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Results for “Genomic profiling”

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At least 703 records · Page 39Linked to original sources
PubMed2005

Rational design of a Corynebacterium glutamicum pantothenate production strain and its characterization by metabolic flux analysis and genome-wide transcriptional profiling.

A "second-generation" production strain was derived from a Corynebacterium glutamicum pantothenate producer by rational design to assess its potential to synthesize and accumulate the vitamin pantothenate by batch cultivation. The new pantothenate production strain carries a deletion of the ilvA gene to abolish isoleucine synthesis, the promoter down-mutation P-ilvEM3 to attenuate ilvE gene expression and thereby increase ketoisovalerate availability, and two compatible plasmids to overexpress the ilvBNCD genes and duplicated copies of the panBC operon. Production assays in shake flasks revealed that the P-ilvEM3 mutation and the duplication of the panBC operon had cumulative effects on pantothenate production. During pH-regulated batch cultivation, accumulation of 8 mM pantothenate was achieved, which is the highest value reported for C. glutamicum. Metabolic flux analysis during the fermentation demonstrated that the P-ilvEM3 mutation successfully reoriented the carbon flux towards pantothenate biosynthesis. Despite this repartition of the carbon flux, ketoisovalerate not converted to pantothenate was excreted by the cell and dissipated as by-products (ketoisocaproate, DL-2,3,-dihydroxy-isovalerate, ketopantoate, pantoate), which are indicative of saturation of the pantothenate biosynthetic pathway. Genome-wide expression analysis of the production strain during batch cultivation was performed by whole-genome DNA microarray hybridization and agglomerative hierarchical clustering, which detected the enhanced expression of genes involved in leucine biosynthesis, in serine and glycine formation, in regeneration of methylenetetrahydrofolate, in de novo synthesis of nicotinic acid mononucleotide, and in a complete pathway of acyl coenzyme A conversion. Our strategy not only successfully improved pantothenate production by genetically modified C. glutamicum strains but also revealed new constraints in attaining high productivity.

Bacterial Proteins↗
PubMed2002

Expression profiling and comparative sequence derived insights into lipid metabolism.

Expression profiling and genomic DNA sequence comparisons are increasingly being applied to the identification and analysis of the genes that are involved in lipid metabolism. Not only has genome-wide expression profiling aided in the identification of novel genes that are involved in important processes in lipid metabolism such as sterol efflux, but also the utilization of information from these studies has added to our understanding of the regulation of pathways that participate in the process. Coupled with these gene expression studies, cross-species comparison (a technique used to search for sequences that are conserved through evolution) has proven to be a powerful tool to identify important noncoding regulatory sequences and novel genes that are relevant to lipid biology. An example of the value of this approach was the recent chance discovery of a new apolipoprotein gene (that which encodes apolipoprotein AV) that has dramatic effects on triglyceride metabolism in mice and humans.

Animals↗
PubMed2004

Expression profiling and comparative genomics identify a conserved regulatory region controlling midline expression in the zebrafish embryo.

Differential gene transcription is a fundamental regulatory mechanism of biological systems during development, body homeostasis, and disease. Comparative genomics is believed to be a rapid means for the identification of regulatory sequences in genomes. We tested this approach to identify regulatory sequences that control expression in the midline of the zebrafish embryo. We first isolated a set of genes that are coexpressed in the midline of the zebrafish embryo during somitogenesis stages by gene array analysis and subsequent rescreens by in situ hybridization. We subjected 45 of these genes to Compare and DotPlot analysis to detect conserved sequences in noncoding regions of orthologous loci in the zebrafish and Takifugu genomes. The regions of homology that were scored in nonconserved regions were inserted into expression vectors and tested for their regulatory activity by transient transgenesis in the zebrafish embryo. We identified one conserved region from the connective tissue growth factor gene (ctgf), which was able to drive expression in the midline of the embryo. This region shares sequence similarity with other floor plate/notochord-specific regulatory regions. Our results demonstrate that an unbiased comparative approach is a relevant method for the identification of tissue-specific cis-regulatory sequences in the zebrafish embryo.

Animals↗
PubMed2005

Acute myeloid leukemia with a complex aberrant karyotype is a distinct biological entity characterized by genomic imbalances and a specific gene expression profile.

Acute myeloid leukemia (AML) with a complex aberrant karyotype is a distinct biological entity. It is characterized by: (1) a sharp increase in incidence above age 50; (2) a characteristic pattern of chromosomal gain and, especially, loss, that is, of 5q14q33, 7q32q35, and 17p13, translating into reduced expression of genes in these regions; (3) a unique gene expression pattern including up-regulation of genes involved in DNA repair; (4) a high incidence of TP53 deletions and/or mutations; and (5) an overall unfavorable prognosis. Further unraveling the biology of AML with a complex aberrant karyotype by gene expression profiling may provide deeper insights into the pathogenesis of as well as the reasons for chemoresistance in this AML subtype. These data may be the basis for developing targeted therapeutic strategies to increase the cure rate in patients with AML and a complex aberrant karyotype.

Acute Disease↗
PubMed2026

Genome-wide H3K4me3 profiling of circulating immune cells reveals dynamic epigenetic reprogramming during acute critical COVID-19.

INTRODUCTION: Severe COVID-19 is associated with innate immune dysregulation resembling sepsis-induced immunoparalysis. Epigenetic mechanisms, particularly changes in H3K4me3 enrichment at gene promoters, have been observed in immune tolerance and monocyte dysfunction in sepsis. Whether comparable H3K4me3 alterations occur during acute critical COVID-19 illness has not been investigated. METHODS: In this prospective single-center study, 46 hospitalized COVID-19 patients were enrolled, of whom 27 were treated in the intensive care unit (ICU group) and 19 on the normal ward (non-ICU group). Genome-wide H3K4me3 ChIP-seq was performed on PBMCs at hospital admission (T1) in the total cohort and after seven days (T2) in the ICU group. Monocyte HLA-DR expression and ex vivo TLR-stimulated cytokine secretion were assessed as functional immune readouts. RESULTS: Among 706 differentially bound consensus peaks with promoter association between ICU and non-ICU groups, 704 showed increased H3K4me3 occupancy in ICU patients, predominantly at neutrophil effector gene loci, supported by pathway enrichment of neutrophil degranulation and innate immune activation. Monocyte HLA-DR expression and ex vivo TLR-stimulated IL-6 secretion were persistently reduced throughout the first week of ICU treatment. Longitudinal profiling in the ICU group revealed a shift from an interferon-driven chromatin signature at admission toward sustained innate immune activation and ECM remodeling at day seven. CONCLUSION: This study provides the first genome-wide H3K4me3 characterization of circulating immune cells during acute critical COVID-19, demonstrating that epigenetic reprogramming is an active and dynamic process that mirrors the functional immune dysregulation observed in these patients.

Humans↗
PubMed2005

Promoter addresses: revelations from oligonucleotide profiling applied to the Escherichia coli genome.

BACKGROUND: Transcription is the first step in cellular information processing. It is regulated by cis-acting elements such as promoters and operators in the DNA, and trans-acting elements such as transcription factors and sigma factors. Identification of cis-acting regulatory elements on a genomic scale requires computational analysis. RESULTS: We have used oligonucleotide profiling to predict regulatory regions in a bacterial genome. The method has been applied to the Escherichia coli K12 genome and the results analyzed. The information content of the putative regulatory oligonucleotides so predicted is validated through intra-genomic analyses, correlations with experimental data and inter-genome comparisons. Based on the results we have proposed a model for the bacterial promoter. The results show that the method is capable of identifying, in the E.coli genome, cis-acting elements such as TATAAT (sigma70 binding site), CCCTAT (1 base relative of sigma32 binding site), CTATNN (LexA binding site), AGGA-containing hexanucleotides (Shine Dalgarno consensus) and CTAG-containing hexanucleotides (core binding sites for Trp and Met repressors). CONCLUSION: The method adopted is simple yet effective in predicting upstream regulatory elements in bacteria. It does not need any prior experimental data except the sequence itself. This method should be applicable to most known genomes. Profiling, as applied to the E.coli genome, picks up known cis-acting and regulatory elements. Based on the profile results, we propose a model for the bacterial promoter that is extensible even to eukaryotes. The model is that the core promoter lies within a plateau of bent AT-rich DNA. This bent DNA acts as a homing segment for the sigma factor to recognize the promoter. The model thus suggests an important role for local landscapes in prokaryotic and eukaryotic gene regulation.

Escherichia coli↗
PubMed2005

The human HERC family of ubiquitin ligases: novel members, genomic organization, expression profiling, and evolutionary aspects.

The HERC family of ubiquitin ligases is characterized by the presence of a HECT domain and one or more RCC1-like domains. We report the identification of two novel members, HERC4 and HERC6, and subdivide the family into one group of two large and one group of four small members according to protein size and domain structure. The small members share a similar genomic organization, three of them mapping to chromosomal region 4q22, indicating strong evolutionary cohesions. Phylogenetic analysis reveals that the HERC ancestor emerged in nematodes and that the family expanded throughout evolution. The mRNA expression pattern of the small human members was found to be diverse in selected tissues and cells; overexpressed proteins display a similar cytosolic distribution. These data indicate that the HERC family members exhibit similarities in many aspects, but also sufficient differences indicating functional diversity.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Molecular-timetable methods for detection of body time and rhythm disorders from single-time-point genome-wide expression profiles.

Detection of individual body time (BT) via a single-time-point assay has been a longstanding unfulfilled dream in medicine, because BT information can be exploited to maximize potency and minimize toxicity during drug administration and thus will enable highly optimized medication. To achieve this dream, we created a "molecular timetable" composed of >100 "time-indicating genes," whose gene expression levels can represent internal BT. Here we describe a robust method called the "molecular-timetable method" for BT detection from a single-time-point expression profile. The power of this method is demonstrated by the sensitive and accurate detection of BT and the sensitive diagnosis of rhythm disorders. These results demonstrate the feasibility of BT detection based on single-time-point sampling, suggest the potential for expression-based diagnosis of rhythm disorders, and may translate functional genomics into chronotherapy and personalized medicine.

Animals↗
PubMed2006

Genomic organization, expression profile, and characterization of the new protein PRA1 domain family, member 2 (PRAF2).

PRA1 domain family, member 2 (PRAF2) is a new 19 kDa protein with four putative transmembrane (TM) domains. PRAF2 (formerly designated JM4) belongs to a new protein family, which plays a role in the regulation of intracellular protein transport. Recently, PRAF2 was found to interact with the chemokine receptor CCR5. In order to further study the function and regulation of PRAF2, we determined its genomic structure and its protein expression pattern in normal and cancerous human tissues. PRAF2 encodes a 178-residue protein, whose sequence is related to PRAF1 (PRA1/prenylin) and PRAF3 (JWA/GTRAP3-18). The human PRAF2 gene contains three exons separated by two introns and is located on human chromosome Xp11.23. The recombinant PRAF2 protein was readily expressed in Schneider 2 (S2) insect cells, and the native protein was detected in human tissues with strong expression in the brain, small intestine, lung, spleen, and pancreas. The protein was undetectable in tissue of the testes. Strong PRAF2 protein expression was also found in human tumor tissues of the breast, colon, lung, and ovary, with a weaker staining pattern in normal tissues of the same patient. Our studies show for the first time that the CCR5-interacting PRAF2 protein is expressed in several human tissues with a possible function in ER/Golgi transport and vesicular traffic.

Biological Transport↗
PubMed2004

Prediction of similarly acting cis-regulatory modules by subsequence profiling and comparative genomics in Drosophila melanogaster and D.pseudoobscura.

MOTIVATION: To date, computational searches for cis-regulatory modules (CRMs) have relied on two methods. The first, phylogenetic footprinting, has been used to find CRMs in non-coding sequence, but does not directly link DNA sequence with spatio-temporal patterns of expression. The second, based on searches for combinations of transcription factor (TF) binding motifs, has been employed in genome-wide discovery of similarly acting enhancers, but requires prior knowledge of the set of TFs acting at the CRM and the TFs' binding motifs. RESULTS: We propose a method for CRM discovery that combines aspects of both approaches in an effort to overcome their individual limitations. By treating phylogenetically footprinted non-coding regions (PFRs) as proxies for CRMs, we endeavor to find PFRs near co-regulated genes that are comprised of similar short, conserved sequences. Using Markov chains as a convenient formulation to assess similarity, we develop a sampling algorithm to search a large group of PFRs for the most similar subset. When starting with a set of genes involved in Drosophila early blastoderm development and using phylogenetic comparisons of Drosophila melanogaster and D.pseudoobscura genomes, we show here that our algorithm successfully detects known CRMs. Further, we use our similarity metric, based on Markov chain discrimination, in a genome-wide search, and uncover additional known and many candidate early blastoderm CRMs. AVAILABILITY: Software is available via http://arep.med.harvard.edu/enhancer

Algorithms↗
PubMedDate not supplied

Optimized blood cell profiling method for genomic biomarker discovery using high-density microarray.

High-quality biomarkers for disease progression, drug efficacy and toxicity liability are essential for improving the efficiency of drug discovery and development. The identification of drug-activity biomarkers is often limited by access to and the quantity of target tissue. Peripheral blood has increasingly become an attractive alternative to tissue samples from organs as source for biomarker discovery, especially during early clinical studies. However, given the heterogeneous blood cell population, possible artifacts from ex vivo activations, and technical difficulties associated with overall performance of the assay, it is challenging to profile peripheral blood cells directly for biomarker discovery. In the present study, Applied BioSystems' blood collection system was evaluated for its ability to isolate RNA suitable for use on the Affymetrix microarray platform. Blood was collected in a TEMPUS tube and RNA extracted using an ABI-6100 semi-automated workstation. Using human and rat whole blood samples, it was demonstrated that the RNA isolated using this approach was stable, of high quality and was suitable for Affymetrix microarray applications. The microarray data were statistically analysed and compared with other blood protocols. Minimal haemoglobin interference with RNA labelling efficiency and chip hybridization was found using the TEMPUS tube and extraction method. The RNA quality, stability and ease of handling requirement make the TEMPUS tube protocol an attractive approach for expression profiling of whole blood to support target and biomarker discovery.

Animals↗
PubMed2004

Genome-wide expression profiling of RNA interference of hepatitis B virus gene expression and replication.

Small interfering RNA (siRNA) has been used repeatedly to down-regulate viral gene expression and inhibit viral replication in mammalian cells. In this study, we showed that siRNAs specific for two conserved regions within the hepatitis B S antigen (HBsAg) gene can inhibit antigen production in two human liver cell lines which constitutively produce and secrete HBsAg. The inhibitory effect was concentration dependent for both PLC/PRF/5 and 2.2.15 cells. Decreases in the corresponding viral transcript levels were observed. The inhibitory effect was observed within 24 h and was still evident 7 days after the initial treatment with siRNA. A significant reduction in virion production was also observed for the 2.2.15 cells. A critical consideration in this study was the specificity of the siRNA-mediated inhibition. To address this, we first examined the effects on cell growth and viability. These were not affected in either cell line. cDNA microarrays were also used to examine genome-wide changes in gene regulation. No significant off-target gene regulation was observed in either cell line. Our findings thus indicate that siRNA can specifically mediate the down-regulation of viral gene expression leading to a reduction in virion production.

Gene Expression Profiling↗
PubMed2026

Parallel single-cell host immune profiling and pathogen genomic characterization in Klebsiella-associated sepsis: a pilot study.

OBJECTIVES: Sepsis is a life-threatening syndrome characterized by profound immune dysregulation and substantial biological heterogeneity. Here, we conducted a pilot study to explore host immune remodeling in Klebsiella-associated sepsis by combining single-cell RNA sequencing of peripheral blood mononuclear cells with whole-genome sequencing of the corresponding bloodstream isolates. METHODS: In this prospective observational pilot study, we analyzed peripheral blood mononuclear cells (PBMCs) from two patients with Klebsiella-associated sepsis and two healthy controls (HC) using single-cell RNA sequencing. PBMC composition, differential transcriptional responses, and pathway analysis were assessed across immune subsets. The corresponding bloodstream isolates were characterized by phenotypic antimicrobial susceptibility testing and whole-genome sequencing. RESULTS: Compared to HC, septic patients showed expansion of the myeloid compartment and contraction of the NK/T compartment. High-resolution analysis suggested shifts within lymphoid populations. At the transcriptional level, sepsis was associated with compartment-specific enrichment of interferon-related and host-defence pathways, as well as oxidative phosphorylation, ATP synthesis, and mitochondrial electron transport signatures across multiple PBMC subsets. Classical monocytes exhibited a coordinated decrease in MHC class II-related transcripts. The sepsis-associated isolates were identified as Klebsiella pneumoniae and Klebsiella variicola and were notable for overall antimicrobial susceptibility, limited resistomes, and absence of canonical hypervirulence determinants. CONCLUSION: Our data provide a preliminary description of immune remodeling during Klebsiella-associated sepsis and suggest that severe clinical disease may be associated with isolates lacking classical multidrug-resistance or hypervirulence features. These findings should be interpreted as preliminary and hypothesis-generating and require validation in larger cohorts with detailed clinical severity assessment.

Female↗
PubMed2006

Genome-wide replication profiles of S-phase checkpoint mutants reveal fragile sites in yeast.

The S-phase checkpoint kinases Mec1 and Rad53 in the budding yeast, Saccharomyces cerevisiae, are activated in response to replication stress that induces replication fork arrest. In the absence of a functional S-phase checkpoint, stalled replication forks collapse and give rise to chromosome breakage. In an attempt to better understand replication dynamics in S-phase checkpoint mutants, we developed a replication origin array for budding yeast that contains 424 of 432 previously identified potential origin regions. As expected, mec1-1 and rad53-1 mutants failed to inhibit late origin activation. Surprisingly however, 17 early-firing regions were not replicated efficiently in these mutants. This was not due to a lack of initiation, but rather to problems during elongation, as replication forks arrested in close proximity to these origins, resulting in the accumulation of small replication intermediates and eventual replication fork collapse. Importantly, these regions were not only prone to chromosome breakage in the presence of exogenous stress but also in its absence, similar to fragile sites in the human genome.

Cell Cycle Proteins↗
PubMed2002

Genome-wide transcriptional profiling in a histidine kinase mutant of Helicobacter pylori identifies members of a regulon.

To identify putative members of a regulon controlled by the H. pylori sensory histidine kinase HP0164 (HK0164), we constructed HK0164 null mutant H. pylori strains and analyzed bacterial gene transcription using DNA arrays. Seven genes were differentially expressed in multiple HK0164 mutant strains compared to their expression in control strains. Strain-dependent variations in differential expression were also detected. These results indicate that the signal transduction circuit utilizing HK0164 controls the transcription of at least seven genes in H. pylori.

Gene Expression Regulation, Bacterial↗
PubMed2001

Global profiling of gene expression in cancer using genomics and proteomics.

Global profiling of gene expression at the genomic level using DNA microarrays and at the protein level using a variety of technologies have followed separate paths. Studies of gene expression for several types of tumors, using DNA microarrays, have been published recently that are informative with respect to delineating distinct patterns of gene expression among subsets of related tumors. Proteomics-based profiling uniquely allows delineation of global changes in protein expression patterns resulting from transcriptional and post-transcriptional control, post-translational modifications and shifts in proteins between different cellular compartments. Some of the current technologies for proteome profiling and the application of proteomics to the analysis of tumor tissues are reviewed. Given that comprehensive expression profiles obtained using genomics and proteomics are highly complementary, a combined approach to profiling may well uncover expression patterns that could not be predicted using a single approach.

Gene Expression Profiling↗
PubMed2000

Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray.

cDNA microarray technology has been increasingly used to monitor global gene expression patterns in various tissues and cell types. However, applications to mammalian development have been hampered by the lack of appropriate cDNA collections, particularly for early developmental stages. To overcome this problem, a PCR-based cDNA library construction method was used to derive 52,374 expressed sequence tags from pre- and peri-implantation embryos, embryonic day (E) 12.5 female gonad/mesonephros, and newborn ovary. From these cDNA collections, a microarray representing 15,264 unique genes (78% novel and 22% known) was assembled. In initial applications, the divergence of placental and embryonic gene expression profiles was assessed. At stage E12.5 of development, based on triplicate experiments, 720 genes (6.5%) displayed statistically significant differences in expression between placenta and embryo. Among 289 more highly expressed in placenta, 61 placenta-specific genes encoded, for example, a novel prolactin-like protein. The number of genes highly expressed (and frequently specific) for placenta has thereby been increased 5-fold over the total previously reported, illustrating the potential of the microarrays for tissue-specific gene discovery and analysis of mammalian developmental programs.

Amino Acid Sequence↗