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PubMed2001

Determining significant fold differences in gene expression analysis.

A typical use for RNA expression microarrays is comparing the measurement of gene expression of two groups. There has not been a study reproducing an entire experiment and modeling the distribution of reproducibility of fold differences. Our goal was to create a model of significance for fold differences, then maximize the number of ESTs above that threshold. Multiple strategies were tested to filter out those ESTs contributing to noise, thus decreasing the requirements of what was needed for significance. We found that even though RNA expression levels appears consistent in duplicate measurements, when entire experiments are duplicated, the calculated fold differences are not as consistent. Thus, it is critically important to repeat as many data points as possible, to ensure that genes and ESTs labeled as significant are truly so. We were successfully able to use duplicated expression measurements to model the duplicated fold differences, and to calculate the levels of fold difference needed to reach significance. This approach can be applied to many other experiments to ascertain significance without a priori assumptions.

Expressed Sequence Tags↗
PubMed1999

Comparative gene-expression analysis.

The study of differences in gene-expression patterns is one of the most promising approaches for understanding mechanisms of differentiation and development. In addition, the identification of disease-related target molecules opens new avenues for rational pharmaceutical intervention. Recent technical advances and improvements are accelerating the analysis of gene-expression profiles at the transcript level. The knowledge and comprehension of currently applied methods is one of the central criteria for an efficient and successful gene-screening approach.

Expressed Sequence Tags↗
PubMed2005

Experimental comparison and cross-validation of the Affymetrix and Illumina gene expression analysis platforms.

The growth in popularity of RNA expression microarrays has been accompanied by concerns about the reliability of the data especially when comparing between different platforms. Here, we present an evaluation of the reproducibility of microarray results using two platforms, Affymetrix GeneChips and Illumina BeadArrays. The study design is based on a dilution series of two human tissues (blood and placenta), tested in duplicate on each platform. The results of a comparison between the platforms indicate very high agreement, particularly for genes which are predicted to be differentially expressed between the two tissues. Agreement was strongly correlated with the level of expression of a gene. Concordance was also improved when probes on the two platforms could be identified as being likely to target the same set of transcripts of a given gene. These results shed light on the causes or failures of agreement across microarray platforms. The set of probes we found to be most highly reproducible can be used by others to help increase confidence in analyses of other data sets using these platforms.

Adult↗
PubMed2003

The use of gene expression analysis to gain insights into signaling mechanisms of metastatic medulloblastoma.

Metastasis is the leading cause of treatment failure in medulloblastoma. Understanding the genetic regulation of metastasis may aid in the development of novel treatments. We therefore performed in silico analysis of the mRNA expression of 83 medulloblastomas compiled from two independent microarray studies by focusing on 135 genes most frequently linked to metastasis in other tumors. We then asked whether expression of these genes correlated with metastasis in the medulloblastoma array data sets. We found the platelet-derived growth factor receptor alpha, early growth response protein 1 and insulin-like growth factor 2 genes as well as several genes associated with MYCC and ERBB2 overexpressed by at least 2-fold in metastatic tumors in both array data sets. We conclude that these genes may interact to promote prometastatic signaling in medulloblastoma.

Cerebellar Neoplasms↗
PubMed1998

Human gap junction protein connexin31: molecular cloning and expression analysis.

We have isolated and characterized a human genomic clone containing the complete coding region of connexin31 (Cx31). Similar to rodent Cx31, the coding region of human Cx31 is completely contained within the second exon and consists of 810 nucleotides. The deduced human Cx31 polypeptide consists of 270 amino acids with a predicted molecular mass of 30.818 kDa. Its sequence is most similar to mouse Cx31 (82.6% identical amino acids) and rat (83.0% identical amino acids), but shows considerably fewer potential sites of phosphorylation. After Northern blot hybridization, two Cx31 transcripts of 2.2 and 1.8 kb were detected in total RNA of the human keratinocyte cell line HaCaT and two transcripts of 2.2 and 1.9 kb in total RNA of E6/E7 transfected human keratinocytes (HEK cells). Using affinity-purified rabbit antibodies to mouse Cx31, immunofluorescence analysis demonstrated relatively weak expression of human Cx31 in HaCaT and HEK cells. The Cx31 gene exists as a single copy gene in the human genome and was mapped to the chromosomal region 1p34-p36 by analyzing human-mouse somatic cell hybrids.

Amino Acid Sequence↗
PubMed1996

PCR cloning and expression analysis of a cDNA encoding a pectinacetylesterase from Vigna radiata L.

A cDNA clone encoding a pectinacetylesterase (PAE) was isolated from 3-day-old mung bean seedlings using PCR-based techniques. Degenerate oligonucleotide primers were designed according to the N-terminus and internal peptides from the purified PAE. The full-length clone of 1453 bp codes for a signal peptide of 24 amino acids and a mature protein of 375 amino acids. The Mr and the pI of the cDNA-deduced amino acid sequence agree with the values estimated for the purified enzyme. No significant sequence identity between the PAE and any known protein could be found in the databases. Northern analysis revealed developmentally regulated expression of the mRNA in mung been seedlings.

Amino Acid Sequence↗
PubMed2003

Integrating regulatory motif discovery and genome-wide expression analysis.

We propose motif regressor for discovering sequence motifs upstream of genes that undergo expression changes in a given condition. The method combines the advantages of matrix-based motif finding and oligomer motif-expression regression analysis, resulting in high sensitivity and specificity. motif regressor is particularly effective in discovering expression-mediating motifs of medium to long width with multiple degenerate positions. When applied to Saccharomyces cerevisiae, motif regressor identified the ROX1 and YAP1 motifs from Rox1p and Yap1p overexpression experiments, respectively; predicted that Gcn4p may have increased activity in YAP1 deletion mutants; reported a group of motifs (including GCN4, PHO4, MET4, STRE, USR1, RAP1, M3A, and M3B) that may mediate the transcriptional response to amino acid starvation; and found all of the known cell-cycle regulation motifs from 18 expression microarrays over two cell cycles.

Algorithms↗
PubMed2001

Cloning and expression analysis of NhL1, a gene encoding an extracellular lipase from the fungal pea pathogen Nectria haematococca MP VI (Fusarium solani f. sp. pisi) that is expressed in planta.

The filamentous fungus Nectria haematococca (anamorph Fusarium solani f. sp. pisi) resides in soil, and attacks pea seedlings in the area of the underground epicotyl and upper tap root, causing foot rot disease. We detected lipase activity during in vitro growth of N. haematococca. Subsequently, a lipase gene was cloned and functionally characterised by heterologous expression in Saccharomyces cerevisiae. The full-length cDNA of 1152 bp was cloned using a 3' RACE-PCR approach coupled with cDNA library screening. The genomic clone, comprising an ORF of 999 bp interrupted by two introns of 56 and 64 bp, was isolated from a newly constructed lambda phage library. Analysis of the deduced protein sequence revealed the presence of a typical signal peptide at the N-terminus, and of the three conserved amino acids forming the active site of lipases. The lipase of N. haematococca has a low degree of similarity to the lipases from Humicola lanuginosa (37.2%), Rhizomucor miehei (21.6%), Rhizopus delemar (23.1%), Rhizopus niveus (25.9%), and to mono- and diacylglycerol lipase from Penicillium camembertii (30.8%), and very high similarity (94.6%) to a lipase from Fusarium heterosporum. The lipase from N. haematococca shows maximal activity at 37 degrees C and pH 8.0. Based on Southern analysis, the lipase clone represents a single-copy gene in N. haematococca. Expression analysis was performed by RT-PCR. In vitro, the lipase gene shows a low basal expression, but is highly inducible by lipase substrates, and repressed by glucose. During plant infection, transcripts of this fungal lipase gene were detected 4, 8, and 10 days after infection.

Amino Acid Sequence↗
PubMed2004

A comparative expression analysis of four MRX genes regulating intracellular signalling via small GTPases.

The X chromosomal mental retardation genes have attained high interest in the past. A rough classification distinguishes syndromal mental retardation (MRXS) and nonsyndromal mental retardation (MRX) conditions. The latter are suggested to be responsible for human specific development of cognitive abilities. These genes have been shown to be engaged in chromatin remodelling or in intracellular signalling. During this analysis, we have compared the expression pattern in the mouse of four genes from the latter class of MRX genes: Ophn1, Arhgef6 (also called alphaPix), Pak3, and Gdi1. Ophn1, Pak3, and Gdi1 show a specific neuronal expression pattern with a certain overlap that allows to assign these signalling molecules to the same functional context. We noticed the highest expression of these genes in the dentate gyrus and cornu ammonis of the hippocampus, in structures engaged in learning and memory. A completely different expression pattern was observed for Arhgef6. In the CNS, it is expressed in ventricular zones, where neuronal progenitor cells are located. But Arhgef6 expression is also found in other non-neural tissues. Our analysis provides evidence that these signalling molecules are involved in different spatio-temporal expression domains of common signalling cascades and that for most tissues considerable functional redundancy of Rho-mediated signalling pathways exists.

Animals↗
PubMed2005

Gene expression analysis of Escherichia coli grown in miniaturized bioreactor platforms for high-throughput analysis of growth and genomic data.

Combining high-throughput growth physiology and global gene expression data analysis is of significant value for integrating metabolism and genomics. We compared global gene expression using 500 ng of total RNA from Escherichia coli cultures grown in rich or defined minimal media in a miniaturized 50-microl bioreactor. The microbioreactor was fabricated out of poly(dimethylsiloxane) (PDMS) and glass and equipped to provide on-line, optical measurements. cDNA labeling for microarray hybridizations was performed with the GeniconRLS system. From these experiments, we found that the expression of 232 genes increased significantly in cells grown in minimum medium, including genes involved in amino acid biosynthesis and central metabolism. The expression of 275 genes was significantly elevated in cells grown in rich medium, including genes involved in the translational and motility apparatuses. In general, these changes in gene expression levels were similar to those observed in 1,000-fold larger cultures. The increasing rate at which complete genomic sequences of microorganisms are becoming available offers an unprecedented opportunity for investigating these organisms. Our results from microscale cultures using just 500 ng of total RNA indicate that high-throughput integration of growth physiology and genomics will be possible with novel biochemical platforms and improved detection technologies.

Bioreactors↗
PubMed2001

Whole-genome expression analysis: challenges beyond clustering.

Measuring the expression of most or all of the genes in a biological system raises major analytic challenges. A wealth of recent reports uses microarray expression data to examine diverse biological phenomena - from basic processes in model organisms to complex aspects of human disease. After an initial flurry of methods for clustering the data on the basis of similarity, the field has recognized some longer-term challenges. Firstly, there are efforts to understand the sources of noise and variation in microarray experiments in order to increase the biological signal. Secondly, there are efforts to combine expression data with other sources of information to improve the range and quality of conclusions that can be drawn. Finally, techniques are now emerging to reconstruct networks of genetic interactions in order to create integrated and systematic models of biological systems.

Gene Expression Profiling↗
PubMed1999

Normalization of array hybridization experiments in differential gene expression analysis.

For detecting and confirming differentially expressed genes it is necessary to have a trustworthy reference. So called 'housekeeping genes' are frequently used for this purpose as internal standard. However, if the influence of new experimental conditions is to be analyzed it is not safe to assume a priori that the expression of these genes is not affected. Therefore two synthetic poly(A)-RNAs were generated by PCR and in vitro transcription. They were used as external standards for normalization of northern blots and cDNA arrays where non-regulated genes as internal reference were not available.

Ampicillin Resistance↗
PubMed1997

Switching of gene expression: analysis of the factors that spatially and temporally regulate plant gene expression.

In this chapter, we have reviewed the present research and understanding of several families of transcription factors in plants. From this information, it appears there is good conservation between the types of transcription factors in plants and animals. However, there are several types of factors which have been isolated in plants that remain to be documented in animals (e.g., HD-Zip and GT). These as well as the presence of two types of TATA-binding proteins (TBPs) in plants suggest that although transcription in eukaryotes is highly conserved, fundamental differences may exist. Despite the differences, the modes of regulating transcription are well conserved. Figure 3 summarizes these modes of regulation. In recent years, the role of chromatin structure as well as subcellular localization have been the focus of a vast amount of research in mammals, Drosophila and yeast. However, very little research in these areas has been done in plants. Isolation of genes such as Curly leaf suggest a conservation of genes that influence the formation of heterochromatin-like structures. Whether or not this gene influences chromatin/heterochromatin structure in plants, however, remains to be tested. The study of nuclear localization of factors such as COP1 and KN1 is now leading to models for regulating nuclear transport as well as intercellular transport of transcription factors. Further study of the inter- and intracellular movement of these and other transcription factors may provide information on new modes of regulating transcription. In addition to understanding the role chromatin structure and subcellular localization of transcription factors may have on transcription initiation, the biological role of many plant transcription factors remains to be identified. Several approaches may be taken to understand the mechanisms by which transcription factors influence biochemical and physiological processes in the plant. These steps include 1) identification of the DNA-binding sites of the factors as well as the promoter regions which contain these sites. Presently, this approach is limiting in that not many non-coding regions have been sequenced and characterized in detail. Furthermore, the presence of a putative binding site within a promoter does not necessarily indicate that the factor will bind to the site in vivo. 2) Analysis of the binding affinity for a particular factor to a binding site in comparison to other related factors, via in vitro competition assays and quantitative titrations. This will provide information on how strongly these factors are binding to the sites, but without knowledge of all the factors present in a single cell it is difficult to recreate the in vivo conditions. 3) Generation of transgenic plants or microinjection of DNA/RNA to express a particular factor ectopically, reduce expression of the factor via antisense expression, and creation of dominant negative mutants by overexpression of key dimerization domains may provide information concerning what biological pathways these factors influence. 4) Isolation of mutations in particular transcription factors has been extremely informative in floral development. However, this approach usually entails isolation of a mutant due to a phenotype and eventual mutated locus. The cloning of the locus may or may not involve a transcription factor. 5) Many plant transcription factors have been isolated via sequence similarity to other previously identified and/or characterized transcription factors. However, the biological role of may of these factors is not known. In addition to ectopic expression of these factors by creating transgenic plants, isolation of a loss-of-function mutation may provide valuable information concerning the role of this factor in vivo. Many loss-of-function mutations in MADS box genes have led to a better understanding of how the MADS domain proteins interact with one another as well as how they influence floral development. (ABSTRACT TRUNCATED)

Cell Compartmentation↗
PubMed2003

Influence of histochemical stains on quantitative gene expression analysis after laser-assisted microdissection.

Laser-assisted microdissection (LAM) allows isolation of specific cell populations for molecular studies. The combination of LAM and of real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) enables generation of quantitative cell-specific gene expression data. Histochemical stains used to identify cells desired for LAM should provide acceptable morphology and not interfere with RNA or with subsequent molecular analysis techniques. To determine a reliable stain for analysing RNA, using the housekeeping gene, RPL13A, we performed quantitative gene expression analysis of laser microdissected cells from prostatic frozen tissues. The frozen sections were histochemically stained with hematoxylin, methyl green, toluidine blue O and May-Grunwald. After laser microdissection real-time quantitative RT-PCR was performed. Methyl green yielded more RT-PCR product than did the other dyes. The lowest yield of amplification was obtained after May-Grunwald staining. Therefore we recommend methyl green for general use in gene expression analysis, especially when handling small amounts of RNA.

Ethidium↗
PubMed2005

YPED: a proteomics database for protein expression analysis.

We have developed the Yale Protein Expression Database (YPED) to address the storage, retrieval, and integrated analysis of proteomics data generated by Yale's Keck Protein Chemistry and Mass Spectrometry Facility. YPED is Web-accessible and currently handles sample requisition, result reporting and sample comparison for ICAT, DIGE and MUDPIT samples. Sample descriptions are compatible with the evolving MIAPE standards. Peptides and proteins identified using Sequest or Mascot are validated with the Trans-Proteomic Pipeline developed at the Institute of Systems Biology and data from the resulting XML file are stored in the database. Researchers can view, subset and download their data through a secure Web interface.

Databases, Protein↗
PubMed1994

Isolation of a receptor tyrosine kinase (DTK) from embryonic stem cells: structure, genetic mapping and analysis of expression.

Analysis of receptor tyrosine kinases expressed during mouse embryonic stem cell differentiation resulted in the cloning of a receptor designated developmental tyrosine kinase (DTK). The 850 amino acid mature receptor protein comprises an extracellular domain with two immunoglobulin-like motifs and two fibronectin type III modules, a 25 amino acid transmembrane domain and a cytoplasmic region with a catalytic kinase domain. In embryonic stem cells growing in the presence of leukemia inhibitory factor DTK is abundantly expressed and this level of expression is maintained in differentiating embryonic stem cells and cystic embryoid bodies. In mid-gestational embryos (E14.5), DTK RNA is expressed in many tissues including brain, eye, thymus, lung, heart, gut, liver, testis and limbs. In contrast, expression of DTK in adult mice becomes restricted to brain, portions of the gastrointestinal tract, bladder, testis and ovary. There is enrichment of transcripts encoding DTK in purified fetal liver hematopoietic stem cells, when compared with unfractionated fetal liver. The DTK gene maps to mouse chromosome 2, band F.

Amino Acid Sequence↗