• Molecular & Cellular Toxicology
• /
• 제1권2호
• /
• pp.92-98
• /
• 2005

DNA microarray analysis of gene expression in toxicogenomics typically requires relatively large amounts of total RNA. This limits the use of DNA microarray when the sample available is small. To confront this limitation, different methods of linear RNA amplification that generate antisense RNA (aRNA) have been optimized for microarray use. The target preparation strategy using amplified RNA in DNA microarray protocol can be divided into direct-incorporation labeling which resulted in cDNA targets (Cy-dye labeled cDNA from aRNA) and indirect-labeling which resulted in cRNA targets (i.e. Cy-dye labeled aRNA), respectively. However, despite the common use of amplified targets (cDNA or cRNA) from aRNAs, no systemic assessment for the use of amplified targets and bias in terms of hybridization performance has been reported. In this investigation, we have compared the hybridization performance of cRNA targets with cDNA targets from aRNA on a 10 K cDNA microarrays. Under optimized hybridization conditions, we found that 43% of outliers from cDNA technique and 86% from the outlier genes were reproducibly detected by both targets hybridization onto cDNA microarray. This suggests that the cRNA labeling method may have a reduced capacity for detecting the differential gene expression when compared to the cDNA target preparation. However, further validation of this discordant result should be pursued to determine which techniques possesses better accuracy in identifying truly differential genes.

• 생명과학회지
• /
• 제16권2호
• /
• pp.240-244
• /
• 2006

본 실험에서는 토마토의 종자를 기내 배양하여 얻어진 1g 이하의 무균 잎 조직을 이용하여 미토콘드리아를 분리 정제하여 MitoTracker를 이용하여 세포생물학적으로 확인하였고, 이들의 mt를 이용하여 미토콘드리아 DNA와 RNA를 추출과 검정을 하였다. 또한 고농도의 이온성을 이용하여 미토콘드리아와 mtDNA 및 mtRNA을 추출할 수 있었으며, 식물의 여러 종류에도 사용되어질 수 있을 것이다. mtDNA는 PCR 분석에 의하여 plastid DNA와 혼재되어 있지 않음을 확인하였다. mtRNA는 RT-PCR 분석을 통하여 plastid RNA와 흔재되어 있지 않음을 확인할 수 있었다.

• BMB Reports
• /
• 제53권9호
• /
• pp.453-457
• /
• 2020

The right-handed double-helical structure of DNA (B-DNA), which follows the Watson-Crick model, is the canonical form of DNA existing in normal physiological settings. Even though an alternative left-handed structure of DNA (Z-DNA) was discovered in the late 1970s, Z-form nucleic acid has not received much attention from biologists, because it is extremely unstable under physiological conditions, has an ill-defined mechanism of its formation, and has obscure biological functions. The debate about the physiological relevance of Z-DNA was settled only after a class of proteins was found to potentially recognize the Z-form architecture of DNA. Interestingly, these Z-DNA binding proteins can bind not only the left-handed form of DNA but also the equivalent structure of RNA (Z-RNA). The Z-DNA/RNA binding proteins present from viruses to humans function as important regulators of biological processes. In particular, the proteins ADAR1 and ZBP1 are currently being extensively re-evaluated in the field to understand potential roles of the noncanonical Z-conformation of nucleic acids in host immune responses and human disease. Despite a growing body of evidence supporting the biological importance of Z-DNA/RNA, there remain many unanswered principal questions, such as when Z-form nucleic acids arise and how they signal to downstream pathways. Understanding Z-DNA/RNA and the sensors in different pathophysiological conditions will widen our view on the regulation of immune responses and open a new door of opportunity to develop novel types of immunomodulatory therapeutic possibilities.

• 생명과학회지
• /
• 제19권3호
• /
• pp.305-310
• /
• 2009

플라스미드 pGKV21에는 229-nt single-strand DNA initiation (ssi) signal이 존재한다. Asymmetric PCR 기법으로 합성된 229-nt ssDNA 단편을 이용하여 실제로 RNA polymerase에 의한 priming ability와 protein interaction을 확인하였다. in vitro primer RNA 합성 실험 결과, 229-nt ssDNA 단편은 filamentous M13 phage의 주형 DNA에서와 비슷한 효율로 시발체 RNA를 합성하였으며, 이 반응은 strand-specific하게 이루어졌다. DNase I footprinting과 gel retardation 실험 결과, RNA polymerase와 SSB 단백질은 229-nt ssDNA 단편에 stable interaction을 하며, 시발체 RNA를 합성하였다. 또한, in vivo 조건 하에서 RNA polymerase의 저해제인 rifampicin을 처리하여 세포내에 ssDNA 중간체가 집적되는 정도를 비교하여 본 결과, 플라스미드 pGKV21은 rifampicin-sensitive RNA polymerase가 상보가닥 합성에 관여 함을 보여 주었다.

• Fisheries and Aquatic Sciences
• /
• 제7권4호
• /
• pp.192-203
• /
• 2004

The effects of temperatures, starvation, and kind of foods on growth, RNA/DNA ratio and protein contents during metamorphosis and early juvenile stage of olive flounder (Paralichthys olivaceus) were examined. During metamorphosis, warm-acclimated fish showed higher RNA and DNA content than those of the cold-acclimated fish, excepting H stage (28 DAH) at which the ratio was higher at cold temperature. RNA/DNA ratio during metamorphosis showed similar values at two temperatures tested. However, after 42 DAH warm-acclimated juveniles had higher DNA content compared with cold-acclimated fish, resulted in marked decreases in RNA/DNA ratios. Higher RNA content at H stage of cold-acclimated fish was consistent with an increase in protein content. Growth of fish rearing at warm temperature was higher than those of fish at cold temperature during all experiments. In starvation experiment, contents of DNA, RNA and protein significantly decreased. Even though there were no significant differences in total length (TL) and body weight between the live mysid-fed and artificial pellet-fed fish at 35 mm TL, both RNA/DNA and protein/DNA ratios of the former group was significantly higher than those of the latter due primarily to lower DNA content of the live mysid-fed group. The results from this study suggest that temperature, starvation and kind of foods should be considered when RNA/DNA ratio applied to assessing the cultured larval and juvenile fish condition.

• 한국어류학회지
• /
• 제22권4호
• /
• pp.267-272
• /
• 2010

문치가자미 Pleuronectes yokohamae의 RNA/DNA비 일주기 변화를 조사하기 위하여 부화후 89일, 전장 $23.2{\pm}0.2mm$의 치어를 48시간 동안 일정한 시간 간격으로 채집하였다. RNA/DNA비는 주간(0800, 1100, 1400, 1700)에 높았고 야간(2000, 2300, 0200, 0500 hours)에는 낮게 나타났다. 이번 연구결과에서 문치가자미의 생화학적 일주기 변화를 확인하였고, 이와 같은 RNA/DNA비의 일주기적 변화를 고려하여 시료 채집시간 결정을 포함한 시료채집 계획작성과 data분석이 필요할 것으로 생각된다.

• 미생물학회지
• /
• 제21권4호
• /
• pp.229-237
• /
• 1983

Aspergillus nidulans의 tRNA 유전자의 구성과 발현기착을 연구하기 위하여 우선 Aspergillus의 총 tRNA 유전자를 cloning 하였다. Aspergillus의 핵 DNA롱 포자로 부터 분리해 내고 본질 형성에서 분리한 BamHI과 T4 DNA ligase를 사용하여 pBR322플라스미드에 재조합시켜서 cloning하였다. 15벤의 transformation을 하여 30,000개 의 transformants 얻었고, 이 중 Aspergillus DNA를 가지고 있는 colony는 5,300켜개였다. In vivo에 서 S2p로 표지 된 total tRNA를 probe로 하여 colony hybridization 실험 결과, 105개의 total tRNA유전자 clone을 얻었다. 위의 결과와 cohybridization 실험 결과를 분석해 보면, Asprgillus의 tRNA 유전자는 yeast의 그것보다는 좀 더 밀집되어 존재한다고 생각된다.

• BMB Reports
• /
• 제38권2호
• /
• pp.198-205
• /
• 2005

Resveratrol (RES) and genistein (GEN) are the dietary natural products known to possess chemopreventive property and also the ability to repair DNA damage induced by mutagens/carcinogens. It is believed that the therapeutic activity of these compounds could be primarily due to their interaction with nucleic acids but detailed reports are not available. We here explore the interaction of these drugs with nucleic acids considering DNA and RNA as a potential therapeutic target. The interaction of RES and GEN has been analysed in buffered solution with DNA [saline sodium citrate (SSC)] and RNA [tris ethylene diammine tetra acetic acid (TE)] using UV-absorption and Fourier transform infrared (FTIR) spectroscopy. The UV analysis revealed lesser binding affinity with nucleic acids at lower concentration of RES (P/D = 5.00 and 10.00), while at higher drug concentration (P/D = 0.75, 1.00 and 2.50) hyperchromic effect with shift in the ${\lambda}_{max}$ is noted for DNA and RNA. A major RES-nucleic acids complexes was observed through base pairs and phosphate backbone groups with K = $35.782\;M^{-1}$ and K = $34.25\;M^{-1}$ for DNA-RES and RNA-RES complexes respectively. At various concentrations of GEN (P/D = 0.25, 0.50, 0.75, 1.00 and 2.50) hyperchromicity with shift in the ${\lambda}_{max}$ from 260 $\rightarrow$ 263 om and 260 $\rightarrow$ 270 nm is observed for DNA-GEN and RNA-GEN complexes respectively. The binding constant (from UV analysis) for GEN-nucleic acids complexes could not be obtained due to GEN absorbance overlap with that of nucleic acids at 260 nm. Nevertheless a detailed analysis with regard to the interaction of these drugs (RES/GEN) with DNA and RNA could feasibly be understood by FTIR spectroscopy. The NH band of free DNA and RNA which appeared at $3550-3100\;cm^{-1}$ and $3650-2700\;cm^{-1}$ shifted to $3450-2950\;cm^{-1}$ and $3550-3000\;cm^{-1}$ in DNA-RES and RNA-RES complexes respectively. Similarly shifts corresponding to $3650-3100\;cm^{-1}$ and $3420-3000\;cm^{-1}$ have been observed in DNA-GEN and RNA-GEN complexes respectively. The observed reduction in NH band of free nucleic acids upon complexation of these drugs is an indication of the involvement of the hydroxyl (OH) and imino (NH) group during the interaction of the drugs and nucleic acids (DNA/RNA) through H-bonded formation. The interaction of RES and GEN with bases appears in the order of G $\geq$ T > C > A and A > C $\geq$ T > G. Further interaction of these natural compounds with DNA and RNA is also supported by changes in the vibrational frequency (shift/intensity) in symmetrical and asymmetrical stretching of aromatic rings of drugs in the complex spectra. No appreciable shift is observed in the DNA and RNA marker bands, indicating that the B-DNA form and A-family conformation of RNA are not altered during their interaction with RES and GEN.

• 생명과학회지
• /
• 제8권2호
• /
• pp.203-207
• /
• 1998

본 연구는 옥수수에서 분리한 미토콘드리아에서 NADH-dehydrogenase 유전자 (subunit 4)의 cDNA를 RT-PCR의 방법을 사용하여 조제 한 ㅜ 염기서열 수행한 경과 특이한 점을 감지 할 수 있었다. 일반적인 RNA cditing은 C에서 U로 또는 U에서 C로 치환되는 현장으로 옥수수의 NAD4유전자에서도 이러한 editing 형상이 일어나는 것을 발견하였다. 또는 T가 G로 그리고 G 가 A로 변화되는 특이한 부분들이 생성되는 것을 관찰하였다. 이러한 RNA ediring은 주로 exon 1과 exon 4 에 많이 일어나며, 염기 치환되는 부분들은 에서늬 NAD4유전자의 RNA edting site들과 일피하지 않은 점으로 미루어 보아 RNA editing 현상은 무작의로 생성된다고 본다.된다고 본다.

• 생명과학회지
• /
• 제7권3호
• /
• pp.167-171
• /
• 1997

번역개시 및 인산화의 조절에 관여하는 RNA와 단백질의 결합 및 인식기작을 연구하기 위해서[$\alpha$^{-32}$P] UMP-labeled HIV Rev-responsive element(RRE) RNA를 이용한 affinity screening에 이해서 Hela ZA-PII cDNA library로부터 이중선RNA결합단백질의 cDNA (RBFII)를 분리하였다. RBFII의 cDNA에 대한 염기서열을 결정하였으며 기존에 연구된 바 있는 RBFII(RBF 또는 TRBP로 보고되었으며 본 연구에서는 RBFII와 구분하기 위해 RBFI으로 명명)과 대부분의 경우 공통적인 ORF를 지니는 것으로 나타났다. 그러나 5’말단에서는 공통적인 ORF 가 RBFI의 경우 21개의 아미노산을 의미하는 63 nt가 Lac-Z의 N-말단에 연결된데 비해서 특이한 46개 아미노기를 의미하는 138nt가 존재함이 밝혀졌다. 5’-말단에 처음 나타나는 ATG 및 부근의 염기서열을 분석해 볼 때 양 cDNA는 5’말단이 완전하지 않은 것으로 사료된다.