中国红豆杉细胞紫杉醇合成期特异表达新基因TS1-FL的部分cDNA克隆

采用mRNA差异显示技术(DDRT—PCR)比较了悬浮培养的中国红豆杉细胞合成紫杉醇前后的基因表达差异,并从紫杉醇合成期细胞中分离出一个特异表达的cDNA克隆TS1．TS1长度为638bp,序列相似性分析结果表明它代表一新基因序列(Genbank登录号：AF426429),将此新基因命名为TS1-FL．开放读框分析结果表明Tsl拥有一个不完整的开放读框,蛋白质同源性检索没有发现与TS1翻译序列有较大同源性的蛋白质．对此基因的进一步研究可揭示它在紫杉醇生物合成中所扮演的角色．     

基于cDNA宏阵列的系统聚类分析猪发育阶段的基因表达谱

取杜洛克猪胚胎第33,45,55，65．75天的背最长肌样本．用cDNA Macroarray分析方法和聚类分析技术分析了327个EST在骨骼肌内不同发育阶段的基因表达谱．结果表明有98条EST在不同发育时期显著差异表达．第33天和第45天两阶段基因表达状态相似．第55天和第65天基因表达状态相似．而第75天的基因表达与第55天和第65天两个阶段的基因表达具有相近的聚类关系．表达状态相近，基因功能相似的基因大都被聚类在一起．     

人16周胎儿脑cDNA文库的构建及鉴定

本文从 16周的胎儿脑中抽提总 m RNA,经过一系列酶促反应后合成 c DNA,分级分离柱除去小片段 DNA后克隆到λgt10载体中 ,转染宿主菌 C60 0 hfl,文库包装效率为 4 .2× 10 6pfu/μg,得到的原始克隆数为 2 .1× 10 6,c DNA插入片段平均大于 1.2 kpb.根据已知序列设计引物 ,从这个 c DNA文库中扩增出血管内皮生长因子 ( VEGF)的全长 c DNA基因     

人nov基因全长cDNA克隆、测序及其组织表达谱分析

采用特异性mRNA富集技术，从人早期胚胎中得到人nov基因(novH)的cDNA克隆，序列分析表明，该基因cDNA序列由2389个碱基对组成，包含一段长1071bp的开放阅读框(ORF)，3′端含有加尾信号AATAAA和ply(A)尾巴。Northern杂交显示，在Hela细胞和肾上腺组织中有表达的novHmRNA，大小约为2．5kb；多组织RNA点杂交分析结果进一步表明，novH基因在肾上腺和主动脉中的表达水平相对较高，在成人肾、肝、肺和小肠组织中亦有少量的表达。     

Cholera toxin-induced modulation of gene expression: elucidation via cDNA microarray for rational cell-based sensor design

Cell-based biosensors utilize functional changes in cellular response to identify the biological threats in a physiological relevant manner. Cell-based sensors have been used for a wide array of applications including toxicological assessment and drug-screening. In this paper, we utilize DNA arrays to identify differential gene expression events induced by toxin exposure for the purpose of developing a reporter gene assay system compatible with insertion into a cell-based sensor platform. HT29, an intestine epithelial cell line, was used as a cell model to study the cholera toxin (CT)-induced host cell modulation using DNA array analysis. A false positive model was generated from analysis of housekeeping genes in untreated control experiments to characterize our system and to minimize the number of false positives in the data. Threshold probability scores (−3.72), which gives <0.02% false positives for up/down regulation from the false positive model, were used to identify 73 and 25 known genes/expression tag sequences (ESTs) that were up- and down-regulated, respectively, in cells exposed 23 nM of CT. Using quantitative multiplex PCR assay, the gene expression levels for several genes shown to be modulated according to the microarray experiments, such as apolipoprotein D (Apol D), E-cadherin, and cyclin A2, were confirmed. The differential expression of genes encoding cytochrome P450, glutathione transferase (GST), and MGAT2 were noteworthy and consistent with previous studies. Our study provides an approach to analyze cDNA microarray data with defined false positive rates. The utility of cDNA microarray information for the design of cell-based sensor using a reporter gene approach is discussed.     

结肠癌诱导分化基因表达差异的消减cDNA文库的建立

为保存和分析抑制性消减杂交 (SSH)获得的结肠癌经全反式维甲酸和 1 ,2 5-(OH) 2 D3联合诱导分化前、后的差异cDNA ,把消减产物与TA载体连接并转化到大肠杆菌中建立消减cDNA文库 .结果表明 :诱导前消减cDNA文库的容量为 1 .0 5× 1 0 4 pfu ,诱导后消减cDNA文库的容量为 1 .49× 1 0 4 pfu .本实验为进一步研究结肠癌的发病机制和诱导分化的机制提供了物质基础 .     

Electrochemical detection of a breast cancer susceptible gene using cDNA immobilized chitosan-co-polyaniline electrode

An electrochemical breast cancer biosensor based on a chitosan-co-polyaniline (CHIT-co-PANI) copolymer coated onto indium-tin-oxide (ITO) was fabricated by immobilizing the complementary DNA (cDNA) probe (42 bases long) associated with the breast cancer susceptible gene BRCA1. Both the CHIT-co-PANI/ITO and the cDNA/CHIT-co-PANI/ITO electrodes were characterized with Fourier transform infrared (FTIR) spectroscopy, atomic force microscopy (AFM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). For the cDNA/CHIT-co-PANI/ITO electrode, the amperometric current decreased linearly with an increasing logarithm of molar concentration of the single-stranded target DNA (ssDNA) within the range of 0.05-25 fmol. The bioelectrode exhibited a sensitivity of 2.104 μA/fmol with a response time of 16 s. The cDNA/CHIT-co-PANI/ITO electrode had a shelf life of about six months, even when stored at room temperature.     

Noise Analysis of Duplicated Data on Microarrays Using Mixture Distribution Modeling

We propose a technique for estimating gene expression values for duplicated data on cDNA microarrays. In the scatter plots, the distribution is constructed from a mixture of normal two-dimensional distributions, which represent fluctuations in gene expression values due to noise. An expectation-maximization (EM) algorithm is used for estimating the modeling parameters. The probability that duplicated data is shifted by noise is calculated using Bayesian estimation. Six data sets of rice cDNA microarray assays were used to test the proposed technique. Genes in the data sets were subjected to clustering based on probability of true value. Clustering successfully identified candidate genes regulated by circadian rhythms in rice.     

Gene Amplification and High-Level Expression of Human Pro-Urokinase cDNA in Chinese Hamster Ovary Cells

Human Pro-Urokinase (Pro-UK) is expressed in CHO-DHFR- cells at high efficiency by co-transfecting the mouse dhfr gene and Pro-UK cDNA under the control of the SV40 late promoter. After gene co-amplification, the product level could reach 2-3 μg/106 cells/24 h in the presence of 5×10-6 mol/ L MTX, and the levels can be further raised to 3. 5-4 μg/106 cells/24 h by PMA superinduction. The copy number of Pro-UK cDNA in the genomes of host cells is about 200-300 copies/cell. The Western blot analysis shows that the recombinant Pro-UK has similar molecular weight to its natural counterpart, and also the amidolytic activity of the product is determined by S-2444 assay.