Msp-1 polymorphism detected with a cDNA probe for the P-450 I family on chromosome 15
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目的研究极低频电磁场暴露对小鼠脑和肝组织c-fos mRNA水平的影响.方法将小鼠暴露于50 Hz、0.2 mT及50 Hz、6.0 mT的电磁场中,持续2周或4周;采用竞争性RT-PCR方法检测小鼠脑和肝脏c-fos mRNA水平.结果50 Hz、0.2 mT及50 Hz、6.0 mT电磁场暴露2周后,小鼠脑组织c-fos mRNA的水平上升为(0.017 8±0.007 6)amol/120 ng cDNA和(0.009 2±0.004 2)amol/120 ng cDNA,与对照组[(0.001 2±0.000 5)amol/120 ng cDNA]比较,差异有显著性(P＜0.05);肝组织c-fos mRNA的水平上升为(0.0117±0.005 5)amol/120ng cDNA和(0.014 8±0.016 2)amol/120 ng cDNA,与对照组[(0.000 5±0.000 5)amol/120 ng cDNA]比较,差异有显著性(P＜0.05).0.2 mT和6.0 mT电磁场暴露4周后,小鼠脑组织c-fos mRNA的水平上升为(0.010 0±0.005 4)amol/120 ng cDNA和(0.019 8±0.007 9)amol/120 ng cDNA,与对照组[(0.001 5±0.000 8)amol/120ng cDNA]比较,差异有显著性(P＜0.05);肝组织c-fos mRNA的水平分别上升为(0.017 3±0.012 2)amol/120ng cDNA和(0.013 3±0.009 0)amol/120 ng cDNA,而对照组无表达.结论50 Hz电磁场暴露引起小鼠脑和肝脏c-fos基因转录水平明显上调.
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Simultaneous amplification of 5′ and 3′ cDNA ends based on template-switching effect and inverse PCR
A new approach for simultaneously amplifying the 5′ and 3′ ends of a desired cDNA is described. The method combines the template-switching effect with inverse PCR, which generates the flanking 5′ and 3′ regions of a certain cDNA with very low or no background. It requires only minimal amounts of total RNA for the synthesis of first-strand cDNA, while the same cDNA can be used to amplify flanking sequences of any cDNA species present in the sample. This method is reliable and easy to perform, which is very useful for isolating cDNA species of rare transcripts.
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A number of cDNA clones in summary encoding 700 amino acid residues from the N-end of rat liver elongation factor 2 (EF-2) and including 49 nucleotides of the 5'-untranslated mRNA region have been obtained. EF-2 cDNA clones were isolated from gradually constructed small (1000-5000 clones) specific cDNA libraries using the primer extension method for synthesis of the first cDNA chain. The complete primary structure of cDNA and protein EF-2 from rat liver was derived taking into account the primary structure of the 3'-terminal region EF-2 cDNA previously reported [(1986) Proc. Natl. Acad. Sci. USA 83, 4978-4982]. Comparison of this cDNA with hamster cDNA has shown that (i) the base sequences had a 89.7% homology while that of the 5'-untranslated region was 73%; (ii) there are two amino acid replacement in rat liver EF-2 as compared with hamster EF-2.
Protein primary structure
Elongation factor
Primer (cosmetics)
Primer extension
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To study the effects of extremely low frequency electromagnetic fields (ELF EMFs) on c-fos gene expression in mouse brain and liver tissues.Mice were exposed to 50 Hz sinusoidal 0.2 mT or 6.0 mT electromagnetic field for 2 weeks or 4 weeks. Competitive RT-PCR method was used to measure c-fos mRNA level.After exposure to 0.2 mT or 6.0 mT field for 2 weeks, c-fos mRNA levels in brain tissue [(0.0178 +/- 0.0076) amol/120 ng cDNA and (0.0092 +/- 0.0042) amol/120 ng cDNA respectively] were higher than that of control level [(0.0012 +/- 0.0005) amol/120 ng cDNA] (P < 0.05). In liver tissue c-fos mRNA levels [(0.0117 +/- 0.0055) amol/120 ng cDNA and (0.0148 +/- 0.0162) amol/120 ng cDNA respectively] were also higher than that of control level [(0.0005 +/- 0.0005) amol/120 ng cDNA] (P < 0.05). After exposure to 0.2 mT or 6.0 mT field for 4 weeks, c-fos mRNA levels in brain tissue [(0.0100 +/- 0.0054) amol/120 ng cDNA and (0.0198 +/- 0.0079) amol/120 ng cDNA respectively] were higher than that of control level [(0.0015 +/- 0.0008) amol/120 ng cDNA] (P < 0.05). In liver tissue the exposure induced much higher expression level [(0.0173 +/- 0.0122) amol/120 ng cDNA and (0.0133 +/- 0.0090) amol/120 ng cDNA respectively] while no expression was found in the control.Exposure to 50 Hz electromagnetic fields may induce up-regulation of c-fos transcription in mouse brain and liver tissue.
Liver tissue
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Abstract We constructed a “cDNA bank” of human colorectal cancer and surrounding normal tissues with our unique mRNA assay system. Total nucleic acids extracted from patients’ tissues were applied to 96-well plates, where poly(dT) sequences of oligonucleotides were immobilized. After hybridization, the cDNA was reverse-transcribed on the plate with the captured mRNA as a template, followed by synthesis of double-stranded (ds) cDNA. The resulting sense cDNA was removed from the plate, then used in PCR for analysis of various genes. The sense strand of the cDNA was repeatedly synthesized by using the immobilized antisense cDNA as a template even from plates used once and stored at 4 °C for as long as 6 months. Furthermore, the results of PCR could be easily compared among different specimens if the same amount of total mRNA were applied to the plate for the ds cDNA synthesis. This demonstrated that the cDNA bank constructed from clinical materials provides almost unlimited supplies of cDNA for multiple gene analysis of cancer.
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Molecular probe
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水母雪莲（Saussurea medusa Maxim）为名贵珍稀中药材，其主要药用成分为类黄酮，尤其是3-脱氧类黄酮。目前关于雪莲的研究主要集中在采用细胞培养生产类黄酮等方面，但对于雪莲类黄酮生物合成的分子机制了解甚少，极大限制了这一珍贵资源的利用。本研究采用水母雪莲红色系愈伤组织及悬浮细胞为材料，构建cDNA文库，从中克隆水母雪莲类黄酮次生代谢中的相关基因并对这些基因进行了深入的生物信息学分析、转基因研究初步确定其功能，以期了解雪莲类黄酮次生代谢的分子机制，为提高类黄酮的合成奠定基础。主要结果如下： 1. 成功地构建了水母雪莲红色系愈伤组织与悬浮细胞cDNA文库，原始文库滴度达到4×106pfu/ml，扩增文库滴度接近1011 pfu/ml，重组率达98%。PCR检测插入片段，均在0.5kb到3kb之间，1kb以上占62%。从文库中检测到了chs、dfr及Myb转录因子SmP，文库覆盖度达到要求且为PCR筛选文库提供了可能。 2. 采用部分简并引物，通过RT-PCR克隆了水母雪莲查尔酮异构酶基因Smchi特异探针，并根据这一探针序列设计特异引物，采用TD-PCR法筛选cDNA文库，获得Smchi cDNA序列，全长831bp，编码一个232氨基酸残基的蛋白。根据cDNA序列克隆了Smchi DNA序列，结果表明Smchi基因无内含子。Smchi cDNA序列与翠菊chi基因高度同源，ORF区域同源性高达84%，但推测氨基酸序列则只有79.3%。Smchi mRNA具有复杂的二级结构。SmCHI具有典型的Chalcone结构域，其二级结构与苜蓿CHI蛋白十分相似，7个α-螺旋与8个延伸链由随机结构联系起来。但其活性中心的第三个关键氨基酸残基N115为M115所取代，这一取代可能导致该蛋白无生物活性，也可能使它具有一般CHI不同的功能。构建Smchi正义、反义真核表达载体，通过农杆菌介导导入烟草，获得转正义、反义Smchi基因的烟草。转基因烟草花色未改变，但叶片总黄酮发生了显著的变化，50%转正义基因烟草总黄酮含量显著提高，最高比对照提高6倍，70%转反义基因烟草总黄酮含量显著下降，最多达85.1%，初步证明Smchi具有功能，并能有效调控烟草类黄酮次生代谢。因此，SmCHI可能是不同于已知CHI的一类新的CHI蛋白，它催化的反应可能与花色素合成无关，其反应机制也可能有所不同。 3. 伴随Smchi的克隆获得了一个黄烷酮3-羟化酶类似基因Smf3h的cDNA，全长1334bp，编码一个343aa的蛋白。根据这一cDNA序列克隆了Smf3h DNA序列，全长1630bp，结果表明该基因由4个外显子和3个内含子组成。Smf3h mRNA具有十分复杂的二级结构。 推测蛋白氨基酸同源性分析表明，SmF3H属于2OG-FeII_Oxy家族，与同一家族的的颠茄H6H的同源性为45%，与拟南芥F3H的同源性为40%，但对SmF3H、典型F3H及典型H6H推测蛋白二级结构、活性中心关键氨基酸残基的位置与相对距离、软件进行功能预测分析，发现SmF3H与F3H更相似。构建Smf3h的正义与反义真核表达载体，通过农杆菌介导导入烟草，但只获得一批转正义基因的烟草，反义基因导致烟草不能再生而未获得转反义基因烟草。转基因烟草花色未改变，叶片总黄酮也与对照相似，初步确认Smf3h与烟草类黄酮生物合成无关，而是一个既不属于f3h也不属于h6h的功能未确定的新基因。 4. 采用与克隆Smchi基因相似的方法，从cDNA文库中克隆了SmP基因cDNA，全长969bp，编码一个256 aa的蛋白质。根据cDNA序列克隆了SmP基因的DNA序列，结果表明，SmP基因无内含子。SmP基因cDNA 一级结构及mRNA二级结构预测分析表明，该基因A+T含量很高（63%），所形成二级结构以A-T配对为主，其稳定性可能较差。SmP推测蛋白序列具有R2R3-Myb转录因子的典型特征，在N-端具有两个Myb DNA-binding Domain，其二级结构与鸡Myb转录因子1A5J十分相似，与其他基因如水稻OsMYB、番茄ThMYB的同源区域主要集中在这一结构域，分别为71.3%和70.8%;C-端富含丝氨酸，与烟草NtMYB、葡萄VlMYB等类黄酮调控因子相似，都呈寡聚体分布，并具有相同的保守磷酸化位点S170与S206。构建SmP基因真核表达载体，通过农杆菌介导导入烟草，获得大量转基因烟草。转基因烟草花色未发生改变，但51%的转基因烟草叶片总黄酮含量都显著提高（0.5-6倍），表明SmP具有促进烟草类黄酮生物合成的功能，但所调控的支路与花色素合成无关。初步试验结果表明，转SmP基因烟草对蚜虫具有很高的抗性，可有效地抑制蚜虫在烟草上的生长，抑制率最高可达92%-100%。这一抗性与烟草中类黄酮的积累可能具有直接的联系，但还需要进一步的试验证明。
5. 与美国俄亥俄州立大学Erich Grotewold 博士实验室合作，完成了微型EST库50个克隆的测序并进行了分析，从中获得了水母雪莲花色素合酶基因SmANS及醛脱氢酶基因SmALDH的特异探针。根据SmANS特异探针设计引物，采用PCR从这50个克隆中筛选获得了SmANS的cDNA序列，全长1229bp，编码一个356aa的蛋白质。SmANS在cDNA水平上与同属的翠菊ANS基因高度同源，但同源区域集中在ORF区域，达到80%，mRNA 预测二级结构十分复杂;推测氨基酸序列与翠菊ANS同源性达到82.9%。SmANS属于2OG-FeII_Oxy家族，在2OG-FeII_Oxy结构域高度保守，与翠菊、甜橙ANS保守结构域同源性达到94%。预测蛋白二级结构以α-螺旋-β-折叠为主，由7个主螺旋和11个主β-折叠及随机结构连接而成，并具有2OG-FeII_Oxy家族活性中心的三个保守的组氨酸残基（His84、His235、His291）和一个天冬氨酸残基（Asp237）。 6. 根据微型EST库中获得的SmALDH特异探针设计引物，采用PCR从这50个克隆中筛选获得了SmALDH基因cDNA 序列，全长1664bp，编码一个491aa的蛋白质。SmALDH基因cDNA具有独特的碱基组成，3/-UTR富含A+T，占该区域碱基总量的80%，5/-UTR的A+T和G+C各占50%，比ORF区域（52%）还低，因此其mRNA二级结构中5/-UTR可以单独形成自身二级结构并且十分稳定，这可能影响基因的表达。这一现象在水稻、玉米等植物中也存在。SmALDH在cDNA水平上在ORF区域与拟南芥、藏红花、水稻等具有较高同源性，分别为64.03%、63.89%、63.72%，但在推测蛋白氨基酸序列水平上同源性反而较低，分别为54.9%、54.3%、54.0%。SmALDH缺少线粒体定位信号，为胞质醛脱氢酶，具有一个Aldedh 保守结构域，还具有与1OF7-H相似的以α-螺旋-β-折叠为主的二级结构，由10个主螺旋和15个主β-折叠及随机结构连接而成。由于ALDH在植物细胞乙醇发酵中具有解除醛类物质毒害的功能，因此SmALDH基因的克隆为改造细胞自身以适应发酵培养条件，解决水母雪莲细胞大规模培养中需氧问题提供了可能。
MYB
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目的研究极低频电磁场暴露对小鼠脑和肝组织c-fos mRNA水平的影响.方法将小鼠暴露于50 Hz、0.2 mT及50 Hz、6.0 mT的电磁场中,持续2周或4周;采用竞争性RT-PCR方法检测小鼠脑和肝脏c-fos mRNA水平.结果50 Hz、0.2 mT及50 Hz、6.0 mT电磁场暴露2周后,小鼠脑组织c-fos mRNA的水平上升为(0.017 8±0.007 6)amol/120 ng cDNA和(0.009 2±0.004 2)amol/120 ng cDNA,与对照组[(0.001 2±0.000 5)amol/120 ng cDNA]比较,差异有显著性(P＜0.05);肝组织c-fos mRNA的水平上升为(0.0117±0.005 5)amol/120ng cDNA和(0.014 8±0.016 2)amol/120 ng cDNA,与对照组[(0.000 5±0.000 5)amol/120 ng cDNA]比较,差异有显著性(P＜0.05).0.2 mT和6.0 mT电磁场暴露4周后,小鼠脑组织c-fos mRNA的水平上升为(0.010 0±0.005 4)amol/120 ng cDNA和(0.019 8±0.007 9)amol/120 ng cDNA,与对照组[(0.001 5±0.000 8)amol/120ng cDNA]比较,差异有显著性(P＜0.05);肝组织c-fos mRNA的水平分别上升为(0.017 3±0.012 2)amol/120ng cDNA和(0.013 3±0.009 0)amol/120 ng cDNA,而对照组无表达.结论50 Hz电磁场暴露引起小鼠脑和肝脏c-fos基因转录水平明显上调.
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This protocol describes how to prepare fluorescently labeled cDNA for hybridization to microarrays. It consists of two steps: first, a mixture of anchored oligo(dT) and random hexamers is used to prime amine-modified cDNA synthesis by reverse transcriptase using a modified deoxynucleotide with a reactive amine group (aminoallyl-dUTP) and an RNA sample as a template. Second, the cDNA is purified and exchanged into bicarbonate buffer so that the amine groups in the cDNA react with the dye N -hydroxysuccinimide (NHS) esters, covalently joining the dye to the cDNA. The dye-coupled cDNA is purified again, and the amount of dye incorporated per microgram of cDNA is determined.
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