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PubMed2007

Porcine kallikrein gene family: genomic structure, mapping, and differential expression analysis.

Kallikreins belong to a family of serine proteases that are widespread throughout living organisms, expressed in diverse tissue-specific patterns, and known to have highly diverse physiological functions. The 15 human and 24 mouse kallikreins have been implicated in pathophysiology of brain, kidney, and respiratory and reproductive systems and often are used as cancer biomarkers. To better elucidate the structure and evolutionary origin of this important gene family in the pig, we have constructed a contiguous BAC clone-derived physical map of the porcine kallikrein gene region and have fully sequenced a BAC clone containing 13 kallikrein genes, 11 of which are novel. Radiation hybrid mapping assigns this kallikrein-gene-rich region to porcine chromosome 6. Phylogenetic and percent identity plot-based analyses revealed strong structure and order conservation of kallikreins among four mammalian species. Reverse transcriptase-polymerase chain reaction-based expression analysis of porcine kallikreins showed a complex expression pattern across different tissues with the thymus being the only tissue expressing all 13 kallikrein genes. [The sequence data described in this paper has been submitted to GenBank under Accession No. AC149292].

Animals↗
PubMed2006

Automated target preparation for microarray-based gene expression analysis.

DNA microarrays have rapidly evolved toward a platform for massively paralleled gene expression analysis. Despite its widespread use, the technology has been criticized to be vulnerable to technical variability. Addressing this issue, recent comparative, interplatform, and interlaboratory studies have revealed that, given defined procedures for "wet lab" experiments and data processing, a satisfactory reproducibility and little experimental variability can be achieved. In view of these advances in standardization, the requirement for uniform sample preparation becomes evident, especially if a microarray platform is used as a facility, i.e., by different users working in the laboratory. While one option to reduce technical variability is to dedicate one laboratory technician to all microarray studies, we have decided to automate the entire RNA sample preparation implementing a liquid handling system coupled to a thermocycler and a microtiter plate reader. Indeed, automated RNA sample preparation prior to chip analysis enables (1) the reduction of experimentally caused result variability, (2) the separation of (important) biological variability from (undesired) experimental variation, and (3) interstudy comparison of gene expression results. Our robotic platform can process up to 24 samples in parallel, using an automated sample preparation method that produces high-quality biotin-labeled cRNA ready to be hybridized on Affymetrix GeneChips. The results show that the technical interexperiment variation is less pronounced than with manually prepared samples. Moreover, experiments using the same starting material showed that the automated process yields a good reproducibility between samples.

Animals↗
PubMed2004

Genome-wide gene expression analysis for induced ischemic tolerance and delayed neuronal death following transient global ischemia in rats.

Genome-wide gene expression analysis of the hippocampal CA1 region was conducted in a rat global ischemia model for delayed neuronal death and induced ischemic tolerance using an oligonucleotide-based DNA microarray containing 8,799 probes. The results showed that expression levels of 246 transcripts were increased and 213 were decreased following ischemia, corresponding to 5.1% of the represented probe sets. These changes were divided into seven expression clusters using hierarchical cluster analysis, each with distinct conditions and time-specific patterns. Ischemic tolerance was associated with transient up-regulation of transcription factors (c-Fos, JunB Egr-1, -2, -4, NGFI-B), Hsp70 and MAP kinase cascade-related genes (MKP-1), which are implicated cell survival. Delayed neuronal death exhibited complex long-lasting changes of expression, such as up-regulation of proapoptotic genes (GADD153, Smad2, Dral, Caspase-2 and -3) and down-regulation of genes implicated in survival signaling (MKK2, and PI4 kinase, DAG/PKC signaling pathways), suggesting an imbalance between death and survival signals. Our study provides a differential gene expression profile between delayed neuronal death and induced ischemic tolerance in a genome-wide analysis, and contributes to further understanding of the complex molecular pathophysiology in cerebral ischemia.

Animals↗
PubMed2006

Gene expression analysis of dendritic/Langerhans cells and Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a neoplastic disorder that results in clonal proliferation of cells with a Langerhans cell (LC) phenotype. The pathogenesis of LCH is still poorly understood. In the present study, serial analysis of gene expression (SAGE) was applied to LCs generated from umbilical cord blood CD34+ progenitor cells to identify LC-specific genes and the expression of these genes in LCH was investigated. Besides the expression of several genes known to be highly expressed in LCs and LCH such as CD1a, LYZ, and CD207, high expression of genes not previously reported to be expressed in LCs, such as GSN, MMP12, CCL17, and CCL22, was also identified. Further analysis of these genes by quantitative RT-PCR revealed high expression of FSCN1 and GSN in all 12 LCH cases analysed; of CD207, MMP12, CCL22, and CD1a in the majority of these cases; and CCL17 in three of the 12 cases. Immunohistochemistry confirmed protein expression in the majority of cases. The expression of MMP12 was most abundant in multi-system LCH, which is the LCH type with the worst prognosis. This suggests that expression of MMP12 may play a role in the progression of LCH. These data reveal new insight into the pathology of LCH and provide new starting points for further investigation of this clonal proliferative disorder.

Antigens, CD↗
PubMed1999

[Use of high-density filters ("DNA arrays") for gene expression analysis in human tissues].

Most technical strategies for the analysis of gene expression in tissues are able to study only one protein or RNA product at the same time. A new recent method referred to as < > or < > is able to analyze simultaneously several hundreds of different genes. The DNA array is a nylon membrane on which are spotted equal amounts of cDNAs corresponding to different genes. This filter is hybridized with a < > probe synthesized with mRNA derived from the tissue analyzed. The result gives a global profile of gene expression within the tissue and allows quantitative and comparative analysis between different tissues or cell types.

DNA, Complementary↗
PubMed2006

A framework for gene expression analysis.

MOTIVATION: Global gene expression measurements as obtained, for example, in microarray experiments can provide important clues to the underlying transcriptional control mechanisms and network structure of a biological cell. In the absence of a detailed understanding of this gene regulation, current attempts at classification of expression data rely on clustering and pattern recognition techniques employing ad-hoc similarity criteria. To improve this situation, a better understanding of the expected relationships between expression profiles of genes associated by biological function is required. RESULTS: It is shown that perturbation expansions familiar from biological systems theory make precise predictions for the types of relationships to be expected for expression profiles of biologically associated genes, even if the underlying biological factors responsible for this association are not known. Classification criteria are derived, most of which are not usually employed in clustering algorithms. The approach is illustrated by using the AtGenExpress Arabidopsis thaliana developmental expression map.

Algorithms↗
PubMed1995

Molecular and expression analysis of the Rhizobium meliloti phosphoenolpyruvate carboxykinase (pckA) gene.

The pckA gene of Rhizobium meliloti, encoding phosphoenolpyruvate carboxykinase, was isolated from a genomic cosmid library by complementation of the succinate growth phenotype of a Pck- mutant. The gene region was mapped by subcloning and Tn5 insertion mutagenesis. The DNA sequence for a 2-kb region containing the structural gene and its promoter was determined. The pckA gene encodes as 536-amino-acid protein that shows homology with other ATP-dependent Pck enzymes. The promoter was identified following primer extension analysis and is similar to sigma 70-like promoters. Expression analysis with a pckA::lacZ gene fusion indicated that the pckA gene was strongly induced at the onset of stationary phase in complex medium. When defined carbon sources were tested, the expression level of the pckA gene was found to be high when cells were grown in minimal media with succinate or arabinose as the sole carbon source but almost absent when glucose, sucrose, or glycerol was the sole carbon source. Glucose and sucrose were not found to strongly repress pckA induction by succinate.

Amino Acid Sequence↗
PubMed2003

Gene expression analysis on small numbers of invasive cells collected by chemotaxis from primary mammary tumors of the mouse.

BACKGROUND: cDNA microarrays have the potential to identify the genes involved in invasion and metastasis. However, when used with whole tumor tissue, the results average the expression patterns of different cell types. We have combined chemotaxis-based cell collection of the invasive subpopulation of cells within the primary tumor with array-based gene expression analysis to identify the genes necessary for the process of carcinoma cell invasion. RESULTS: Invasive cells were collected from live primary tumors using microneedles containing chemotactic growth factors to mimic chemotactic signals thought to be present in the primary tumor. When used with mammary tumors of rats and mice, carcinoma cells and macrophages constitute the invasive cell population. Microbeads conjugated with monoclonal anti-CD11b (Mac-1alpha) antibodies were used to separate macrophages from carcinoma cells. We utilized PCR-based cDNA amplification from small number of cells and compared it to the quality and complexity of conventionally generated cDNA to determine if amplified cDNA could be used with fidelity for array analysis of this cell population. These techniques showed a very high level of correlation indicating that the PCR based amplification technique yields a cDNA population that resembles, with high fidelity, the original template population present in the small number of cells used to prepare the cDNA for use with the chip. CONCLUSIONS: The specific collection of invasive cells from a primary tumor and the analysis of gene expression in these cells are is now possible. By further comparing the gene expression patterns of cells collected by invasion into microneedles with that of carcinoma cells obtained from the whole primary tumor, the blood, and whole metastatic tumors, genes that contribute to the invasive process in carcinoma cells may be identified.

Animals↗
PubMed2005

Expression analysis of the steroidogenic acute regulatory protein (STAR) gene in developing porcine conceptuses.

Steroidogenesis in porcine non-conceptus tissue is regulated by the STAR-dependent transport of cholesterol from the outer to inner mitochondrial membrane. Previous serial analysis of gene expression (SAGE) identified a STAR mRNA transcript in the porcine peri-implantation conceptus during trophectoderm elongation and increased conceptus estrogen synthesis between gestational day 11 and 12. To assess a potential role for STAR in the modulation of conceptus steroidogenesis via cholesterol transport, the conceptus STAR transcript was PCR cloned and temporal expression of mRNA and protein were examined. Northern analysis of day 12 corpora lutea and pig conceptus RNA detected multiple STAR transcripts in both tissues and identified the cloned transcript as the longest variant. The transcript had a 99% similarity to a truncated ovarian STAR transcript. The conceptus STAR transcript was temporally regulated during elongation but trace expression was present in day 6 blastocysts and day 25 conceptuses. Differential regulation of STAR mRNA was concomitant with the presence of the stimulatory transcription factor steroidogenic factor 1, and absence of the inhibitory transcription factor dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1, transcripts. In contrast to peak STAR mRNA expression at the filamentous stage, Western blot analyses revealed STAR protein levels were highest in tubular conceptuses. These data confirm the presence of STAR mRNA and protein during porcine conceptus elongation and suggest regulation of STAR at two levels, transcriptionally, in part, through differential regulation of transcription factors, and post-transcriptionally, evidenced by the disparity of protein to RNA in filamentous conceptuses.

Animals↗
PubMed2000

High-throughput gene expression analysis for drug discovery.

The ability to rapidly survey and compare gene expression levels between reference and test samples is moving the drug discovery process towards a more genomic orientation. The success of the Human Genome Project and related private genomics initiatives, combined with new technologies to probe, image and access expression data, are responsible for this transformation. This article reviews the history, status and future direction of high-throughput gene expression analysis. It describes classical approaches, explains the development of methods such as differential display for discovering novel genes, and discusses how microarray technology is exploiting collections of known sequences to pinpoint drug targets.

Journal Article↗
PubMed2003

Application of genome-wide expression analysis to identify molecular markers useful in monitoring industrial fermentations.

Genome-wide expression analysis of an industrial strain of Saccharomyces cerevisiae identified the YOR387c and YGL258w homologues as highly inducible in zinc-depleted conditions. Induction was specific for zinc deficiency and was dependent on Zap1p. The results indicate that these sequences may be valuable molecular markers for detecting zinc deficiency in industrial fermentations.

Culture Media↗
PubMed2006

Gene expression analysis using human cancer xenografts to identify novel predictive marker genes for the efficacy of 5-fluorouracil-based drugs.

The development of a diagnostic method for predicting the therapeutic efficacy or toxicity of anticancer drugs is a critical issue. We carried out a gene expression analysis to identify genes whose expression profiles were correlated with the sensitivity of 30 human tumor xenografts to 5-fluorouracil (5-FU)-based drugs (tegafur + uracil [UFT], tegafur + gimeracil + oteracil [S-1], 5'-deoxy-5-fluorouridine [5'-DFUR], and N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine [capecitabine]), as well as three other drugs (cisplatin [CDDP], irinotecan hydrochloride [CPT-11], and paclitaxel) that have different modes of action. In the present study, we focused especially on the fluoropyrimidines. The efficacy of all anticancer drugs was assayed using human tumor xenografts in nude mice. The mRNA expression profile of each of these xenografts was analyzed using a Human Focus array. Correlation analysis between the gene expression profiles and the chemosensitivities of seven drugs identified 39 genes whose expression levels were correlated significantly with multidrug sensitivity, and we suggest that the angiogenic pathway plays a pivotal role in resistance to fluoropyrimidines. Furthermore, many genes showing specific correlations with each drug were also identified. Among the candidate genes associated with 5-FU resistance, the dihydropyrimidine dehydrogenase mRNA expression profiles of the tumors showed a significant negative correlation with chemosensitivity to all of the 5-FU based drugs except for S-1. Therefore, the administration of S-1 might be an effective strategy for the treatment of high dihydropyrimidine dehydrogenase-expressing tumors. The results of the present study may enhance the prediction of tumor response to anticancer drugs and contribute to the development of tailor-made chemotherapy.

Animals↗
PubMed1998

Sequence and expression analysis of bovine pigment epithelium-derived factor.

PEDF, a member of the serpin superfamily of proteins related through their highly conserved folded conformation, has neurotrophic properties, including promotion of neurite-outgrowth and neuronal survival. Previously, we have purified and characterized PEDF protein from extracellular matrixes of bovine eyes. Here, we show the cDNA sequence and expression analysis of bovine PEDF. Northern analysis of RNA from bovine retinal pigment epithelium (RPE) and neural retina using a human PEDF cDNA fragment reveals expression of the PEDF gene only for RPE. Sequence analysis of a cDNA clone isolated from bovine RPE predicts a polypeptide of 416 amino acid residues that shares 88.6% and 85% amino acid identity with human and mouse PEDF, respectively. It has an N-terminal signal peptide, a consensus glycosylation site and homology with serpins including the conserved residues required for maintaining the serpin tertiary structure. Cell-free expression of the bovine PEDF cDNA by in vitro transcription and translation yields a precursor polypeptide of 45,000-Mr that immunoprecipitates with an antibody to human PEDF. Expression analysis in stably transfected baby hamster kidney cells shows that the recombinant bovine protein is secreted to the culture media as a mature 50,000-Mr protein, which induces neurite-outgrowth on retinoblastoma cells, like the naturally-occurring PEDF protein. Thus, the bovine PEDF cDNA isolated here codes for a functional soluble secreted PEDF glycoprotein.

Amino Acid Sequence↗
PubMed2002

Expression analysis of the tylosin-biosynthetic gene cluster: pivotal regulatory role of the tylQ product.

Expression analysis by RT-PCR, applied to the entire tyl cluster, revealed that the pattern of transcription is more complex than expected. For example, the five tylG polyketide synthase genes are not necessarily cotranscribed or even coregulated. Among the regulatory genes, tylQ has emerged as a key factor. Although several genes (including the positive regulator, tylS) were possibly expressed constitutively, only tylQ was silent during secondary metabolism. Analysis of engineered strains, in which tylQ was disrupted or overexpressed, showed that the TylQ protein is a transcriptional repressor that blocks tylosin biosynthesis by controlling expression of the activator, tylR. Before tylosin production can be triggered, tylQ must be switched off, or at least downregulated.

Base Sequence↗
PubMed2006

A system for receptor functional analysis based on c-fos mRNA expression: analysis of GnRH receptors as a test system.

This report describes the establishment of a system for assessing receptor activation by RT-PCR-based detection of c-fos mRNA induction. In this system, COS-7 cells were transiently transfected with GnRH receptor expression plasmid, and ligand-induced c-fos expression was quantified by the RT-competitive PCR method. The results were compared with those of a conventional inositol phosphate (IP) assay. Changes in c-fos expression levels were observed in a dose- and ligand-dependent manner. Similar tendencies were observed in ligand selectivity between c-fos expression and IP production. The novel system developed and established in the present study is sensitive by using RT-PCR and convenient because it requires only basic methods of cell culture and molecular biology. It also has the merit that it does not need any specific measuring devices or radioactive substances. Given the ability of c-fos to respond to diverse stimuli, the present system may be applicable for various receptors for bioactive substances in addition to GnRH receptor, and useful for various purposes including screening ligands for orphan receptors.

Animals↗
PubMed1998

Cloning, expression analysis and chromosomal localization of the human nuclear receptor gene GCNF.

Germ cell nuclear factor (GCNF) is an orphan member of the nuclear receptor gene superfamily. We report the cloning of a cDNA encoding a new variant of human GCNF from human testis and its expression analysis. Southern blot analysis of the human genomic DNA indicates that the GCNF gene is not closely related to other members within the nuclear receptor superfamily. Chromosomal localization of the GCNF gene shows that the gene is located on chromosome 9 at the locus q33-34.1. In situ hybridization analysis of GCNF expression in the testis shows that human GCNF is expressed exclusively in germ cells.

Adult↗
PubMed2002

Laser capture microdissection and microarray expression analysis of lung adenocarcinoma reveals tobacco smoking- and prognosis-related molecular profiles.

Recent expression profile analyses revealed that lung adenocarcinomas can be divided into several subgroups with diverse pathological features. Because cellular heterogeneity of tumors can confound these analyses, we used laser capture microdissection and microarray expression analysis to characterize the molecular profiles of lung adenocarcinomas. We found 45 genes delineating smokers and nonsmokers that were located at chromosomal loci frequently altered in non-small cell lung cancers, and 27 genes, which were differentially expressed between survivors and nonsurvivors 5 years after surgery. These results are consistent with the hypothesis that the abnormal expression of genes involved in maintaining the mitotic spindle checkpoint and genomic stability, e.g., hBUB3, hZW10, and APC2, contribute to the molecular pathogenesis and tumor progression of tobacco smoke-induced adenocarcinoma of the lung.

Adenocarcinoma↗