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At least 199 records · Page 11Linked to original sources
PubMed2002

Analysis of far-red light-regulated genome expression profiles of phytochrome A pathway mutants in Arabidopsis.

Phytochrome A (phyA) is the primary photoreceptor responsible for various far-red (FR) light-mediated responses. Previous studies have identified multiple phyA signaling mutants, including both positive and negative regulators of the phyA-mediated responses. How these defined intermediates act to mediate FR light responses is largely unknown. Here a cDNA microarray was used to examine effects of those mutations on the far-red light control of genome expression. Clustering analysis of the genome expression profiles supports the notion that phyA signaling may entail a network with multiple paths, controlling overlapping yet distinct sets of gene expression. FHY1, FAR1 and FHY3 most likely act upstream in the phyA signaling network, close to the phyA photoreceptor itself. FIN219, SPA1 and REP1 most likely act somewhere more downstream in the network and control the expression of smaller sets of genes. Further, this study also provides genomics evidence for the partial functional redundancy between FAR1 and FHY3. These two homologous proteins control the expression of a largely overlapping set of genes, and likely act closely together in the phyA-mediated FR light responses.

Arabidopsis↗
PubMed2003

Construction and validation of a Sinorhizobium meliloti whole genome DNA microarray: genome-wide profiling of osmoadaptive gene expression.

Based on the complete Sinorhizobium meliloti genome sequence we established DNA microarrays as a comprehensive tool for systematic genome-wide gene expression analysis in S. meliloti 1021. For these PCR fragment-based microarrays, called Sm6kPCR, a collection of probes for the 6207 predicted protein-coding genes consisting of 6046 gene-specific PCR fragments and 161 70 mer oligonucleotides was arrayed in high density on glass slides. To obtain these PCR fragments primer pairs were designed to amplify internal gene-specific DNA fragments of 80-350 bp. Additionally, these primers were characterized by a 5' extension that allowed for reamplification using standard primers after the first amplification employing the specific primers. In order to ascertain the quality of the Sm6kPCR microarrays and to validate gene expression studies in S. meliloti parallel hybridizations based on RNA samples obtained from cells cultured under identical conditions were performed. In addition, gene expression in S. meliloti in response to an osmotic upshift imposed by the addition of 0.38 M NaCl was monitored. 137 genes were identified showing significant changes in gene expression resulting from the osmotic upshift. From these genes 52 were induced and 85 genes were repressed. Among the genes displaying different RNA levels some functional groups could be identified that are particularly remarkable. Repression was observed for 8 genes related to motility and chemotaxis, 7 genes encoding amino acid biosynthesis enzymes and 15 genes involved in iron uptake whereas 14 genes involved in transport of small molecules and 4 genes related to polysaccharide biosynthesis were induced.

Chromosome Mapping↗
PubMed2005

Characterization of the genome composition of Bartonella koehlerae by microarray comparative genomic hybridization profiling.

Bartonella henselae is present in a wide range of wild and domestic feline hosts and causes cat-scratch disease and bacillary angiomatosis in humans. We have estimated here the gene content of Bartonella koehlerae, a novel species isolated from cats that was recently identified as an agent of human endocarditis. The investigation was accomplished by comparative genomic hybridization (CGH) to a microarray constructed from the sequenced 1.93-Mb genome of B. henselae. Control hybridizations of labeled DNA from the human pathogen Bartonella quintana with a reduced genome of 1.58 Mb were performed to evaluate the accuracy of the array for genes with known levels of sequence divergence. Genome size estimates of B. koehlerae by pulsed-field gel electrophoresis matched that calculated by the CGH, indicating a genome of 1.7 to 1.8 Mb with few unique genes. As in B. quintana, sequences in the prophage and the genomic islands were reported absent in B. koehlerae. In addition, sequence variability was recorded in the chromosome II-like region, where B. koehlerae showed an intermediate retention pattern of both coding and noncoding sequences. Although most of the genes missing in B. koehlerae are also absent from B. quintana, its phylogenetic placement near B. henselae suggests independent deletion events, indicating that host specificity is not solely attributed to genes in the genomic islands. Rather, the results underscore the instability of the genomic islands even within bacterial populations adapted to the same host-vector system, as in the case of B. henselae and B. koehlerae.

Bartonella↗
PubMed2003

Blood genomic expression profile for neuronal injury.

This study determined whether stroke and other types of insults produced a gene expression profile in blood that correlated with the presence of neuronal injury. Adult rats were subjected to ischemic stroke, intracerebral hemorrhage, status epilepticus, and insulin-induced hypoglycemia and compared with untouched, sham surgery, and hypoxia animals that had no brain injury. One day later, microarray analyses showed that 117 genes were upregulated and 80 genes were downregulated in mononuclear blood cells of the "injury" (n = 12) compared with the "no injury" (n = 9) animals. A second experiment examined the whole blood genomic response of adult rats after global ischemia and kainate seizures. Animals with no brain injury were compared with those with brain injury documented by TUNEL and PANT staining. One day later, microarray analyses showed that 37 genes were upregulated and 67 genes were downregulated in whole blood of the injury (n = 4) animals compared with the no-injury (n = 4) animals. Quantitative reverse transcription-polymerase chain reaction confirmed that the vesicular monoamine transporter-2 increased 2.3- and 1.6-fold in animals with severe and mild brain injury, respectively, compared with no-injury animals. Vascular tyrosine phosphatase-1 increased 2.0-fold after severe injury compared with no injury. The data support the hypothesis that there is a peripheral blood genomic response to neuronal injury, and that this blood response is associated with a specific blood mRNA gene expression profile that can be used as a marker of the neuronal damage.

Animals↗
PubMed2004

Pharmacogenomic analysis of cytogenetic response in chronic myeloid leukemia patients treated with imatinib.

PURPOSE: To better understand the molecular basis of cytogenetic response in chronic myeloid leukemia patients treated with imatinib, we studied gene expression profiles from a total of 100 patients from a large, multinational Phase III clinical trial (International Randomized Study of IFN-alpha versus STI571). EXPERIMENTAL DESIGN: Gene expression data for >12,000 genes were generated from whole blood samples collected at baseline (before imatinib treatment) using Affymetrix oligonucleotide microarrays. Cytogenetic response was determined based on the percentage of Ph(+) cells from bone marrow following a median of 13 months of treatment. RESULTS: A genomic profile of response was developed using a subset of individuals that exhibited the greatest divergence in cytogenetic response; those with complete response (0% Ph(+) cells; n = 53) and those with minimal or no response (>65% Ph(+) cells; n = 13). A total of 55 genes was identified that were differentially expressed between these two groups. Using a "leave-one-out" strategy, we identified the optimum 31 genes from this list to use as our genomic profile of response. Using this genomic profile, we were able to distinguish between individuals that achieved major cytogenetic response (0-35% Ph(+) cells) and those that did not, with a sensitivity of 93.4% (71 of 76 patients), specificity of 58.3% (14 of 24 patients), positive predictive value of 87.7%, and negative predictive value of 73.7%. CONCLUSIONS: Interestingly, many of the genes identified appear to be strongly related to reported mechanisms of BCR-ABL transformation and warrant additional research as potential drug targets. The validity and clinical implications of these results should be explored in future studies.

Antineoplastic Agents↗
PubMed2002

Genome-wide profiling of DNA methylation reveals transposon targets of CHROMOMETHYLASE3.

DNA methylation has been implicated in a variety of epigenetic processes, and abnormal methylation patterns have been seen in tumors. Analysis of methylation patterns has traditionally been conducted either by using Southern analysis after cleavage with methyl-sensitive restriction endonucleases or by bisulfite sequencing. However, neither method is practical for analyzing more than a few genes. Here, we describe a simple technique for genome-wide mapping of DNA methylation patterns. Fragmentation by a methyl-sensitive restriction endonuclease is followed by size fractionation and hybridization to microarrays. We demonstrate the utility of this method by characterizing methylation patterns in Arabidopsis methylation mutants. This analysis reveals that CHROMOMETHYLASE3 (CMT3), which was previously shown to maintain CpXpG methylation, preferentially methylates transposons, even when they are present as single copies within the genome. Methylation profiling has potential applications in disease research and diagnostic screening.

Arabidopsis↗
PubMed2006

Whole genome expression profiling of glucose-dependent insulinotropic peptide (GIP)- and adrenocorticotropin-dependent adrenal hyperplasias reveals novel targets for the study of GIP-dependent Cushing's syndrome.

CONTEXT: The mechanisms responsible for the ectopic adrenal expression of glucose-dependent insulinotropic peptide (GIP) receptor (GIPR) in GIP-dependent Cushing's syndrome (CS) are unknown. Chronic adrenal stimulation by ACTH in Cushing's disease or GIP in GIP-dependent ACTH-independent macronodular adrenal hyperplasia both lead to the induction of genes implicated in adrenal proliferation and steroidogenesis. OBJECTIVE: The objective of the study was to identify genes differentially expressed specifically in GIP-dependent CS that could be implicated in the ectopic expression of GIPR. METHODS: We used the Affymetrix U133 plus 2.0 microarray oligochips to compare the whole genome expression profile of adrenal tissues from five cases of GIP-dependent bilateral ACTH-independent macronodular adrenal hyperplasia with CS, one case of GIP-dependent unilateral adenoma with CS, five cases of ACTH-dependent hyperplasias, and a pool of adrenals from 62 normal individuals. RESULTS: After data normalization and statistical filtering, 723 genes with differential expression were identified, including 461 genes or sequences with a known functional implication, classified in eight dominant functional classes. Specific findings include repression of perilipin, the overexpression of 13 G protein-coupled receptors, and the potential involvement of Rho-GTPases. We also isolated 94 probe sets potentially linked to the formation of GIP-dependent nodules adjacent to the diffuse hyperplasia. These included probe sets related to the linker histone H1 and repression of RXRa and CCND2. The expression profiles for eight genes were confirmed by real-time RT-PCR. CONCLUSION: This study identified an extensive series of potentially novel target candidate genes that could be implicated in the molecular mechanisms of ectopic expression of the GIPR as well as in the multistep progression of GIP-dependent CS.

Adolescent↗
PubMed2006

Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues.

BACKGROUND: Formalin-fixed paraffin-embedded (FFPE) tissues represent the largest source of archival biological material available for genomic studies of human cancer. Therefore, it is desirable to develop methods that enable whole genome amplification (WGA) using DNA extracted from FFPE tissues. Multiple-strand Displacement Amplification (MDA) is an isothermal method for WGA that uses the large fragment of Bst DNA polymerase. To date, MDA has been feasible only for genomic DNA isolated from fresh or snap-frozen tissue, and yields a representational distortion of less than threefold. RESULTS: We amplified genomic DNA of five FFPE samples of normal human lung tissue with the large fragment of Bst DNA polymerase. Using quantitative PCR, the copy number of 7 genes was evaluated in both amplified and original DNA samples. Four neuroblastoma xenograft samples derived from cell lines with known N-myc gene copy number were also evaluated, as were 7 samples of non-small cell lung cancer (NSCLC) tumors with known Skp2 gene amplification. In addition, we compared the array comparative genomic hybridization (CGH)-based genome profiles of two NSCLC samples before and after Bst MDA. A median 990-fold amplification of DNA was achieved. The DNA amplification products had a very high molecular weight (> 23 Kb). When the gene content of the amplified samples was compared to that of the original samples, the representational distortion was limited to threefold. Array CGH genome profiles of amplified and non-amplified FFPE DNA were similar. CONCLUSION: Large fragment Bst DNA polymerase is suitable for WGA of DNA extracted from FFPE tissues, with an expected maximal representational distortion of threefold. Amplified DNA may be used for the detection of gene copy number changes by quantitative realtime PCR and genome profiling by array CGH.

Carcinoma, Non-Small-Cell Lung↗
PubMed2001

Hundredfold productivity of genome analysis by introduction of microtemperature-gradient gel electrophoresis.

Genome profiling, which employs temperature-gradient gel electrophoresis (TGGE) for DNA analysis, has recently been developed in identifying species by genotype. However, the performance of this technology like the general applications of TGGE was, though highly informative, limited in its ability due to methodological reasons. This study demonstrates that minimization of the gel for TGGE, to around one-tenth of its conventional size (approximately 2 cm), can be successfully introduced, resulting in a hundredfold higher performance (total evaluation of time, cost, and degree of parallel operations) than that of the conventional. Reproducibility was evaluated from the measures of the pattern similarity scores (PaSS) between band patterns (genome profiles) obtained with the conventional TGGE, and that with micro-TGGE (microTGGE) developed here, after extracting a set of featuring points from genome profiles. Size minimization, which leads to the reduction of the amount of samples required (cost-saving), is another great advantage, enhancing the employment of multicolor fluorescence technology. Since the further development of microbe-related fields such as epidemiology and microbial ecology inevitably require knowledge based on the identification of a great number of species and strains, microbe-related fields will receive the most optimal benefits from the technological improvements attained here.

Bacillus↗
PubMed2005

Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans.

A key challenge of functional genomics today is to generate well-annotated data sets that can be interpreted across different platforms and technologies. Large-scale functional genomics data often fail to connect to standard experimental approaches of gene characterization in individual laboratories. Furthermore, a lack of universal annotation standards for phenotypic data sets makes it difficult to compare different screening approaches. Here we address this problem in a screen designed to identify all genes required for the first two rounds of cell division in the Caenorhabditis elegans embryo. We used RNA-mediated interference to target 98% of all genes predicted in the C. elegans genome in combination with differential interference contrast time-lapse microscopy. Through systematic annotation of the resulting movies, we developed a phenotypic profiling system, which shows high correlation with cellular processes and biochemical pathways, thus enabling us to predict new functions for previously uncharacterized genes.

Animals↗
PubMed2002

Expression profile and genome location of cDNA clones from an infant human trabecular meshwork cell library.

PURPOSE: To delineate the profile of genes expressed in infant human trabecular meshwork and identify candidate genes for glaucoma. METHODS: Human trabecular meshwork cell cultures were established from six young donors. A cDNA library was made from the combined trabecular meshwork mRNA. The end-sequence of random clones was determined by direct sequencing. These sequences were then analyzed by a National Center for Biotechnology Information (NCBI, Bethesda, MD) database search. Nucleotide searches were performed using the BLASTN (ver. 2.1.3; against the nonredundant nucleic acid sequence) and dbEST databases (both provided by NCBI in the public domain at www.ncbi.nlm.nih.gov). RESULTS: Sequences from 1118 clones from this nonamplified trabecular meshwork cDNA library were categorized. Of these, 877 expressed sequence tags (ESTs) (78.7%) were known genes. One hundred thirty-nine ESTs (12.5%) showed close identity to EST sequences reported in the public domain database (dbEST). Thirteen ESTs (1.2%) showed no significant similarity to known genes or ESTs in the public databases and were thus defined as novel ESTs. The most abundant genes expressed by the human trabecular meshwork included ferritin H, eukaryotic translation elongation factor 1-alpha, ferritin L, fibronectin, and TIMP-1. Ferritin H was the most abundant transcript, making up more than 4% of the genes expressed by the human trabecular meshwork. Extracellular matrix proteins were also highly expressed. The chromosome location of the trabecular meshwork ESTs is reported. CONCLUSIONS: A profile of genes expressed by human trabecular meshwork is presented. Thirteen novel ESTs were identified. The combined information obtained from expression analysis and chromosomal localization of trabecular meshwork cDNAs should be valuable in identifying candidate genes for glaucoma.

Cells, Cultured↗
PubMed2002

Genomic scale profiling of autoimmune inflammation in the central nervous system: the nervous response to inflammation.

Using gene microarray technology, we found that inflammation in the central nervous system (CNS) not only induced the expression of many immune-related genes, but also significantly altered the gene expression profile of neural cells. Two unique groups of CNS genes were identified. The first group includes genes encoding ion channels, neural transmitters and growth factors. The second group includes genes that are important for nervous tissue regeneration. Additionally, a distinct pattern of gene expression was also identified in recovering animals. Thus, during autoimmune inflammation, the CNS actively responds to immune attacks by activating its own defense and repair genes.

Animals↗
PubMed2006

Whole genome expression profiling using DNA microarray for determining biocompatibility of polymeric surfaces.

There is an ever increasing need to find surfaces that are biocompatible for applications like medical implants and microfluidics-based cell culture systems. The biocompatibility of five different surfaces with different hydrophobicity was determined using gene expression profiling as well as more conventional methods to determine biocompatibility such as cellular growth rate, morphology and the hydrophobicity of the surfaces. HeLa cells grown on polymethylmethacrylate (PMMA) or a SU-8 surface treated with HNO3-ceric ammonium nitrate (HNO3-CAN) and ethanolamine showed no differences in growth rate, morphology or gene expression profiles as compared to HeLa cells grown in cell culture flasks. Cells grown on SU-8 treated with only HNO3-CAN showed almost the same growth rate (36 +/- 1 h) and similar morphology as cells grown in cell culture flasks (32 +/- 1 h), indicating good biocompatibility. However, more than 200 genes showed different expression levels in cells grown on SU-8 treated with HNO3-CAN compared to cells grown in cell culture flasks. This shows that gene expression profiling is a simple and precise method for determining differences in cells grown on different surfaces that are otherwise difficult to find using conventional methods. It is particularly noteworthy that no correlation was found between surface hydrophobicity and biocompatibility.

Biocompatible Materials↗
PubMed2002

Whole-genome expression profiles identify gene batteries in Drosophila.

Microarray studies make it possible to obtain gene expression data on a whole-genome scale. Arbeitman et al. present microarray data generated at multiple time points throughout Drosophila melanogaster development, and identify new genes engaged in a broad spectrum of processes, including the patterning of the early embryo and senescence in adults.

Animals↗
PubMed2006

Systematic identification of pseudogenes through whole genome expression evidence profiling.

The identification of pseudogenes is an integral and significant part of the genome annotation because of their abundance and their impact on the experimental analysis of functional genes. Most of the computational annotation systems are not optimized for systematic pseudogene recognition, often annotating pseudogenes as functional genes, and users then propagate these errors to subsequent analyses and interpretations. In order to validate gene annotations and to identify pseudogenes that are potentially mis-annotated, we developed a novel approach based on whole genome profiling of existing transcript and protein sequences. This method has two important features: (i) equally detects both processed and non-processed pseudogenes and (ii) can identify transcribed pseudogenes. Applying this method to the human Ensembl gene predictions, we discovered that 2011 (9% of total) Ensembl genes in the categories of known and novel might be pseudogenes based on expression evidence. Of these, 1200 genes are found to have no existing evidence of transcription, and 811 genes are found with transcription evidence but contain significant translation disruption. Approximately 40% of the 2011 identified pseudogenes presented a multi-exon structure, representing non-processed pseudogenes. We have demonstrated the power of whole genome profiling of expression sequences to improve the accuracy of gene annotations.

Computational Biology↗
PubMed2006

Alteration of hTERT full-length variant expression level showed different gene expression profiles and genomic copy number changes in breast cancer.

We analyzed hTERT splicing patterns with respect to telomerase activity in breast cancer. Using a cDNA microarray in 22 cell lines, we observed the difference in expression profiling based on the different levels of full-length variant expression with 71 selected genes. Using 33 known genes that act with the telomerase complex, we performed unsupervised clustering with all cell lines, and found a clustering tendency related to the full-length variant expression level. Using array-based CGH, highly altered genomic copy number changes were found more often in MCF-7 (159 genes) than in MDA-MB-231 (109 genes) and MDA-MB-435 (49 genes), suggesting more genomic changes in MCF-7 cells. On comparing MCF-7 with MDA-MB231 and MDA-MB-435 cell lines, we identified 8 genes with different copy numbers, including dystroglycan, which is located in the p12-21.2 area of chromosome 3. In conclusion, alterations in the level of the full-length variant of hTERT showed different gene expression profiles and genomic copy number changes in breast cancer, which require further study into their cause-and-effect relationship.

Alternative Splicing↗
PubMed2004

The use of Alu-PCR to distinguish between typical pulmonary carcinoids versus classic midgut carcinoids.

Although histologically 'typical' pulmonary and 'classic' midgut carcinoids are similar, the small intestine tumors are more aggressive than their pulmonary counterpart. We believe that in contrast to pulmonary carcinoids arising from Kulchitsky cells, 'classical' midgut carcinoids develop from crypt stem cells that differentiate into endocrine ('classical') or exocrine-endocrine ('non-classical' adenocarcinoid) tumors. The different progenitor cells may determine different malignant potentials between these types of carcinoids. To identify genetic differences between 'classical' midgut and typical pulmonary carcinoids using an Alu-PCR genomic profiling method, we reviewed 54 cases of carcinoid tumors that were surgically removed at Hartford Hospital from 1996 through 2001. Histologically these cases were selected into three groups: i) foregut or pulmonary carcinoids, ii) 'classic' midgut carcinoids of small intestine and iii) multiple typical pulmonary carcinoids. Genomic-profiling of DNA from these cases was performed using an Alu-PCR method. Metastases were observed in 18/20 'classical' intestinal carcinoid tumors, 3/30 pulmonary carcinoids, and 0/4 multiple pulmonary carcinoids. These results confirm that pulmonary carcinoids behave in a more benign fashion than intestinal carcinoid tumors. Using Alu-PCR to profile tumor cell genomic DNA, we showed that 68% of small intestine carcinoids and 58% of pulmonary carcinoids had allelic banding patterns suggestive of either amplification or deletion of gene sequences. Alu-PCR demonstrated loss or gain of genetic sequences that were unique for each examined group. These findings strongly suggest that pulmonary carcinoids differ from their intestinal counterparts.

Alu Elements↗
PubMed2003

Structure-based active site profiles for genome analysis and functional family subclassification.

In previous work, structure-based functional site descriptors, fuzzy functional forms (FFFs), were developed to recognize structurally conserved active sites in proteins. These descriptors identify members of protein families according to active-site structural similarity, rather than overall sequence or structure similarity. FFFs are defined by a minimal number of highly conserved residues and their three-dimensional arrangement. This approach is advantageous for function assignment across broad families, but is limited when applied to detailed subclassification within these families. In the work described here, we developed a method of three-dimensional, or structure-based, active-site profiling that utilizes FFFs to identify residues located in the spatial environment around the active site. Three-dimensional active-site profiling reveals similarities and differences among active sites across protein families. Using this approach, active-site profiles were constructed from known structures for 193 functional families, and these profiles were verified as distinct and characteristic. To achieve this result, a scoring function was developed that discriminates between true functional sites and those that are geometrically most similar, but do not perform the same function. In a large-scale retrospective analysis of human genome sequences, this profile score was shown to identify specific functional families correctly. The method is effective at recognizing the likely subtype of structurally uncharacterized members of the diverse family of protein kinases, categorizing sequences correctly that were misclassified by global sequence alignment methods. Subfamily information provided by this three-dimensional active-site profiling method yields key information for specific and selective inhibitor design for use in the pharmaceutical industry.

Algorithms↗