• Childhood Kidney Diseases
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• 제3권2호
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• pp.209-216
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• 1999

목적 : 신성 요붕증(Nephrogenic diabetes insipidus, NDI)은 바소프레신(arginine vasoporessin, AVP)에 대한 신세뇨관의 저항성으로 인하여 요농축의 장애를 특징으로 하는 드문 유전성 질환이다. 반성유전형 신성 요붕증은 바소프레신 V2수용체(AVPR2)의 장애에 기인하며, NDI 환자에서 지금까지 다양한 AVPR2의 돌연변이가 보고되었다. 저자들은 임상적으로 반성 유전형 신성 요붕증으로 진단된 가계에서 AVPR2 유전자의 돌연변이를 발견하기 위하여 분자유전학적 검사를 실시하였다. 방법 : 대상환자의 백혈구에서 추출한 DNA로 AVPR2유전자를 polymerase chain reaction-single strand conformational polymorphism(PCR-SSCP)분석하여 이상이 발견된 부분은 클론닝하여 염기서열을 분석하였다. 같은 PCR 산물을 Hae III로 처리하여 PCR-RFLP(restriction fragement length polymorphism) 분석을 하였다. 결과 : AVPR2 유전자를 PCR-SSCP 분석하였을 때 PCR 산물의 정상인과 이동거리의 차이가 발견되어 환아에서 돌연변이가 있고 환아의 어머니는 보인자임을 예측하였고, 염기서열을 분석하여 675번째 염기 A가 G로 치환됨으로 tryptophan이 cysteine으로 바뀌는 R202C 점돌연변이를 발견하였다. 같은 PCR 산물을 PCR-RFLP 분석을 하였을 때 돌연변이로 인한 Hae III의 인지부위의 상실을 확인하였고 환아의 어머니가 이종접합보유자 (heterozygote)임을 확인하였다. 결론 : 저자들은 임상적으로 신성 요붕증으로 확인된 환아와 어머니의 V2 수용체 유전자를 분석하여 R202C 돌연변이를 확인하였다. 신성 요붕증은 진단이 지연되면 성장장애, 정신박약과 사망을 초래할 수 있는 심각한 질환이나, 태생기 또는 신생아기에 진단하면 후유증을 예방할 수 있으므로 조기진단 및 보인자 발견에 분자유전학적 진단 방법을 적극 활용하여야 하겠다.

• Journal of Microbiology and Biotechnology
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• 제19권5호
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• pp.439-446
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• 2009

The biosynthesis study of antibiotics saframycin (SFM) in Streptomyces lavendulae and safracin (SAC) in Pseudomonas fluorescens demonstrated that 3-hydroxy-S-methyl-O-methyltyrosine (3hSmOmTyr), a nonproteinogenic amino acid, is the precursor of the tetrahydroisoquinoline molecular core. In the biosynthetic gene cluster of SAC/SFM, sacD/sfmD encodes a protein with high homology to each other but no sequence similarity to other known enzymes; sacF/sfmM2 and sacG/sfmM3 encode methyltransferases for C-methylation and O-methylation; and sacE/sfinF encodes a small protein with significant sequence similarity to the MbtH-like proteins, which are frequently found in the biosynthetic pathways of non ribosomal peptide antibiotics and siderophores. To address their function, the biosynthetic cassette of 3h5mOmTyr was heterologously expressed in S. coelicolor and P. putida, and an in-frame deletion and complementation in trans were carried out. The results revealed that (i) SfmD catalyzes the hydroxylation of aromatic rings; (ii) sacD/sacF/sacG in the SAC gene cluster and sfmD/sfmM2/sfmM3 in the SFM cluster are sufficient for the biosynthesis of 3h5mOmTyr; and (iii) the mbtH-like gene is not required for the biosynthesis of the 3h5mOmTyr precursor.

• Parasites, Hosts and Diseases
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• 제37권4호
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• pp.257-263
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• 1999

Identification of the genes responsible for the recovery of virulence in brain-passaged Acanthamoeba culbertsoni was attempted via mRNA differential display polymerase chain reaction (mRNA DD-PCR) analysis. In order to identify the regulatory changes in transcription of the virulence related genes by the brain passages, mRNA DD-PCR was performed which enabled the display of differentially transcribed mRNAs after the brain passages. Through mRNA DD-PCR analysis. 96 brain-passaged amoeba specific amplicons were observed and were screened to identify the amplicons that failed to amplify in the non-brain-passaged amoeba mRNAs. Out of the 96 brain-passaged amoeba specific amplicons, 12 turned out to be amplified only from the brain-passaged amoeba mRNAs by DNA slot blot hybridization. The clone, A289C, amplified with an arbitrary primer of UBC ＃289 and the oligo dT$_{11}$-C primer, revealed the highest homology (49.8％) to the amino acid sequences of UPD-galactose lipid transferase of Erwinia amylovora, which is known to act as an important virulence factor. The deduced amino acid sequences of an insert DNA in clone A289C were also revealed to be similar to cpsD, which is the essential gene for the expression of type III capsule in group B streptococcus. Upregulated expression of clone A289C was verified by RNA slot blot hybridization. Similar hydrophobicity values were also observed between A289C (at residues 47-66) and the AmsG gene of E. amylovora (at residues 286-305: transmembrane domains). This result suggested that the insert of clone A289C might play the same function as galactosyl transferase controlled by the AmsG gene in E. amylovora.a.

• 생명과학회지
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• 제20권5호
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• pp.683-690
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• 2010

효모 Saccharomyces diastaticus 는 세포 외로 분비되는 glucoamylase I, II, III 동위효소 중 하나를 생산하여 전분을 가수분해하여 포도당을 생성할 수 있다. Glucoamylase I, II, III는 STA1, STA2, STA3 유전자에 의해 각각 암호화된다. 효모 Saccharomyces 속이 포자가 형성되는 시기에 세포 내에서 특이적으로 발현된다고 알려진 glucoamylase (SGA)의 분자생물학적 및 생화학적 연구를 수행하기 위한 일환으로 S. diastaticus YIY 345 형질전환체의 배양 상등액으로부터 SGA 정제를 시도하였다. 황산암모늄 침전, DEAE-Sephadex A-50, CM-Sephadex C-50, Sephadex G-200 chromatography 등의 정제과정을 거쳐서 비특이 활성이 174배 증가된 0.22 mg의 순수한 SGA를 얻었다. HPLC와 SDS-PAGE 분석을 통해 이 효소는 63, 68 kDa의 단위체로 구성된 이합체임을 확인할 수 있었다. Con-A Sepharose 친화성 크로마토그피와 탈당쇄 효소를 처리한 결과로부터 SGA는 N-연결형 당쇄로 수식되었으며 단백질 부분은 59 kDa이었다. 정제한 SGA와 세포 외 분비성 glucoamylase의 효소학적 특성을 조사하고 비교한 결과 SGA의 최적 pH와 온도는 각각 5.5와 $45^{\circ}C$로 나타났으며 세포 외 분비성 glucoamylase는 5.0과 $50^{\circ}C$로 나타났다. SGA는 세포 외로 분비되는 glucoamylase에 비해 열처리 및 SDS에 대해 더 민감한 반응성을 나타내었다.

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.84-91
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• 2006

A Saccharomyces cerevisiae pgs1 nulI mutant, which is deficient with phosphatidyl glycerol (PG) and cardiolipin (CL) biosynthesis, grows well on most fermentable carbon sources, but fails to grow on non-fermentable carbon sources such as glycerol, ethanol, and lactate. This mutant also cannot grow on galactose medium as the sole carbon source. We found that the incorporation of $[^{14}C]-galactose$, which is the first step of the galactose metabolic pathway (Leloir pathway), into the pgs 1 null mutant cell was extremely repressed. Exogenously expressed PGS1 (YCpPGS1) under indigenous promoter could completely restore the pgs1 growth defect on non-fermentable carbon sources, and dramatically recovered $[^{14}C]-galactose$ incorporation into the pgs1 mutant cell. However, PGS1 expression under the GALl promoter $(YEpP_{GAL1}-PGS1myc)$ could not complement pgs1 mutation, and the GAL2-lacZ fusion gene $(YEpP_{GAL2}-lacZ)$ also did not exhibit its $\beta-galactosidase$ activity in the pgs1 mutant. In wild-type yeast, antimycin $A(1\;{\mu}g/ml)$, which inhibits mitochondrial complex III, severely repressed not only the expression of the GAL2-lacZ fusion gene, but also uptake of $[^{14}C]-galactose$. However, exogenously expressed PGS1 partially relieved these inhibitory effects of antimycin A in both the pgs1 mutant and wild-type yeast, although it could not basically restore the growth defect on galactose by antimycin A. These results suggest that the PGSI gene product has an important role in utilization of galactose by Gal genes, and that intact mitochondrial function with PGS1 should be required for galactose incorporation into the Leloir pathway. The PGS1 gene might provide a clue to resolve the historic issue about the incapability of galactose with deteriorated mitochondrial function.

• Asian Pacific Journal of Cancer Prevention
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• 제17권8호
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• pp.4149-4154
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• 2016

Background: Cervical carcinoma is one of the main causes of mortality in women worldwide as well as in India. It occurs as a result of various molecular events that develop from the combined influences of an individual's genetic predisposition and external agents such as smoking and menstrual hygiene, for example. However, infection with human papillomavirus (HPV) is the established major risk factor. The aim of the current study was to investigate p16 CpG island methylation and establish any correlation with mRNA expression in north Indian population. Materials and Methods: We analyzed 196 woman volunteer out of which 98 were cases and 98 healthy controls. For the analysis of methylation pattern, DNA extracted from blood samples was modified with a bisulfate kit and used as template for methylation specific PCR (MSP). Quantitative real-time PCR (QRT-PCR) was performed to check mRNA expression. Results: Correlation between methylation status of p16 gene and poor menstrual hygiene was significant (p=0.006), high parity cases showed methylation of p16 gene (p=0.031) with increased risk up to 1.86 times for cervical cancer and smoking was a strong risk factor associated with cervical cancer. We analyzed methylation pattern and found 60.3% methylation in cases with low mRNA expression level (0.014) as compare to controls (1.24). It was also observed that promoter methylation of p16 gene was significantly greater in FIGO stage III. Conclusions: We conclude that p16 methylation plays an important role in cervical cancer in the north Indian population and its methylation decreases mRNA expression. It can be used as an important and consistent blood biomarker in cervical cancer patients.

• 미생물학회지
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• 제36권1호
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• pp.1-7
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• 2000

발광 박테리아인 Photobacterium 종들의 lux 오페론 하부 영역에서 riboflavin 생합성에 관여하는 유전자들(ribⅠ,Ⅱ,Ⅲ,Ⅳ)이 발견되었다. Photobacterium phosphoreum의 lux 유전자와 rib 유전자를 포함하는 intergenic 영역의 단일사슬 DNA가 P. phosphoreum의 mRNA에 의하여 S1 nuclease digestion에서 손상받지 않았으며, ribⅠ에 의하여 암호화되는 P. phosphoreum의 riboflavin synthase의 활성도가 lux-specific한 효소들인 luciferase 혹은 fatty acid reductase 활성도와 같이 bioluminescence intensity의 발현과 함께 대수기 말기에서 증가하는 박테리아 발광반응의 특이한 조절 체계인 'autoinduction' 양상을 보였다. 또한 P. leiognathi의 luxB로부터 ribⅡ까지 포함하는 DNA를 강력한 lux 프로모터와 reporter(chloramphenicol acetyl transferase, CAT) 유전자 사이에 삽입하고 접합(conjugation)의 방법으로 P. leiognathi에 유전자 전이(gene transfer)시켜 CAT reporter 유전자의 발현을 P. leiognathi에서 조사한 바, 그 유전자의 발현 정도에 큰 차이가 없었을 뿐만 아니라 이 구조에서 lux 프로모터를 제거하게 되면 CAT reporter 유전자의 발현이 전혀 나타나지 않았다. 이들 실험 결과들은 lux 유전자와 rib 유전자의 intergenic영역에 lux 오페론의 전사 종결 구조(transcriptional terminator)가 존재하지 않으며 ribflavin 생합성 유전자들이 그들 고유의 프로모터에 의하여 전사되는 것이 아니라 lux 오페론의 프로모터에 의하여 발현됨을 나타내는 것으로, 이는 Photobacterium 종들에서 lux 유전자와 rib 유전자들은 공동의 발현 조절 체계를 갖는 것으로 요약된다.

• Reproductive and Developmental Biology
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• 제30권2호
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• pp.125-133
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• 2006

This study was conducted to detect the apoptosis incidence in blastocysts and to compare the abundance of Bax, Bcl2L1, VEGF and FGFR2 in in vitro fertilized (IVF), parthenogenetic (PAT) and nuclear transfer (NT) embryos. Oocytes matured for 40 hr were enucleated and reconstructed with confluenced fetal fibroblasts (FFs) derived from a ${\sim}45$ day fetus. Reconstructed eggs were then fused with 2 DC pulses (2.0 kV/cm, $30{\mu}sec$) and cultured with $7.5{\mu}g/ml$ cytochalasin B for 3 hr. Parthenotes (PAT) were produced with the same electric strength and culture for NT eggs. The embryos were cultured in NCSU-23 medium at $39^{\circ}C,\;5%\;CO_2,\;5%\l;O_2$ in air. In 3 runs, set of 10 embryos at the 4-cell to blastocyst stages were used to extract total RNA for analyzing the gene expression patterns of pro-apoptotic (Bax), anti-apoptotic (Bcl2L1), vasculogenesis (VEGF), implantation (FGFR2III) using real-time quantitative PCR. Cleavage and blastocyst rates were significantly higher (P<0.05) in IVF and PAT ($79.3{\pm}8.5\;and\;25.5{\pm}6.1,\;and\;85.0{\pm}6.4\;and\;38.6{\pm}5.5$, respectively)than NT counterparts ($65.1{\pm}5.2\;and\;15.6{\pm}3.0$, respectively). Significantly higher (P<0.05) total cells were observed in IVF controls and PAT ($34.7{\pm}5.8\;and\;38.1{\pm}4.1$) than NT embryos ($24.8{\pm}3.2$). Apoptosis index was significantly lower (P<0.05) in IVF than NT embryos. The Relative abundances (RA) of Bax and VEGF were significantly higher (P<0.05) at blastocyst stage in NT than IVF control. The RA of Bcl2L1 and FGFR2III were significantly higher (P<0.05) at blastocyst stage in IVF than NT. The present study observed the abnormal gene expressions in NT embryos at various developmental stages, suggesting certain clues to find out the cause of the low efficiency of NT to term.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• 제23권2호
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• pp.246-252
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• 2010

In order to improve the availability of phytase and probiotics together, a phytase gene from Aspergillus ficuum has been expressed in Lactobacillus. In this study, the transformed Lactobacillus with phytase gene was fed to pigs to determine its effect on pig production, feed conversion and gut microbes. Forty eight, 60-day-old, castrated pigs (Duroc${\times}$Landrace${\times}$Pietrain) were assigned to 6 groups, 8 pigs for each group. Group 1 was the control, group 2 was added with chlortetracycline (500 mg/kg), group 3 was added with the transformed Lactobacillus (500 mg/kg) with 20% (w/w) of calcium monohydrogen phosphate (CMP, $CaHPO_{4}$) removed, group 4 was added with the natural Lactobacillus (500 mg/kg) with 20% (w/w) of CMP removed, group 5 was added with the transformed Lactobacillus (500 mg/kg) with 40% (w/w) of CMP removed, group 6 was added with phytase (500 mg/kg) with 40% (w/w) of CMP removed. The results showed: i) the average daily gain (ADG) was improved in groups 2, 3 and 4 (p<0.05); ii) the diarrhea rates in the groups added with Lactobacillus were lower than in the other groups (p<0.05), in which the transformed Lactobacillus had more effect on reducing digestive disease; iii) the transformed Lactobacillus was most effective in improving the digestibilities of crude protein (CP), calcium (Ca), phosphorus (P), compared with the other groups (p<0.05); iv) Lactobacillus could increase lactic acid bacterium number and ammonia concentrations, and decrease pH values and E. coli number in pig feces (p<0.05); v) the phytase activity in the feces of pigs fed with the transformed Lactobacillus was 133.32 U/g, which was higher than in group 4 (9.58 U/g, p<0.05), and was almost the same as group 6 (135.94 U/g); vi) the transformed Lactobacillus could increase serum concentrations of IgA, triglyceride, and glutamic oxaloacetic transaminase activity (p<0.05), and had no significant effect on other serum indexes (p>0.05).