Expression profiling by oligonucleotide microarrays spotted on coated polymer slides

We have developed a ready-to-spot polymer microarray slide, which is coated with a uniform layer of reactive electrophilic groups using anthraquinone-mediated photo-coupling chemistry. The slide coating reduces the hydrophobicity of the native polymer significantly, thereby enabling robust and efficient one-step coupling of spotted 5' amino-linked oligonucleotides onto the polymer slide. The utility of the coated polymer slide in gene expression profiling was assessed by fabrication of spotted oligonucleotide microarrays using a collection of 5' amino-linked 70-mer oligonucleotide probes representing 96 yeast genes from Operon. Two-colour hybridizations with labelled cDNA target pools derived from standard grown and heat-shocked wild type yeast cells could reproducibly measure heat shock induced expression of seven different heat shock protein (HSP) genes. Moreover, the observed fold changes were comparable to those reported previously using spotted cDNA arrays and high-density 25-mer oligonucleotide arrays from Affymetrix. The low hybridization signals obtained from the DeltaSSA4 mutant cDNA target, together with the high signal detected in two-colour hybridizations with heat-shocked wild type yeast relative to the DeltaSSA4 mutant strain implies that unspecific binding of cDNA target to the SSA4-specific 70-mer oligonucleotide probes is negligible. Combined, our results indicate that the coated polymer microarray slide represents a robust and cost-effective array platform for pre-spotted oligonucleotide arrays.     

Genome-wide analysis of lipoprotein expression in Escherichia coli MG1655

To gain insight into the cell envelope of Escherichia coli grown under aerobic and anaerobic conditions, lipoproteins were examined by using functional genomics. The mRNA expression levels of each of these genes under three growth conditions--aerobic, anaerobic, and anaerobic with nitrate--were examined by using both Affymetrix GeneChip E. coli antisense genome arrays and real-time PCR (RT-PCR). Many genes showed significant changes in expression level. The RT-PCR results were in very good agreement with the microarray data. The results of this study represent the first insights into the possible roles of unknown lipoprotein genes and broaden our understanding of the composition of the cell envelope under different environmental conditions. Additionally, these data serve as a test set for the refinement of high-throughput bioinformatic and global gene expression methods.     

Development of a toxicological gene array and quantitative assessment of this technology

High-density arrays of DNA bound to solid substrates offer a powerful approach to identifying changes in gene expression in response to toxicants. While DNA arrays have been used to explore qualitative changes in gene regulation, less attention has focused on the quantitative aspects of this technology. Arrays containing expressed sequence tags for xenobiotic metabolizing enzymes, proteins associated with glutathione regulation, DNA repair enzymes, heat shock proteins, and housekeeping genes were used to examine gene expression in response to beta-naphthoflavone (beta-NF). Upregulation of cytochrome P4501a1 (Cyp1a1) and 1a2 in mouse liver was maximal 8 h after beta-NF administration. Significant upregulation of Cyp1a2 was noted at beta-NF doses as low as 0.62 and 1.2 mg/kg when gene expression was measured by microarray or Northern blotting, respectively. Maximal Cyp1a2 induction is 5-fold by Northern analysis and 10-fold by microarray. Induction of Cyp1a1 was 15- and 20-fold by Northern and microarray analysis, respectively. The coefficient of variation for spot to spot and slide to slide comparisons was <15%; this variability was smaller than interanimal variability (18-60%). Comparison of mRNA expression in control animals indicated that there are differences in labeling/detection associated with Cy3/Cy5 dyes; accordingly, experiments must include methods for establishing baseline signals for all genes. We conclude that the dynamic range and sensitivity of DNA microarrays on glass slides is comparable to Northern blotting analysis and that variability of the data introduced during spotting and hybridization is less than the interanimal variability.     

Solute transport proteins and the outer membrane protein NmpC contribute to heat resistance of Escherichia coli AW1.7

This study aimed to elucidate determinants of heat resistance in Escherichia coli by comparing the composition of membrane lipids, as well as gene expression, in heat-resistant E. coli AW1.7 and heat-sensitive E. coli GGG10 with or without heat shock. The survival of E. coli AW1.7 at late exponential phase was 100-fold higher than that of E. coli GGG10 after incubation at 60°C for 15 min. The cytoplasmic membrane of E. coli AW1.7 contained a higher proportion of saturated and cyclopropane fatty acids than that of E. coli GGG10. Microarray hybridization of cDNA libraries obtained from exponentially growing or heat-shocked cultures was performed to compare gene expression in these two strains. Expression of selected genes from different functional groups was quantified by quantitative PCR. DnaK and 30S and 50S ribosomal subunits were overexpressed in E. coli GGG10 relative to E. coli AW1.7 upon heat shock at 50°C, indicating improved ribosome stability. The outer membrane porin NmpC and several transport proteins were overexpressed in exponentially growing E. coli AW1.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of membrane properties confirmed that NmpC is present in the outer membrane of E. coli AW1.7 but not in that of E. coli GGG10. Expression of NmpC in E. coli GGG10 increased survival at 60°C 50- to 1,000-fold. In conclusion, the outer membrane porin NmpC contributes to heat resistance in E. coli AW1.7, but the heat resistance of this strain is dependent on additional factors, which likely include the composition of membrane lipids, as well as solute transport proteins.     

Gene expression changes associated with cytotoxicity identified using cDNA arrays

In order to investigate gene expression changes associated with cytotoxicity, we used cDNA arrays to monitor the expression of over 5,000 genes in response to toxic stress in the HepG2 liver cell line. Cells were treated with cytotoxic doses of acetaminophen, caffeine or thioacetamide for nine time points ranging from 1 to 24 h. Samples of mRNA from each time point were used to prepare radiolabeled cDNA, which was hybridized to nylon-membrane-based cDNA arrays. High-stringency washes were applied to reduce cross-hybridization. Analysis of spot intensities revealed that each compound led to approximately 150-250 gene expression changes that were sustained over at least three adjacent time points. The affected genes could be classified into clusters based on their temporal patterns of differential expression. A common set of 44 genes showed similar expression changes in response to all three compounds. Of these changes, 90% could be confirmed by quantitative RT-PCR analysis. The results indicate that detailed array-based time-course studies, coupled with a sensitive and highly specific confirmation assay, provide a powerful means of identifying cytotoxicity-associated gene expression changes. Electronic Publication     

Comparison of gene expression between left atria and left ventricles from non-diseased humans

We examine the reliability and accuracy of gene array technology in analyzing differences in gene expression between human non-diseased left atrium and left ventricle. We have used cDNA gene arrays and validated those data by carefully designed quantitative real-time polymerase chain reaction (PCR). We have identified pitfalls using cDNA gene array technology based on comparisons with other gene array studies and with changes reported for the levels of expression of the genes corresponding to these cDNAs. The high error rate reported here underscores the cautionary comments reported by others in this field.     

Analysis of reading frame and expressional regulation of randomly selected promoter-proximal genes in Escherichia coli

The expression of seventy-seven randomly cloned genes of Escherichia coli was examined following a variety of treatments including heat shock, glucose starvation, phosphate starvation, ammonium starvation or osmotic shock, with the aid of lacZ reporter gene protein fusions on multicopy plasmids. Two of 77 genes (amr and yigL) had not previously been identified as protein encoding open-reading frames (ORFs) in annotations of the E. coli genome database. Thirteen genes exhibited significant changes in expression in response to at least one of the treatments, and six of them appeared to be controlled by more than one sigma (sigma) factor of RNA polymerase. This study thus allows us not only to identify the reading frame of the genomic genes but also to support the hypothesis earlier proposed that a significant proportion of genes in E. coli are involved in adaptations to various stresses to which the organism is likely to be exposed in the environment.     

Expression levels of heat shock factors are not functionally coupled to the rate of expression of heat shock genes

The expression patterns of two mammalian heat shock factors (HSFs) were analysed in cell systems known to reflect an altered heat shock response. For being able to discriminate between the two closely related factors HSF 1 and HSF 2, specific cDNA sequences were cloned and used to generate antisense RNAs as hybridization probes. In general, in various cell lines expression of the two heat shock factors was clearly different. These expression patterns of the HSF genes were not influenced by retinoic acid-induced differentiation of human NT2 and mouse F9 teratocarcinoma cells. Generally, HSF 2 expression was extremely low, whereas the significantly higher expression of HSF 1 revealed cell specific differences. The highest expression rates of both HSFs were observed in 293 cells. To examine whether these high levels are involved in the constitutive expression of heat shock genes in these cells, we analysed the binding pattern of 293 cell proteins to the heat shock elements (HSEs). As with other cells, HSE-binding activity in 293 cells was only observed after heat shock treatment. This points to an HSE-independent way for high level expression of heat shock genes in these cells.     

对虾白斑综合症病毒重组cDNA克隆的构建与分析

取经人工注射感染了对虾白班综合症病毒40-45h的凡纳对虾鳃组织,分离mRNA,以mRNA为模板合成双链cDNA,并克隆于PUC质粒的Not I/Sal I位点,构建了1000余株对虾感染后期鳃细胞的重组cDNA克隆,重组质粒经PCR鉴定插入片段,DNA斑点杂交分析目的片段,测定了20株对虾白斑综合症病毒的重组cDNA克隆的末端DNA序列,并对其进行了包含存在的开放阅读框架,启动区上游序列、编码产物的特性等分析。结果显示,PCR产物在0.3-1.6kb之间;大于1kb的克隆中有31.8%的克隆为白斑综合症病毒的重组cDNA克隆。已测序的不包含同源序列的13株克隆中含有14个开放阅读框,其中11个上游可检出启动子基序,4个可检出启动子调制元件,ORF转译产物的特性基序分析显示,有2个ORFs可检出锌指基序,3个ORFs可检出亮氮酸拉链基序,2个ORFs可检出NTP结合基序,未检定核定位信号基序。     

Inflammatory gene profiles in gastric mucosa during Helicobacter pylori infection in humans

Helicobacter pylori infection is associated with an inflammatory response in the gastric mucosa, ultimately leading to cellular hyperproliferation and malignant transformation. Hitherto, only expression of a single gene, or a limited number of genes, has been investigated in infected patients. cDNA arrays were therefore used to establish the global pattern of gene expression in gastric tissue of healthy subjects and of H. pylori-infected patients. Two main gene expression profiles were identified based on cluster analysis. The data obtained suggest a strong involvement of selected Toll-like receptors, adhesion molecules, chemokines, and ILs in the mucosal response. This pattern is clearly different from that observed using gastric epithelial cell lines infected in vitro with H. pylori. The presence of a "Helicobacter-infection signature," i.e., a set of genes that are up-regulated in biopsies from H. pylori-infected patients, could be derived from this analysis. The genotype of the bacteria (presence of genes encoding cytotoxin-associated Ag, vacuolating cytotoxin, and blood group Ag-binding adhesin) was analyzed by PCR and shown to be associated with differential expression of a subset of genes, but not the general gene expression pattern. The expression data of the array hybridization was confirmed by quantitative real-time PCR assays. Future studies may help identify gene expression patterns predictive of complications of the infection.     

番茄线粒体和内质网小分子热激蛋白基因的分子克隆

以热激处理的番茄（Lycopersicon esculentum Mill．）花为实验材料,构建了cDNA库,运用ＲＴ－ＰＣＲ方法克隆番茄粒体和内质网小分子热激蛋白cDNA,利用这两个保守区片段为探针,筛选cDNA库,获得线粒体和内质网小分子热激蛋白全序列cDNA。;通过分析线粒体和内质网小分子热激蛋白基因对温度的反应,发现小分子热激蛋白基因在番茄花中的热激应答温度低于它们在叶片中的热激应答温度,并且番茄叶片中的线粒体小分子热激蛋白基因还具有低温应答特性。对线粒体和内质网小分子热激蛋白基因的分子结构特点,小分子热激蛋白基因在番茄花中的特别热激应答温度的调控机理以及线粒体小分子热激蛋白的基因在中片中的低温度应答成因进行了讨论。     

Molecular Cloning the Gene of Small Heat Shock Protein in the Mitochondria and Endoplasmic Reticulum of Tomato

A cDNA library was constructed with the heat shocked tomato (Lycopersicon esculentum Mill.) flowers and then was screened with the probes of mitochondrial and endoplasmic reticulum conservative regions that were cloned by using RT-PCR. The complete cDNAs of mitochondrial and endoplasmic reticulum small heat shock protein ( shsp ) were selected out from the cDNA library. Furthermore, the temperature responses of these shsp genes were determined. Northern hybridization showed that the heat response temperatures of both genes in tomato flower were lower than that in leaf and that mitochondria shsp in leaf was cold-inducible. In this paper, the molecular features of the cloned genes, the causes of the uncommon heat response temperatures of sHSP in flower and the cold inducible character of mitochondria shsp gene in leaf were discussed. 