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At least 271 records · Page 15Linked to original sources
PubMed1994

Evolution of the C4 phosphoenolpyruvate carboxylase promoter of the C4 dicot Flaveria trinervia: an expression analysis in the C3 plant tobacco.

The key enzymes of photosynthetic carbon assimilation in C4 plants have evolved from C3 isoforms which were present in the C3 ancestral species. We are interested in the molecular changes responsible for the novel expression pattern of C4 genes and are focussing on phosphoenolpyruvate carboxylase (PEPCase) of the genus Flaveria. The C4 isoform of PEPCase in the C4 plant F. trinervia is encoded by the ppcA subgroup of the PEPcase gene family and is abundantly expressed in the mesophyll cells of leaves. The orthologous ppcA genes of the C3 plant F. pringlei are only weakly expressed and their transcripts do not accumulate in a leaf-specific manner but, rather, are present in all plant organs. To answer the question whether the differences in the expression levels of the ppcA genes from F. pringlei and F. trinervia are caused by changes in the 5' upstream regions of the genes or by C4-specific trans-regulatory factors, varying parts of the 5' flanking region of the ppcA1 genes of both species were fused to the beta-glucuronidase (GUS) gene and inserted in the tobacco genome. GUS expression analysis of transgenic plants revealed that the level of expression of the Flaveria ppcA1 genes are recapitulated in the heterologous C3 plant tobacco. Hence, the 5' upstream region of the ppcA1 gene of F. trinervia contains regulatory cis-elements that are responsible for the C4-specific, abundant expression of this gene. These sequences are located upstream of position -500 relative to the transcription initiation site.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2006

Assessment of representational difference analysis (RDA) to construct informative cDNA microarrays for gene expression analysis of species with limited transcriptome information, using red and green tomatoes as a model.

Microarray technology makes it feasible to analyse the expression of thousands of different gene elements in a single experiment. Most informative are 'whole genome' arrays, where all gene expression products of a single species or variety are represented. Such arrays are now available for a limited number of model species. However, for other, less well-documented species other routes are still necessary to obtain informative arrays. This includes the use of cDNA libraries. To enhance the amount of information that can be obtained from cDNA libraries, redundancy needs to be minimised, and the number of cDNAs relevant for the conditions of interest needs to be increased. Here, we used representational difference analysis (RDA), a mRNA subtraction procedure, as a tool to enhance the efficiency of cDNA libraries to be used to generate microarrays. Tomato was chosen as a model system for a less well-documented species. cDNA libraries for two distinct physiological conditions of tomato fruits, red and green, were made. The libraries were characterized by sequencing and hybridisation analysis. The RDA procedure was shown to be effective in selecting for genes of relevance for the physiological conditions under investigation, and against constitutively expressed genes. At the same time, redundancy was reduced, but complete normalisation was not obtained, and subsequent sequence analysis will be required to obtain non-redundant arrays. Further, known and putative ripening-related cDNAs were identified in hybridisation experiments on the basis of RNA populations as isolated from the green and red stage of ripening.

Fruit↗
PubMed2004

cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type VI.

Peptidylarginine deiminase (PAD) catalyzes the post-translational modification of protein through the conversion of arginine to citrulline in the presence of calcium ions. Human, similar to rodents, has four isoforms of PAD (type I, II, III and IV/V), each of which is distinct in substrate specificity and tissue specific expression. In our large-scale sequencing project, we identified a new human PAD cDNA from a human fetal brain cDNA library. The putative protein encoded by this cDNA is designated hPADVI. Expression analysis of hPADVI showed that it is mainly expressed in adult human ovary and peripheral blood leukocytes. We conclude that hPADVI may be orthologous to mouse ePAD, basing on sequence comparison, chromosome localization and exon-intron structure analysis. PAD-mediated deimination of epithelial cell keratin resulting in cytoskeletal remodeling suggests a possible role for hPADVI in cytoskeletal reorganization in the egg and in early embryo development. This study describes a new important member of the human PAD family.

Amino Acid Sequence↗
PubMed1999

Cloning of rat MLH1 and expression analysis of MSH2, MSH3, MSH6, and MLH1 during spermatogenesis.

The mismatch repair system has been highly conserved in various species. In eukaryotic cells, the Mut S and Mut L homologues play crucial roles in both DNA mismatch repair and meiotic recombination. A full-length rat cDNA clone for rat MLH1 has been constructed using the RT-PCR method. The cDNA has an open reading frame of 2274 nucleotides for a protein of 757 amino acids. We have also obtained partial cDNA clones for MSH3 and MSH6. Northern blot analysis of rat MLH1, MSH2, MSH3, and MSH6 in the testes of rats of different ages showed differential expression of these genes as a function of developmental maturation of the testes. The expression analysis suggests that MSH3 may have a more predominant role in the meiotic recombination process.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Genome expression analysis in yeast reveals novel transcriptional regulation by inositol and choline and new regulatory functions for Opi1p, Ino2p, and Ino4p.

In Saccharomyces cerevisiae, genes encoding phospholipid-synthesizing enzymes are regulated by inositol and choline (IC). The current model suggests that when these precursors become limiting, the transcriptional complex Ino2p-Ino4p activates the expression of these genes, whereas repression requires Opi1p and occurs when IC are available. In this study, microarray-based expression analysis was performed to assess the global transcriptional response to IC in a wild-type strain and in the opi1delta, ino2delta, and ino4delta null mutant strains. Fifty genes were either activated or repressed by IC in the wild-type strain, including three already known IC-repressed genes. We demonstrated that the IC response was not limited to genes involved in membrane biogenesis, but encompassed various metabolic pathways such as biotin synthesis, one-carbon compound metabolism, nitrogen-containing compound transport and degradation, cell wall organization and biogenesis, and acetyl-CoA metabolism. The expression of a large number of IC-regulated genes did not change in the opi1delta, ino2delta, and ino4delta strains, thus implicating new regulatory elements in the IC response. Our studies revealed that Opi1p, Ino2p, and Ino4p have dual regulatory activities, acting in both positive and negative transcriptional regulation of a large number of genes, most of which are not regulated by IC and only a subset of which is involved in membrane biogenesis. These data provide the first global response profile of yeast to IC and reveal novel regulatory mechanisms by these precursors.

Basic Helix-Loop-Helix Proteins↗
PubMed2003

Expression analysis of subtractively enriched libraries (EASEL): a widely applicable approach to the identification of differentially expressed genes.

A variety of methods for high throughput analysis of differential gene expression has been developed over the past years. We have implemented the EASEL technique that adds flexibility, efficiency and wide-applicability to these methods. The EASEL procedure is unique as it integrates several well established techniques and thereby offers a combination of subtractive hybridization of 3' cDNA ends with macroarrays analysis and Serial Analysis of Gene Expression (SAGE). In addition, once a set of interesting, differentially expressed genes is identified, the material required for follow up studies to test the hypothesis that the gene is truly involved in the process of interest is readily available. In this report, we first present a step-by-step validation of the procedure, since several of the incorporated steps had to be tailored to meet specific requirements and implied drastic modifications of the original methods. Secondly, we applied EASEL to the identification of up-regulated gene products in the outflow tract region of the embryonic rat heart. Here we provide evidence that at least two among the differentially expressed genes detected, follistatin-like protein gene and membrane type 1-metallo proteinase gene, are selectively up-regulated in the outflow tract, suggesting their involvement in the development of this region during embryogenesis.

Animals↗
PubMed2006

A comprehensive expression analysis of the WRKY gene superfamily in rice plants during defense response.

To understand the transcriptional regulatory mechanism of host genes during the activation of defense responses in rice, we isolated WRKY transcription factors whose expressions were altered upon attack of the fungal pathogen Magnaporthe grisea, the causal agent of the devastating rice blast disease. A systematic expression analysis of OsWRKYs (Oryza sativa L. WRKYs) revealed that among 45 tested genes the expression of 15 genes was increased remarkably in an incompatible interaction between rice and M. grisea. Twelve of the M. grisea-inducible OsWRKY genes were also differentially regulated in rice plants infected with the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). In experiments with defense signaling molecules, the expression of two genes, OsWRKY45 and OsWRKY62, was increased in salicylic acid (SA)-treated leaves and the expression of three genes, OsWRKY10, OsWRKY82, and OsWRKY85 was increased by jasmonic acid (JA) treatment. OsWRKY30 and OsWRKY83 responded to both SA- and JA treatments. The expression profiles suggest that a large number of WRKY DNA-binding proteins are involved in the transcriptional activation of defense-related genes in response to rice pathogens.

Blotting, Northern↗
PubMed2005

Protein/RNA coextraction and small two-dimensional polyacrylamide gel electrophoresis for proteomic/gene expression analysis of renal cancer biopsies.

A small amount of bioptic tissue ( approximately 5-10mg of fresh tissue) usually does not contain enough material to extract protein and RNA separately, to obtain preparative two-dimensional polyacrylamide gel electrophoresis (2-DE), and to identify a large number of separated proteins by MS. We tested a method, on small renal cancer specimens, for the coextraction of protein and RNA coupled with 2-DE and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) or quadrupole time-of-flight (Q-TOF) analysis. We coextracted 0.28+/-0.05mg of proteins and 2.5+/-0.33microg of RNA for each 10mg of renal carcinoma tissue. Small and large 2-DE gels were compared: they showed a similar number of spots, and it was possible to match each other; using small format gels, one-fifth of the protein amount was required to identify, by Q-TOF analysis, the same number of proteins identifiable in large-format gel using MALDI-TOF analysis. Quality of RNA coextracted with the proteins was tested by real-time PCR on a set of housekeeping genes. They were quantified with high amplification efficiency and specificity. In conclusion, using 5 to 10mg of fresh tissue, it was possible to perform comprehensive parallel proteomic and genomic analysis by high-resolution, small-format 2-DE gels, allowing approximately 300 proteins identification and 1000 genes expression analysis.

Carcinoma, Renal Cell↗
PubMed2002

Large scale gene expression analysis of osteoclastogenesis in vitro and elucidation of NFAT2 as a key regulator.

To understand the molecular events coupling between cell proliferation and differentiation by elucidating genes essential for the process, we conducted a large scale gene expression analysis of an in vitro osteoclastogenesis system consisting of recombinant RANKL and mouse RAW264 cells. The entire process leading to the formation of tartrate resistant acid phosphatase-positive multinucleated cells takes 3 days and plates become fully covered with multinucleated cells at 4 days. Microarray probing at eight time points revealed 635 genes that showed greater than 2-fold differential expression for at least one time point and they could be classified into six groups by the "k-means" clustering analysis. Among a group of 106 early inducible genes (within 2-5 h after RANKL stimulation), four genes including NFAT2 were identified as genes whose enhanced expressions were fairly correlated with an efficient induction of matured osteoclasts. Moreover, cyclosporin A significantly suppressed the multinucleated cell formation accompanying the reduction of the nuclear localization of NFAT2. When the expression of NFAT2 was suppressed by introducing antisense NFAT2, multinucleated cell formation was severely hampered. Functional analysis thus combined with gene analysis by microarray technology elucidated a key role of NFAT2 in osteoclastogenesis in vitro.

Animals↗
PubMed2003

Software packages for quantitative microarray-based gene expression analysis.

Microarray technology enables researchers to investigate the expression of several thousand genes simultaneously. The whole transcriptional response of these genes in normal cells or tissue, in disease condition, as an response to biological, genetical or chemical stimuli or during normal biological processes such as cell cycle or embryonic development can be investigated. This leads to a huge amount of data, from which the relevant information has to be extracted by statistical and computational methods. Several software packages for the analysis of gene expression data are available, both commercially and freely. They differ particularly with regard to the implemented analytical methods, the graphical display and the manageability. In this paper the commercial software packages arraySCOUT, GeneSpring and Spotfire DecisionSite for Functional Genomics are compared and their applicability for analysis of gene expression data is studied. Small artificial and application test datasets are used to compare the computational results of the software packages. As far as possible results are verified with standard statistical software package SAS.

Algorithms↗
PubMed2002

Comparison of fluorescent tag DNA labeling methods used for expression analysis by DNA microarrays.

Gene expression profiling by DNA microarrays has found wide application in many fields of biomedical research. The protocols for this technique are not yet standardized, and for each given step in microarray analysis a number of different protocols are in use. As a consequence, results obtained in different laboratories can be difficult to compare. Of particular importance in this respect are the methods for the preparation of fluorescent cDNA probes that should quantitatively reflect the abundance of different mRNAs in the two samples to be compared. Here we systematically evaluate and compare five different published and/or commercial principles for the synthesis offluorescently labeled probes for microarray analysis (direct labeling, 77 RNA polymerase amplification, aminoallyl labeling, hapten-antibody enzymatic labeling, and 3-D multi-labeled structures). We show that individual labeling methods can significantly influence the expression pattern obtained in a microarray experiment and discuss the respective benefits and limitations of each method.

DNA Probes↗
PubMed2004

Express analysis of explosives, chemical warfare agents and drugs with multicapillary column gas chromatography and ion mobility increment spectrometry.

Description of a gas chromatograph designed for express analysis of explosives (2,4-dinitrotoluene, 2,4,6-trinitrotoluene, pentaerythritol tetranitrate), chemical warfare agents (mustard gas, lewisite, sarin) and drugs (heroin, cocaine hydrochloride, crack) is given. The devices comprises a multicapillary chromatographic column and an ion mobility increment spectrometer (MCC-IMIS). The main analytical characteristics of an IMIS (estimated detection limit (DL), linear dynamic range (LDR), speed of response) and a chromatographic column (separation power, degree of separation, a number of possible peaks at a chromatogram section, divided by analysis time) are determined. The maximum value of DL equal to 5 pg/ml was registered for cis-alpha-LW, and the lowest one of 0.001 pg/ml was for cocaine. The maximum value of LDR equal to 1000 was registered for sarin and the lowest one of 150 was for the ions of lewisite. Speed of response of one compound detection with the IMIS was 0.7 s.

Algorithms↗
PubMed1995

Isolation and expression analysis of tyro3, a murine growth factor receptor tyrosine kinase preferentially expressed in adult brain.

Growth factors and their receptors function in the nervous system to induce proliferation and differentiation of neuronal precursor cells and to support survival of mature neurons. We have isolated a murine growth factor receptor tyrosine kinase using an anti-phosphotyrosine antibody screening procedure and studied the pattern of expression. The deduced amino acid sequence of the kinase has all the characteristics of a growth factor receptor and consists of a putative extracellular domain, a transmembrane domain, and a tyrosine kinase domain. Sequence comparison with known receptor tyrosine kinases indicated that the murine kinase is a mouse homolog of tyro3. tyro3 belongs to the Axl/Ufo growth factor receptor family. In the putative extracellular domain, there are two Ig-like domains and two fibronectin type III repeats which are conserved in other members of the Axl/Ufo family receptors. Northern blot hybridization analysis showed that tyro3 is expressed at high levels in the brain of adult mice, although considerable expression was also observed in the testis. In situ hybridization analysis revealed that high levels of tyro3 are expressed in the cerebral cortex, the lateral septum, the hippocampus, the olfactory bulb, and in the cerebellum. The highest levels of tyro3 expression in the brain are associated with neurons. The preferential expression of tyro3 in specific regions of the adult mouse brain suggests that tyro3 may function as a novel neurotrophic factor receptor.

Amino Acid Sequence↗
PubMed2004

DNA microarray normalization methods can remove bias from differential protein expression analysis of 2D difference gel electrophoresis results.

MOTIVATION: Two-dimensional Difference Gel Electrophoresis (DIGE) measures expression differences for thousands of proteins in parallel. In contrast to DNA microarray analysis, however, there have been few systematic studies on the validity of differential protein expression analysis, and the effects of normalization methods have not yet been investigated. To address this need, we assessed a series of same-same comparisons, evaluating how random experimental variance influenced differential expression analysis. RESULTS: The strong fluctuations observed were reflected in large discrepancies between the distributions of the spot intensities for different gels. Correct normalization for pooling of multiple gels for analysis is, therefore, essential. We show that both dye-specific background levels and the differences in scale of the spot intensity distributions must be accounted for. A variance stabilizing transform that had been developed for DNA microarray analysis combined with a robust Z-score allowed the determination of gel-independent signal thresholds based on the empirical distributions from same-same comparisons. In contrast, similar thresholds holding up to cross-validation could not be proposed for data normalized using methods established in the field of proteomics. AVAILABILITY: Software is available on request from the authors. SUPPLEMENTARY INFORMATION: There is supplementary material available online at http://www.flychip.org.uk/kreil/pub/2dgels/

Algorithms↗
PubMed2000

Expression analysis of thirty one Y chromosome genes in human prostate cancer.

Rapid advances in positional cloning studies have identified most of the genes on the human Y chromosome, thereby providing resources for studying the expression of its genes in prostate cancer. Using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) procedure, we had examined the expression of the Y chromosome genes in a panel of prostate samples diagnosed with benign prostatic hyperplasia (BPH), low and/or high grade carcinoma, and the prostatic cell line, LNCaP, stimulated by androgen treatment. Results from this expression analysis of 31 of the 33 genes, isolated so far from the Y chromosome, revealed three types of expression patterns: i) specific expression in other tissues (e.g., AMELY, BPY1, BPY2, CDY, and RBM); ii) ubiquitous expression among prostate and control testis samples, similar to those of house-keeping genes (e.g., ANT3, XE7,ASMTL, IL3RA, SYBL1, TRAMP, MIC2, DBY, RPS4Y, and SMCY); iii) differential expression in prostate and testis samples. The last group includes X-Y homologous (e.g., ZFY, PRKY, DFFRY, TB4Y, EIF1AY, and UTY) and Y-specific genes (e.g., SRY, TSPY, PRY, and XKRY). Androgen stimulation of the LNCaP cells resulted in up-regulation of PGPL, CSFR2A, IL3RA, TSPY, and IL9R and down regulation of SRY, ZFY, and DFFRY. The heterogeneous and differential expression patterns of the Y chromosome genes raise the possibility that some of these genes are either involved in or are affected by the oncogenic processes of the prostate. The up- and down-regulation of several Y chromosome genes by androgen suggest that they may play a role(s) in the hormonally stimulated proliferation of the responsive LNCaP cells.

Androgens↗
PubMed2002

Mutational and expression analysis of the reelin pathway components CDK5 and doublecortin in gangliogliomas.

Gangliogliomas represent highly differentiated glioneuronal tumors frequently occurring in young patients with focal epilepsies. Dysplastic neurons are a neuropathological hallmark of this neoplasm. Here, we have analyzed two major components of the reelin pathway associated with neuronal migration and cortical cytoarchitecture in gangliogliomas, i.e., cyclin-dependent kinase 5 (CDK5) and doublecortin (DCX). The genomic structure of human CDK5 was identified by an " in silico" cloning approach using the "high throughput genomic sequencing" (htgs) databank, NCBI BLAST 2.1. DNA sequence analysis of CDK5 and DCX was carried out in tissue samples obtained from 23 patients and compared with control DNA from non-affected individuals ( n=100). For gene expression analysis of CDK5 and DCX, a quantitative real time reverse transcription-PCR TaqMan assay was used with mRNA from gangliogliomas ( n=22) and non-lesional central nervous tissue control tissue ( n=7). The human CDK5 gene is located on chromosome 7q36 and contains 12 exons. Its coding sequence reveals 90.1% homology to the mouse counterpart. A novel pseudogene of CDK5 was found on chromosome 8. While the mutational analysis of CDK5 and DCX did not reveal any sequence alterations in gangliogliomas, a lower expression was observed for both genes in tumor compared to control tissue samples. The present data indicate that mutations of CDK5 and DCX genes are not involved in the development of gangliogliomas. A novel pseudogene on chromosome 8 has to be taken into account for future studies on CDK5.

Animals↗
PubMed1998

Cloning, expression analysis, and functional characterization of PKL12, a member of a new subfamily of ser/thr kinases.

We report the cloning of the full-length cDNA of a new murine protein kinase, mPKL12. The sequence reveals a 305-amino-acid protein that contains the characteristic subdomains of the kinase superfamily and particular homology indicating a ser/thr specificity. We have also identified its human homologue gene (94% identical) and the putative homologue proteins from Saccharomyces cerevisiae and Arabidoposis thaliana. These four sequences appear to form a new subfamily of protein kinases, close in size to the theoretical minimal catalytic domain, therefore suggesting that they could be the catalytic unit of a more complex holoenzyme. Using Escherichia coli-purified protein, we have demonstrated that the mPKL12 enzyme possesses an intrinsic kinase activity, capable of phosphorylating enolase and also of promoting autophosphorylation, with a ser/thr specificity. Tissue expression analysis of mPKL12 showed that it is ubiquitously expressed, although at very low levels. RT-PCR analysis of several cell lines also supports this view, therefore suggesting that PKL12 may play a role in a very general cellular function, probably related with the secretory pathway.

Amino Acid Sequence↗