0.003 mM after 4-IPO or 2-AA treatment at 96 hrs exposure, respectively. In conclusion, CYP4B1 based prodrug gene therapy probably have the potential for treatment of colorectal adenocarcinoma.