Factors influencing transient gene expression in electroporated tall fescue protoplasts

For the rapid establishment of optimal conditions for a genetic transformation system for tall fescue, several factors influencing transient gene expression were studied in protoplasts, after the reporter β-glucuronidase gene was introduced by electroporation. In a time-course study of transient gene expression, GUS activity peaked at 24 h after electroporation. Among the different field strength conditions tested, maximum GUS activity was observed at 750 V/cm. Increases in the amount of plasmid DNA to 80 μg/ml led to increased GUS activity. GUS activities increased in linear fashion with increasing protoplast densities up to 2 × 10⁶/ml. Age of suspension cells from which protoplasts were derived influenced transient expression with maximum GUS activity obtained in 3- and 5-day-old suspensions. These results show that monocot and dicot protoplasts respond similarly in electroporation.     

Effects of small interfering RNAs targeting MAPK1 on gene expression profile in HeLa cells as revealed by microarray analysis

The mitogen activated protein kinases (MAPK) signaling cascade plays an important role in cell life. We proved that small interfering RNAs targeting MAPK1 (siRNA-2) could inhibit HeLa cell growth, but the effects of siRNA-2 on gene expression profile were unclear. Using Affymetrix GeneChip HG-U133A 2.0, we identified the long-term changes for 48h in gene expression profile in HeLa cell treated by siRNA-2. The results showed that expressions of 181 genes were altered by siRNA-2 and were divided into two groups: (i) one group showed downregulation by siRNA-2, including the proliferation associated genes, small G protein, cytoskeleton associated protein and extracellular matrix associated protein; and (ii) the other group showed upregulation by siRNA-2, including interferon response genes, OAS family, TRIM family and apoptosis associated genes. The results of Real-time quantitative PCR for MAPK1, NUP188, P38, STAT1, STAT2, MPL and OAS1 were consistent with that of gene chip. Two networks were found to react substantially to the downregulation of MAPK1 by siRNA-2. One of the networks regulates cell growth through cell-cycle control, apoptosis and cytoskeleton. The other network is related to interferon-like response. Our findings suggest that siRNA-mediated downregulation of MAPK1 could be an attractive strategy for cancer therapy.     

Effects of U0126 and fibroblast growth factor on gene expression profile in Ciona intestinalis embryos as revealed by microarray analysis

Fibroblast growth factor (FGF) induces the notochord and mesenchyme in ascidian embryos, via extracellular signal-regulated kinase (ERK) that belongs to the mitogen-activated protein kinase (MAPK) family. A cDNA microarray analysis was carried out to identify genes affected by an inhibitor of MAPK/ERK kinase (MEK), U0126, in embryos of the ascidian Ciona intestinalis. Data obtained from the microarray and in situ hybridization suggest that the majority of genes are downregulated by U0126 treatment. Genes that were downregulated in U0126-treated embryos included Ci-Bra and Ci-Twist-like1 that are master regulatory genes of notochord and mesenchyme differentiation, respectively. The plasminogen mRNA was downregulated by U0126 in presumptive endoderm cells. This suggests that a MEK-mediated extracellular signal is necessary for gene expression in tissues whose specification does not depend on cell-to-cell interaction. Among 85 cDNA clusters that were not affected by U0126, 30 showed mitochondria-like mRNA localization in the nerve cord/muscle lineage blastomeres in the equatorial region. The expression level and asymmetric distribution of these mRNA were independent of MEK signaling.     

Global analysis of hematopoietic and vascular endothelial gene expression by tissue specific microarray profiling in zebrafish

In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp)(y1) zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP+ versus GFP- cells isolated from Tg(fli1:egfp)(y1) embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos.     

Laboratory diet profoundly alters gene expression and confounds genomic analysis in mouse liver and lung

Nutritional studies in laboratory animals have long shown that various dietary components can contribute to altered gene expression and metabolism, but diet alone has not been considered in whole animal genomic studies. In this study, global gene expression changes in mice fed either a non-purified chow or a purified diet were investigated and background metal levels in the two diets were measured by ICP-MS. C57BL/6J mice were raised for 5 weeks on either the cereal-based, non-purified LRD-5001 diet or the purified, casein-based AIN-76A diet, as part of a larger study examining the effects of low dose arsenic (As) in the diet or drinking water. Affymetrix Mouse Whole Genome 430 2.0 microarrays were used to assess gene expression changes in the liver and lung. Microarray analysis revealed that animals fed the LRD-5001 diet displayed a significantly higher hepatic expression of Phase I and II metabolism genes as well as other metabolic genes. The LRD-5001 diet masked the As-induced gene expression changes that were clearly seen in the animals fed the AIN-76A diet when each dietary group was exposed to 100 ppb As in drinking water. Trace metal analysis revealed that the LRD-5001 diet contained a mixture of inorganic and organic As at a total concentration of 390 ppb, while the AIN-76A diet contained approximately 20 ppb. These findings indicate that the use of non-purified diets may profoundly alter observable patterns of change induced by arsenic and, likely, by other experimental treatments, particularly, altering gene and protein expression.     

Arabidopsis gene expression changes during cyst nematode parasitism revealed by statistical analyses of microarray expression profiles

With the availability of microarray technology, the expression profiles of thousands of genes can be monitored simultaneously to help determine the mechanisms of these biological processes. We conducted Affymetrix GeneChip microarray analyses of the Arabidopsis-cyst nematode interaction and employed a statistical procedure to analyze the resultant data, which allowed us to identify significant gene expression changes. Quantitative real-time RT-PCR assays were used to confirm the microarray analyses. The results of the expression profiling revealed 128 genes with altered steady-state mRNA levels following infection by the sugar beet cyst nematode (Heterodera schachtii; BCN), in contrast to only 12 genes that had altered expression following infection by the soybean cyst nematode (H. glycines; SCN). The expression of these 12 genes also changed following infection by BCN, i.e. we did not identify any genes regulated exclusively by SCN. The identification of 116 genes whose expression changes during successful cyst nematode parasitism by BCN suggests a potential involvement of these genes in the infection events starting with successful syncytium induction. Further characterization of these genes will permit the formulation of testable hypotheses to explain successful cyst nematode parasitism.     

Common gene expression strategies revealed by genome-wide analysis in yeast

Background  

Gene expression is a two-step synthesis process that ends with the necessary amount of each protein required to perform its function. Since the protein is the final product, the main focus of gene regulation should be centered on it. However, because mRNA is an intermediate step and the amounts of both mRNA and protein are controlled by their synthesis and degradation rates, the desired amount of protein can be achieved following different strategies.     

Chromosome-specific spatial periodicities in gene expression revealed by spectral analysis

Recent years have seen an unprecedented surge of research activity in studies of gene expression. This extensive work, however, has been almost uniformly focused on genome-wide gene expression and has largely ignored the fundamental fact that every gene has a specific chromosome location. We propose a novel method of spectral analysis for detecting hidden periodicities in gene expression signals ordered along the length of each chromosome. Using this method, we have discovered that each chromosome in rodents and humans has a unique periodic pattern of gene expression. The uncovered spatial periodicities in gene expression are tissue-specific in the sense that the largest differences in humans were observed between two normal tissues (brain and mammary gland) as well as between their tumor counterparts (glioma and breast cancer). The smallest differences resulted from the comparison of tumors (glioma and breast cancer) with their normal counterparts. All such effects do not extend to all chromosomes but are limited to only some of them. The estimated periods and amplitudes are identical for the genes located on the positive and negative DNA strands. While precise molecular mechanisms of chromosome-specific periodicities in gene expression have yet to be unraveled, their universal presence in different tissues adds another dimension to the current understanding of the genome organization.     

Paternal high-fat diet alters triglyceride metabolism-related gene expression in liver and white adipose tissue of male mouse offspring

Obesity is a major public health problem, and its prevalence is progressively increasing worldwide. In addition, accumulating evidence suggests that diverse nutritional and metabolic disturbances including obesity can be transmitted from parents to offspring via transgenerational epigenetic inheritance. The previous reports have shown that paternal obesity has profound impacts on the development and metabolic health of their progeny. However, little information is available concerning the effects of paternal high-fat diet (HFD) exposure on triglyceride metabolism in the offspring. Therefore, we investigated the effects of paternal HFD on triglyceride metabolism and related gene expression in male mouse offspring. We found that paternal HFD exposure significantly increased the body weight, liver and epididymal white adipose tissue (eWAT) weights, and liver triglyceride content in male offspring, despite consuming control diet. In addition, paternal HFD exposure had induced changes in the mRNA expression of genes involved in lipid and triglyceride metabolism in the liver and eWAT. These findings indicate transgenerational inheritance from the paternal metabolic disturbance of triglyceride and support the effects of paternal lifestyle choices on offspring development and health later in life.     

Sexual dimorphism of rat liver gene expression: regulatory role of growth hormone revealed by deoxyribonucleic Acid microarray analysis

GH has diverse physiological actions and regulates the tissue-specific expression of numerous genes involved in growth, metabolism, and differentiation. Several of the effects of GH on somatic growth and gene expression are sex dependent and are regulated by pituitary GH secretory patterns, which are sexually differentiated. The resultant sex differences in plasma GH profiles are particularly striking in rodents and are the major determinant of sex differences in pubertal body growth rates and the expression in liver of several cytochrome P450 (CYP) enzymes that metabolize steroids, drugs, and environmental chemicals of importance to endocrinology, pharmacology, and toxicology. DNA microarray analysis was used to identify rat liver-expressed genes that show sexual dimorphism, and to ascertain the role of GH as a regulator of their sexually dimorphic expression. Adult male and female rats were untreated or were treated with GH by 7-d continuous infusion using an Alzet osmotic minipump. Poly(A) RNA was purified from individual livers and Cy3- and Cy5-labeled cDNA probes cohybridized to Pan Rat Liver and 5K Rat Oligonucleotide microarrays representing 5889 unique rat genes. Analysis of differential gene expression profiles identified 37 liver-expressed, female-predominant genes; of these, 27 (73%) were induced by continuous GH treatment of male rats. Moreover, only three of 30 genes up-regulated in male rat liver by continuous GH treatment did not display female-dominant expression. Further analysis revealed that 44 of 49 male-predominant genes (90%) were down-regulated in the livers of continuous GH-treated male rats compared with untreated male rats, whereas only five of 49 genes that were down-regulated in male rats by continuous GH treatment were not male dominant in their expression. Real-time PCR analysis applied to a sampling of 10 of the sexually dimorphic genes identified in the microarray analysis verified their sex- and GH-dependent patterns of regulation. Taken together, these studies establish that GH-regulated gene expression is the major mechanistic determinant of sexually dimorphic gene expression in the rat liver model.     

Differential gene expression in the developing human macula: microarray analysis using rare tissue samples

The macula is a unique and important region in the primate retina that achieves high resolution and color vision in the central visual field. We recently reported data obtained from microarray analysis of gene expression in the macula of the human fetal retina (Kozulin et al., Mol Vis 15:45–59, 1). In this paper, we describe the preliminary analyses undertaken to visualize differences and verify comparability of the replicates used in that study, report the differential expression of other gene families obtained from the analysis, and show the reproducibility of our findings in several gene families by quantitative real-time PCR.     

Co-ordinated programme of gene expression during asexual intraerythrocytic development of the human malaria parasite Plasmodium falciparum revealed by microarray analysis

Plasmodium falciparum is a protozoan parasite responsible for the most severe forms of human malaria. All the clinical symptoms and pathological changes seen during human infection are caused by the asexual blood stages of Plasmodium. Within host red blood cells, the parasite undergoes enormous developmental changes during its maturation. In order to analyse the expression of genes during intraerythrocytic development, DNA microarrays were constructed and probed with stage-specific cDNA. Developmental upregulation of specific mRNAs was found to cluster into functional groups and revealed a co-ordinated programme of gene expression. Those involved in protein synthesis (ribosomal proteins, translation factors) peaked early in development, followed by those involved in metabolism, most dramatically glycolysis genes. Adhesion/invasion genes were turned on later in the maturation process. At the end of intraerythrocytic development (late schizogony), there was a general shut-off of gene expression, although a small set of genes, including a number of protein kinases, were turned on at this stage. Nearly all genes showed some regulation over the course of development. A handful of genes remained constant and should be useful for normalizing mRNA levels between stages. These data will facilitate functional analysis of the P. falciparum genome and will help to identify genes with a critical role in parasite progression and multiplication in the human host.