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PubMed1999

Genome-scale Gene Expression Analysis and Pathway Reconstruction in KEGG.

The massively parallel hybridization technologies by DNA chips and microarrays make it possible to monitor expression patterns of the whole set of genes in a genome under various conditions. The vast amount of data generated by such technologies necessitates the development of a new database management system that integrates expression data with other molecular biology databases and various analysis tools. We report here an extension of our KEGG (Kyoto Encyclopedia of Genes and Genomes) and DBGET/LinkDB systems for analyzing gene expression data in conjunction with pathway information and genomic information. It is now possible to make use of expression data for the reconstruction of pathways from the complete genome sequences.

Journal Article↗
PubMed2006

Outlier sums for differential gene expression analysis.

We propose a method for detecting genes that, in a disease group, exhibit unusually high gene expression in some but not all samples. This can be particularly useful in cancer studies, where mutations that can amplify or turn off gene expression often occur in only a minority of samples. In real and simulated examples, the new method often exhibits lower false discovery rates than simple t-statistic thresholding. We also compare our approach to the recent cancer profile outlier analysis proposal of Tomlins and others (2005).

Computer Simulation↗
PubMed2004

Large-scale gene expression analysis of human skeletal myoblast differentiation.

To study pathways involved in human skeletal myogenesis, we profiled gene expression in human primary myoblast cells derived from three individuals using both oligonucleotide and cDNA microarrays. Following stringent statistical testing (false-positive rate 0.4%), we identified 146 genes differentially expressed over time. Interestingly, 86 of these genes have not been reported to be involved in myogenesis in mouse cell lines. This demonstrates the additional value of human primary cell cultures in the study of muscle differentiation. Many of the identified genes play a role in muscle regeneration, indicating the close relationship of this process with muscle development. In addition, we found overlap with expression profiling studies in muscle from Duchenne muscular dystrophy patients, confirming ongoing muscle regeneration in Duchenne muscular dystrophy. Further study of these genes can bring new insights into the process of muscle differentiation, and they are candidate genes for neuromuscular disorders with an as yet unidentified cause.

Cell Differentiation↗
PubMed2006

The MYB transcription factor superfamily of Arabidopsis: expression analysis and phylogenetic comparison with the rice MYB family.

MYB proteins are a superfamily of transcription factors that play regulatory roles in developmental processes and defense responses in plants. We identified 198 genes in the MYB superfamily from an analysis of the complete Arabidopsis genome sequence, among them, 126 are R2R3-MYB, 5 are R1R2R3-MYB, 64 are MYB-related, and 3 atypical MYB genes. Here we report the expression profiles of 163 genes in the Arabidopsis MYB superfamily whose full-length open reading frames have been isolated. This analysis indicated that the expression for most of the Arabidopsis MYB genes were responsive to one or multiple types of hormone and stress treatments. A phylogenetic comparison of the members of this superfamily in Arabidopsis and rice suggested that the Arabidopsis MYB superfamily underwent a rapid expansion after its divergence from monocots but before its divergence from other dicots. It is likely that the MYB-related family was more ancient than the R2R3-MYB gene family, or had evolved more rapidly. Therefore, the MYB gene superfamily represents an excellent system for investigating the evolution of large and complex gene families in higher plants. Our comprehensive analysis of this largest transcription factor superfamily of Arabidopsis and rice may help elucidate the possible biological roles of the MYB genes in various aspects of flowering plants.

Amino Acid Sequence↗
PubMed2006

Expression analysis of the fpr (ferredoxin-NADP+ reductase) gene in Pseudomonas putida KT2440.

The ferredoxin-NADP+ reductase (fpr) participates in cellular defense against oxidative damage. The fpr expression in Pseudomonas putida KT2440 is induced by oxidative and osmotic stresses. FinR, a LysR-type transcriptional factor near the fpr gene in the P. putida KT2440 genome, is required for induction of the fpr under both conditions. We have shown that the fpr and finR gene products can counteract the effects of oxidative and osmotic stresses. Interestingly, FinR-independent expression occurs either during a long period of incubation with paraquat or with high concentrations of oxidative stress agent. This result indicates that there may be additional regulators present in the P. putida KT2440 genome. In contrast to in vivo expression kinetics of fpr from the plant pathogen, Pseudomonas syringae, the fpr gene from P. putida KT2440 exhibited unusually prolonged expression after oxidative stress. Transcriptional fusion and Northern blot analysis studies indicated that the FinR is negatively autoregulated. Expression of the fpr promoter was higher in minimal media than in rich media during exponential phase growth. Consistent with this result, the fpr and finR mutants had a long lag phase in minimal media in contrast to wild-type growth characteristics. Antioxidants such as ascorbate could increase the growth rate of all tested strains in minimal media. This result confirmed that P. putida KT2440 experienced more oxidative stress during exponential growth in minimal media than in rich media. Endogenous promoter activity of the fpr gene is much higher during exponential growth than during stationary growth. These findings demonstrate new relationships between fpr, finR, and the physiology of oxidative stress in P. putida KT2440.

Cell Proliferation↗
PubMed2005

Genetical genomics: combining genetics with gene expression analysis.

The biological mechanisms that link genetic variation and its phenotypic outcome stand as a central puzzle in biology. Geneticists have usually approached this problem by trying to identify genetic variants that underlie the trait in question. Ten years ago, microarray technology opened a second front by making it possible to compare expression levels for most active genes under a variety of genetic and environmental conditions. A typical study reveals up- or down-regulation of genes or pathways associated with a phenotype (case/control) or condition (treated/untreated). In the past few years, a number of groups have started to combine gene expression studies with genetic linkage analysis, leading to a new synergy between these approaches. In this strategy, expression levels are treated as quantitative phenotypes and genetic variants that influence gene expression are sought. Several studies have shown that mRNA levels for many genes are heritable, thus amenable to genetic analysis. Quantitative trait loci mapping efforts have led to the initial characterization of genetic regulation in 'cis' probably because of variants in the gene's own regulatory regions, as well as in 'trans', i.e. by loci elsewhere in the genome. The existence of some 'master regulators' that each affects expression levels of hundreds of genes is an important finding that will surely enrich our understanding of regulatory networks. Although this novel field is still developing, understanding the genetic basis of molecular phenotypes such as gene expression is expected to shed light on the intermediate processes that connect genotype to cellular and organismal traits and represents a critical step towards true systems biology.

Animals↗
PubMed2002

Evidence for distinct pathomechanisms in B-cell chronic lymphocytic leukemia and mantle cell lymphoma by quantitative expression analysis of cell cycle and apoptosis-associated genes.

The B-cell lymphoproliferative malignancies B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (MCL) share characteristics, including overlapping chromosomal aberrations with deletions on chromosome bands 13q14, 11q23, 17p13, and 6q21 and gains on chromosome bands 3q26, 12q13, and 8q24. To elucidate the biochemical processes involved in the pathogenesis of B-CLL and MCL, we analyzed the expression level of a set of genes that play central roles in apoptotic or cell proliferation pathways and of candidate genes from frequently altered genomic regions, namely ATM, BAX, BCL2, CCND1, CCND3, CDK2, CDK4, CDKN1A, CDKN1B, E2F1, ETV5, MYC, RB1, SELL, TFDP2, TNFSF10, and TP53. Performing real-time quantitative reverse transcription polymerase chain reaction in a panel of patients with MCL and B-CLL and control samples, significant overexpression and underexpression was observed for most of these genes. Statistical analysis of the expression data revealed the combination of CCND1 and CDK4 as the best classifier concerning separation of both lymphoma types. Overexpression in these malignancies suggests ETV5 as a new candidate for a pathogenic factor in B-cell lymphomas. Characteristic deregulation of multiple genes analyzed in this study could be combined in a comprehensive picture of 2 distinctive pathomechanisms in B-CLL and MCL. In B-CLL, the expression parameters are in strong favor of protection of the malignant cells from apoptosis but did not provide evidence for promoting cell cycle. In contrast, in MCL the impairment of apoptosis induction seems to play a minor role, whereas most expression data indicate an enhancement of cell proliferation.

Adult↗
PubMed2003

Novel approaches to gene expression analysis of active polyarticular juvenile rheumatoid arthritis.

Juvenile rheumatoid arthritis (JRA) has a complex, poorly characterized pathophysiology. Modeling of transcriptosome behavior in pathologic specimens using microarrays allows molecular dissection of complex autoimmune diseases. However, conventional analyses rely on identifying statistically significant differences in gene expression distributions between patients and controls. Since the principal aspects of disease pathophysiology vary significantly among patients, these analyses are biased. Genes with highly variable expression, those most likely to regulate and affect pathologic processes, are excluded from selection, as their distribution among healthy and affected individuals may overlap significantly. Here we describe a novel method for analyzing microarray data that assesses statistically significant changes in gene behavior at the population level. This method was applied to expression profiles of peripheral blood leukocytes from a group of children with polyarticular JRA and healthy control subjects. Results from this method are compared with those from a conventional analysis of differential gene expression and shown to identify discrete subsets of functionally related genes relevant to disease pathophysiology. These results reveal the complex action of the innate and adaptive immune responses in patients and specifically underscore the role of IFN-gamma in disease pathophysiology. Discriminant function analysis of data from a cohort of patients treated with conventional therapy identified additional subsets of functionally related genes; the results may predict treatment outcomes. While data from only 9 patients and 12 healthy controls was used, this preliminary investigation of the inflammatory genomics of JRA illustrates the significant potential of utilizing complementary sets of bioinformatics tools to maximize the clinical relevance of microarray data from patients with autoimmune disease, even in small cohorts.

Journal Article↗
PubMed2004

Expression analysis of the Arabidopsis peroxidase multigenic family.

Class III peroxidases form a numerous multigenic family in higher plants, whose expression is particularly sensitive to internal or external events. Arabidopsis thaliana genome harbours 73 genes encoding peroxidases. Since they exhibit homologies ranging from 28% to 93% at the nucleotide level, the risk of cross-hybridisation may be important when measuring the level of transcripts by blotting techniques, using whole cDNA sequences. We developed a procedure to assess the expression of all peroxidase genes on one membrane, with a high specificity. The method was based on the determination for each gene of a short specific sequence (amplicon) exhibiting at the most 70% homology with any other sequences of the Arabidopsis genome. Amplicons specific for each of the 73 peroxidase genes and two pseudogenes were blotted on a nylon membrane that was hybridised with radiolabelled cDNA libraries prepared from mRNAs of Arabidopsis roots, stems, leaves and flowers. Many genes were expressed at a low level, often in all organs, while sixteen genes were rather strongly expressed, in two to four organs. Some genes with no ESTs reported in databases were found to be expressed and this was confirmed by RT-PCR. Isoelectric focusing analysis revealed that the isoperoxidase pattern was similar in leaves, stems and flowers, but was quite different in roots. To our knowledge, only one similar study has been performed on the cytochrome P450 family, using microarrays, but this is the first work describing the expression profile of a whole large multigenic family using specific macroarrays.

Arabidopsis↗
PubMed2004

Comparative gene expression analysis of murine retina and brain.

PURPOSE: Several high-throughput studies have described gene expression in the central nervous system (CNS), and recently there has been increasing interest in analyzing how gene expression compares in different regions of the CNS. As the retina is often used as a model system to study CNS development and function, we compared retina and brain gene expression using microarray analyses. METHODS: Mouse retina, brain and liver RNA was hybridized to a custom cDNA microarray containing 5,376 genes and ESTs, and the data from the quantified scanned images were analyzed using Bioconductor and SAM. Preferential retina expression was confirmed by real-time PCR. The cellular distribution of genes newly identified as retina enriched genes was determined by immunohistochemistry. RESULTS: Using stringent statistical analyses we identified 733 genes that were preferentially expressed in retina and 389 in brain. The retina-liver hybridizations identified an additional 837 retina enriched genes. The cellular distribution in the retina was determined for two genes that had not previously been reported to be expressed in the retina, the transcription regulatory proteins EWS and PCPB1. Both proteins were found primarily in the inner nuclear layer. Finally, a comparison of the microarray data to publicly available SAGE and EST library databases demonstrated only limited overlap of the sets of retina enriched genes identified by the different methodologies. The preferential retinal expression of a subset of genes from the microarray, which were not identified as differentially expressed by other methods, was confirmed by quantitative PCR. CONCLUSIONS: The finding of differences in the groups of identified retina enriched genes from the various profiling techniques supports the use of multiple approaches to obtain a more complete description of retinal gene expression. Characterization of gene expression profiles of retina and brain may facilitate the understanding of the processes that underlie differences between the retina and other parts of the central nervous system.

Animals↗
PubMed1989

The cDNA structure and expression analysis of the genes for the cysteine proteinase inhibitor cystatin C and for beta 2-microglobulin in rat brain.

Tissue patterns of gene expression were analyzed by measuring mRNA levels and incorporation of radioactive amino acids for cystatin C and beta 2-microglobulin, the two extracellular proteins in the brain with the highest ratio of concentration in cerebrospinal fluid over that in blood plasma. The primary structure of rat cystatin C mRNA from choroid plexus was determined by nucleotide sequencing of cloned cDNA and the tissue patterns of gene expression were analysed by RNA blot analysis and in situ hybridization. Cystatin C was found to be composed of 120 amino acids and to contain a potential site for N-linked glycosylation. The tissue with the highest cystatin C mRNA level was the choroid plexus of the brain. Cystatin C mRNA was also detected in lower levels in other areas of the brain, testis, epididymis, seminal vesicles, prostate, ovary, submandibular gland, and, in trace amounts, in liver. Choroid plexus pieces in culture secreted radioactive cystatin C when incubated with radioactive leucine. Rat beta 2-microglobulin cDNA was cloned and identified by nucleotide sequencing and comparison of the obtained sequence with that of mouse and human beta 2-microglobulin cDNA. Tissue levels of beta 2-microglobulin mRNA in the rat were measured by hybridization to rat beta 2-microglobulin cDNA. The highest levels of beta 2-microglobulin mRNA were observed in liver and choroid plexus. Other parts of the brain and testis contained lower levels of beta 2-microglobulin mRNA.

Amino Acid Sequence↗
PubMed2004

Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue.

Human mesenchymal stem cells (MSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle and neuron. This differentiation potential makes MSC excellent candidates for cell-based tissue engineering. In this study, we have examined phenotypes and gene expression profile of the human adipose tissue-derived stromal cells (ATSC) in the undifferentiated states, and compared with that of bone marrow stromal cells (BMSC). ATSC were enzymatically released from adipose tissues from adult human donors and were expanded in monolayer with serial passages at confluence. BMSC were harvested from the metaphysis of adult human femur. Flowcytometric analysis showed that ATSC have a marker expression that is similar to that of BMSC. ATSC expressed CD29, CD44, CD90, CD105 and were absent for HLA-DR and c-kit expression. Under appropriate culture conditions, MSC were induced to differentiate to the osteoblast, adipocyte, and chondrogenic lineages. ATSC were superior to BMSC in respect to maintenance of proliferating ability, and microarray analysis of gene expression revealed differentially expressed genes between ATSC and BMSC. The proliferating ability and differentiation potential of ATSC were variable according to the culture condition. The similarities of the phenotypes and the gene expression profiles between ATSC and BMSC could have broad implications for human tissue engineering.

Adipocytes↗
PubMed1985

Complex regulation of class II gene expression: analysis with class II mutant cell lines.

Several Ia-negative variants of a homozygous Iad-expressing antigen-presenting B lymphoma cell line, M12, have been obtained by repeated cycles of negative immunoselection after mutagenesis with ethylmethane sulfonate or gamma-irradiation. Two such Iad-negative cell lines, selected with a mixture of alpha I-Ad and alpha I-Ed monoclonal antibodies, failed to present antigen to all cloned Iad-restricted T cells tested, whereas the third cell line, selected with alpha I-Ad reagents only, stimulated I-Ed but not I-Ad-restricted T cells. The mutations in all three cell lines resulted in the absence of RNA specific for the A beta d gene. In addition, two-dimensional gel electrophoresis of immunoprecipitates from one of the I-Ed-negative cell lines demonstrated the presence of intracytoplasmic Ed polypeptides that exhibited significantly decreased amounts of oligosaccharide-induced heterogeneity. The introduction of class II A beta b and A alpha b genes by DNA-mediated transfection resulted in the serologic and functional expression of a class II I-Ab molecule but not the reexpression of the endogenous class II molecules; thus a transacting regulatory element is unlikely to be the target of the mutagenic event. The analysis of these and other Ia variant cell lines may prove useful in understanding the molecular mechanisms that control the expression of class II molecules in B cells.

Animals↗
PubMed2001

Genomic structure, chromosome mapping and expression analysis of the human AXIN2 gene.

Conductin is a Wnt signalling protein and serves as a negative regulator of beta-catenin stability. We have previously isolated the human homolog (AXIN2) of the murine conductin gene and shown that it is mutated in colorectal cancer (CRC) with defective mismatch repair (MMR). Here we report the detailed genomic structure of this gene by analysis of cDNA and genomic clones. The gene spans > or =25 kb containing ten exons ranging from 96 bp to 904 bp. All splice donor and acceptor sites conform to the GT/AG rule. FISH (Fluorescence in situ Hybridization) analysis localized this gene to human chromosome band 17q24 and showed that it exists as a single copy in the human genome. Northern blot analysis from different human organs demonstrated that the AXIN2 gene is highly expressed in human thymus, prostate, testis, small intestine and ovarian tissues but expressed at a lower level in colon. The data reported here provides a framework for further analysis of this important Wnt signalling protein in vertebrate development and tumorigenesis.

Axin Protein↗
PubMed2003

Identification of genes regulated during osteoblastic differentiation by genome-wide expression analysis of mouse calvaria primary osteoblasts in vitro.

Although several independent studies of gene expression patterns during osteoblast differentiation in cultures from calvaria and other in vitro models have been reported, only a small portion of the mRNAs expressed in osteoblasts have been characterized. We have previously analyzed the behavior of several known markers in osteoblasts, using Affymetrix GeneChip murine probe arrays (27,000 genes). In the present study we report larger groups of transcripts displaying significant expression modulation during the culture of osteoblasts isolated from mice calvaria. The expression profiles of 601 such regulated genes, classified in distinct functional families, are presented and analyzed here. Although some of these genes have previously been shown to play important roles in bone biology, the large majority of them have never been demonstrated to be regulated during osteoblast differentiation. Despite the fact that the precise involvement of these genes in osteoblast differentiation and function needs to be evaluated, the data presented herein will aid in the identification of genes that play a significant role in osteoblasts. This will provide a better understanding of the regulation of osteoblast differentiation and maturation.

Animals↗
PubMed1994

Cloning and expression analysis of the cDNA encoding rat Zn-alpha 2-glycoprotein.

We report here the nucleotide (nt) sequence of a rat liver cDNA encoding Zn-alpha 2-glycoprotein (Zn-alpha 2-gp), a plasma protein with a high degree of sequence similarity to class-I major histocompatibility complex (MHC) antigens. The deduced amino acid (aa) sequence contains the coding information for 293 aa residues and shows 60% identity with the aa sequence of human Zn-alpha 2-gp and 35% identity with that corresponding to the extracellular domains of RT1, a rat class-I MHC antigen. Northern blot analysis showed that rat Zn-alpha 2-gp is expressed in liver, but not in a wide number of tissues, including prostate, mammary gland, kidney, intestine, lung, pancreas, ovary, uterus, thyroid, placenta, spleen, brain and heart.

Amino Acid Sequence↗
PubMed2004

Increased expression of Ki-67 in mantle cell lymphoma is associated with de-regulation of several cell cycle regulatory components, as identified by global gene expression analysis.

BACKGROUND AND OBJECTIVES: Mantle cell lymphoma (MCL) is an aggressive disease. Patients with this malignancy have a median survival of 3 years. To better understand disease progression, which is characterized by increased proliferation, we analyzed the gene expression of MCL with different proliferative indices, as determined by immunohistochemical staining for Ki-67. Furthermore, primary and relapsed tumors were compared to identify the possible growth advantages possessed by cells which persist after therapy and which might evolve into a tumor relapse. DESIGN AND METHODS: Twenty-one samples of MCL were analyzed, using the Affymetrix U95Av2 chip, containing probes for approximately 12,000 transcripts. Samples with a high versus low fraction of Ki-67+ cells were compared as were relapsed versus primary tumors. Immunohistochemistry was used to confirm the expression of some gene products. RESULTS: A distinct genetic signature, consisting of 32 genes, was found when comparing Ki-67high with Ki-67low MCL. The signature consisted of genes involved in cellular processes, such as mitotic spindle formation, gene transcription and cell cycle regulation, e.g. components of the p53 and retinoblastoma protein (pRb) pathways. Of note, cyclin D1, the hallmark of MCL, as well as Ki-67 were up-regulated in the samples with a high proliferative index. Comparing primary vs. relapsed tumors, 26 individual genes were found, several involved in cell adhesion. Furthermore, increased expression of transferrin receptor was found in the relapsed tumors. INTERPRETATION AND CONCLUSIONS: A genetic signature distinguishing Ki-67high MCL from Ki-67low was established. The generated signature was used to assign new MCL samples to the high proliferative group, validating the association between these genes and proliferation in MCL.

Cell Cycle Proteins↗