• Imaging Science in Dentistry
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• 제33권1호
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• pp.51-57
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• 2003

Purpose: To investigate the effects of irradiation on the phenotypic expression of the MC3T3-El osteoblastic cell line, particularly an the expression of type I collagen and alkaline phosphatase mRNA. Materials and Methods: Cells were irradiated with a single dose of 0.5, 1, 2, 4, and 8 Gy at a dose rate of 5.38 Gy/min using a cesium 137 irradiator. The specimens were then harvested and RNA extraction was carried out at 1 and 3 days after irradiation. The extracted RNA strands were reverse-transcribed and the resulting cDNA fragments were amplified by PCR. Results: The irradiated cells demonstrated a dose-dependent increase in type I collagen mRNA expression relative to the control group, with a maximum level of type I collagen mRNA expression occurring at 8 Gy. The degree of type I collagen mRNA expression increased significantly at 1 day after irradiation, but little differences were found between the control group and at the 3rd day. The amount of alkaline phosphatase mRNA expression increased significantly at land 3 days after irradiation in the 1 Gy exposed group compared with the control group. Conclusion: The amount of type I collagen and alkaline phosphatase mRNA expression increased significantly 1 day after irradiation when compared with the control group.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제31권5호
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• pp.363-369
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• 2005

Purpose: To determine the role of Insulin-like Growth Factor-I (IGF-I) in the regulation of Vascular Endothelial Growth Factor (VEGF) expression in MG-63 cells and then to find the mechanism b which this regulation occurs. Materials and methods: MG-63 cells were grown to confluence in 60-mm dishes. To determine the effects of IGF-I on expression of VEGF mRNA according to time and concentration, the cells were treated with 10 nM IGF-I, following isolation of total RNA and Northern blot analysis after 1, 2, 4, 8, 12, 24 hours and after 2 hours of treatment with 0.5, 2, 10, 25, 50 nM IGF-I respectively, isolation of total RNA and Northern blot analysis were followed. To determine the mechanism of action of IGF-I, inhibitors such as hydroxyurea $(76.1\;{\mu}g/ml)$, actinomycin D $(2.5\;{\mu}g/ml)$, cycloheximide $(10\;{\mu}g/ml)$ were added 1 hour after treatment of 10 nM IGF-I. Results: 1. the expression of VEGF mRNA was increased with treatment of IGF-I. 2. The expression of VEGF mRNA was increased according to time-and concentration dependent manner of IGF-I. 3. The effect of IGF-I was decreased by hydroxyuera, actinomycin D, but not by cycloheximide. Conclusion: IGF-I regulate the expression of VEGF mRNA in the level of DNA synthesis and transcription. These results could suggest that IGF-I plays an important role in angiogenesis in the process of new bone formation and remodeling.

• BMB Reports
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• 제28권6호
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• pp.538-545
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• 1995

When DNA replication of simian virus 40 (SV40) is initiated on the replication origin, the regions containing the initiation sites of DNA primase, which participates in the transient RNA primer synthesis for formation of Okazaki fragments in the lagging strand, were chosen as the target sites of antisense RNA for studies of the inhibition of SV40 DNA replication. Four recombinant transcription vectors, pUC-PrI, pUC-PrII, pGEM-PrBS, and pGEM-PrSN, coding antisense RNA, were constructed. Four antisense RNAs (named as I, II, BS, and SN) having the size of 18, 19,58, and 123 nts, respectively, were made from the transcription vectors by in vitro transcription. And then, antisense RNA in the concentration of 2${\mu}m$ were added to COS cells transfected with pATSV-W which is a recombinant plasmid containing the SV40 origin of replication. The inhibitory extent of DNA replication was measured by DpnI resistance and was confirmed by measurement of transient RNA primer synthesis. The result shows that six combinations of antisense RNA (I, II, BS, SN, I+SN, and BS+SN) lead to the inhibition of SV40 DNA replication by up to 85%.

• Journal of Microbiology and Biotechnology
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• 제18권1호
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• pp.23-27
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• 2008

Self-splicing group I introns in tRNA anticodon loops have been found in diverse groups of bacteria. In this work, we identified $tRNA^{fMet}$ group I introns in six strains of marine Synechococcus elongatus. Introns with sizes around 280 bp were consistently obtained in all the strains tested. In a phylogenetic analysis using the nucleotide sequence determined in this study with other cyanobacterial $tRNA^{fMet}$ and $tRNA^{Leu}$ intron sequences, the Synechococcus sequence was grouped together with the sequences from other unicellular cyanobacterial strains. Interestingly, the phylogenetic tree inferred from the intronic sequences clearly separates the different tRNA introns, suggesting that each family has its own evolutionary history.

• 미생물학회지
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• 제30권6호
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• pp.466-471
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• 1992

HTLV-I 의 gag-pro 유전자 중첩영역내에서 -1 ribosomal frameshifting 이 일어나는 자리를 결정하기 위하여 gag-pro 중첩영역의 일부를 SP6 promoter 를 가진 백터내에 클로닝하였다. 그 결과 닭의 prelysozyme 에서 유래한 5개의 아미노산을 코드하는 합성유전자와 141 bp 로된 gag-pro 중첩영역의 뒤에 Straphylococcus aureus 의 protein A 유전자단편이 연결된 hybrid 유전자를 보유한 플라스미드를 제작하였다. 이 DNA 클론을 주형으로 SP6 RNA polymerase 의 작용에 의해 한종류의 mRNA 를 다량으로 합성하였다. Invitro 에서 합성된 mRNA 로 무세포계에서 단백질을 합성한 결과 21 kDal 의 단백질이 생성되었고 IgG-Sepharose 를 사용한 affinity chromatography 로 합성된 단백질을 순수하게 정제할 수 있었다. 본연구에서 설명한 in vitro 실험계는 Gag-Pro transframe 단백질의 신속한 정제 및 일차구조의 결정에 유익하게 사용될 것으로 보이며 이와 같은 실험의 결과 mRNA 에서 ribosomal frameshifting 이 일어나는 정확한 site 를 결정할 수 있을 뿐 같은 실험의 결과 mRNA 에서 ribosomal frameshifting 이 일어나는 정확한 site 를 결정할 수 있을 뿐 아니가 pro 유전자의 발현에 필요한 frameshift 를 유도하는 tRNA 의 동정도 가능하게 될 것이다.

• Tuberculosis and Respiratory Diseases
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• 제53권1호
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• pp.36-45
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• 2002

배 경 :포유동물은 고산소 노출되면 이에 적응하기 위해 성숙 폐장의 항산화 효소의 활성도가 증가하는 것으로 알려져 있다. 신생 폐장에서는 이들의 활성도가 뚜렷이 증가하고 이는 고산소 노출에 내성을 보이는 중요한 기전으로 알려져 있다. Peroxiredoxin은 세포내 항산화 효소로 세포내 많은 양이 존재하고 다양한 세포에 분포하고 있다. 그중 Prx I 및 II는 세포질에 존재하는 주된 동종 효소이다.본 연구는 고산소를 투여하여 성숙 백서의 폐장에서 Prx I과 Prx II mRNA 및 단백의 발현을 평가하여 신생 백서의 폐장에서의 발현과 비교하고자 하였다. 방 법 :성숙 백서와 임신 백서로부터 분만하여 얻은 신생 백서를 무작위로 고산소를 시간별로 투여후에 폐조직과 기관지폐포세척액을 얻었다. Prx I 및 Prx II mRNA는 Northernblot 방법으로 구하였으며 Actin mRNA을 내부 기준으로 하여 상대 발현을 평가하였다. Prx I 및 Prx II 단백은 Westernblot 방법으로 구하였으며 Actin 단백을 내부 기준으로 하여 상대 발현을 평가하였다. 결 과 : 성숙 백서에서 고산소 노출 후 24 시간에 Prx I mRNA 발현이 약간의 증가가 유도되었으며 신생 백서에서 고산소 노출 후 24 시간에 Prx I mRNA 발현이 현저한 증가가 유도되었다. 그러나 Prx II mRNA는 고산소 노출 내내 발현이 변화가 없었다. 성숙 및 신생 백서에서 고산소 노출 내내 Prx I 및 Prx II 단백의 발현이 변화가 없었으며 성숙 백서에서 고산소 노출 내내 기관지폐포세척액내 Prx I 및 Prx II 단백의 양의 변화가 없었다. 결 론 : 성숙 및 신생 백서에서 고산소 노출에 의해 Prx I 및 II의 발현이 전사 과정 에서 서로 다르게 조절된다. 성숙 백서보다 신생 백서에서 고산소 노출에 의한 Prx I mRNA의 뚜렷한 발현 증가는 신생 백서가 고산소에 내성을 보이는 하나의 기전으로 생각된다.

• Journal of Microbiology
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• 제41권4호
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• pp.340-344
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• 2003

The group I intron from Tetrahymena thermophila has been demonstrated to employ splicing reactions with its substrate RNA in the trans configuration. Moreover, we have recently shown that the transsplicing group I ribozyme can replace HCV-specific transcripts with a new RNA that exerts anti-viral activity. In this study, we explored the potential use of RNA replacement for cancer treatment by developing trans-splicing group I ribozymes, which could replace tumor-associated RNAs with the RNA sequence attached to the 3' end of the ribozymes. Thymidine phosphorylase (TP) RNA was chosen as a target RNA because it is known as a valid cancer prognostic factor. By performing an RNA mapping strategy that is based on a trans-splicing ribozyme library, we first determined which regions of the TP RNA are accessible to ribozymes, and found that the leader sequences upstream of the AUG start codon appeared to be particularly accessible. Next, we assessed the ribozyme activities by comparing trans-splicing activities of several ribozymes that targeted different regions of the TP RNA. This assessment was performed to verify if the target site predicted to be accessible is truly the most accessible. The ribozyme that could target the most accessible site, identified by mapping studies, was the most active with high fidelity in vitro. Moreover, the specific trans-splicing ribozyme reacted with and altered the TP transcripts by transferring an intended 3' exon tag sequence onto the targeted TP RNA in mammalian cells with high fidelity. These results suggest that the Tetrahymena ribozyme can be utilized to replace TP RNAs in tumors with a new RNA harboring anti-cancer activity, which would revert the malignant phenotype.

• Archives of Pharmacal Research
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• 제27권7호
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• pp.769-775
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• 2004

Hepatic ischemia and reperfusion (l/R) predisposes the liver to secondary stresses such as endotoxemia, possibly via dysregulation of the hepatic microcirculation secondary to an imbalanced regulation of the vascular stress genes. In this study, the effect of hepatic I/R on the hepatic vasoregulatory gene expression in response to endotoxin was determined. Rats were subjected to 90 min of hepatic ischemia and 6 h of reperfusion. Lipopolysaccharide (LPS, 1 mg/kg) was injected intraperitoneally after reperfusion. Plasma and liver samples were obtained 6 h after reperfusion for serum aminotransferase assays and RT-PCR analysis of the mRNA for the genes of interest： endothelin-1 (ET-1), its receptors $ET_A$ and $ET_B$, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), heme oxygenase-1 (HO-1), cyciooxygenase-2 (COX-2), and tumor necrosis factor-a (TNF-${\alpha}$). The activities of serum aminotransferases were significantly increased in the I/R group. This increase was markedly potentiated by LPS treatment. The ET-1 mRNA was increased by LPS alone, and this increase was significantly greater in both the I/R alone and I/R ＋ LPS groups compared to the sham. There were no significant differences in ETA receptor mRNA levels among any of the experimental groups. $ET_B$ mRNA was increased by both LPS alone and I/R alone, with no significant difference between the I/R alone and I/R ＋ LPS groups. The eN OS and HO-1 transcripts were increased by I/R alone and further increased by I/R ＋ LPS. The iNOS mRNA levels were increased by I/R alone, but increased significantly more by both LPS alone and I/R ＋ LPS compared to I/R alone. The TNF-${\alpha}$ mRNA levels showed no change with I/R alone, but were increased by both LPS alone and I/R ＋ LPS. The COX-2 expression was increased significantly by I/R alone and significantly more by I/R ＋ LPS. Taken collectively, significantly greater induction of the vasodilator genes over the constriction forces was observed with I/R ＋ LPS. These results may partly explain the increased susceptibility of ischemic livers to injury as a result of endotoxemia.

• 한국어병학회지
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• 제6권2호
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• pp.93-101
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• 1993

효모의 RNAI유전자는 RNA processing에 관여 하는지 혹은 RNA transport에 관여 하는지 아직까지 유전자의 기능이 정확히 알려져 있지 않은 실정이다. 효모의 RNAI 유전자의 기능을 파악하기 위한 방법으로 본 연구에서는 rna1-1 mutant gene을 cloning하여 이에 대한 DNA sequence를 조사함으로써 RNAI 유전자와 rna1-1 유전자의 차이점을 이해하고자 하였다. rna1-1 marker를 갖는 yeast strain(R49)로 부터 genomic DNA를 추출하여 이를 BglII로 절단하여 genomic southern blotting을 행한 결과 wild type의 경우와 동일하게 3.4 kb에서 hybridization되는 signal을 얻었으며, RNAl 및 rna1-1이 yeast genome내에 single site로 존재함을 보여 주는 결과를 얻었다. mutant strain으로 부터 얻은 3.4 kb의 BglI fragment를 pUC19의 BamHI site에 subcloning하여 transformant들을 얻었고, wild type RNAl 유전자를 probe로 하여 rna1-1 mutant 유전자를 cloning할 수 있었다. pUC19에 cloning된 RNA1유전자 및 rna1-1유전자로부터 다양한 Ba131유도체를 얻어 이들에 대한 염기 서열을 비교한 결과 transcription initation site에서부터 down stream쪽으로 17 아미노산위치에 TCC가 TTC로 대치되어 있었으며 그 결과 serine이 phenylalanine으로 변환되는 결과를 얻었다. Wild type RNAI gene의 5'-region에는 3군데의 TATA-like sequence가 true TATA box인지 확인하기 위하여 Bal3I deletion에 의해 -103nt까지 deletion된 유도체를 얻었으며 ${\Delta}RNAI$, rna1-1, 81-2-6 clone이 rna1-1 allele와 complementation한지 확인하였으나 ${\Delta}RNAI$은 TS-complementation을 하지 못하였다. 따라서 현재까지 TATA-box라고 알려진 부분은 promoter로 작용하지 못함을 확인하였다.

• 생명과학회지
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• 제8권2호
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• pp.126-130
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• 1998

Vibrio unlnificus ATCC 27562의 16S rRNA 유전자를 PCR법과 제한효소절단법으로 분석 하여 얻은 결과는 다음과 같다. 1. 고안된 한쌍의 primer로 PCR을 시행하여 얻은 산물은 약 1.3Kb 였다. 2. PCR산물을 여섯가지의 제한 효소로 절단하여 얻은 단편들은 아래와 같다. BamH I: 어떤 restriction fragment도 만들지 않았다. Alu I : 약 400bp와 200bp의 두가지 fragment를 생산하였다. Sau3A I : 약 70bp에서 450bp 사이에 세가지 fragment를 생산하였다. Hind III : 약 800bp와 500bp의 약간 큰 두가지 fragment를 생산하였다. Sal I : 약 500bp와 750bp의 두가지 fragment를 생산하였다. Sma I : 약 800bp와 470bp의 두가지 fragment를 생산하였다.