Genome-wide analysis relating expression level with protein subcellular localization.
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Biological Transport↗
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Expression analysis of the 5'-upstream region of mouse P/Q-type Ca(2+) channel alpha( lA) subunit gene fused to Escherichia coli lacZ reporter gene in the spinal cord using transgenic mice.
The P/Q-type Ca(2+) channel alpha(1A) subunit is expressed in spinal cord including ventral motor neurons and interneurons and dorsal horn. To identify the transcriptional mechanisms of the mouse alpha(IA) subunit gene in spinal cord, transgenic mice carrying a 0.5, 1.5, 3.0 or 6.3-kb 5'-upstream region fused to the Escherichia coli lacZ reporter gene were examined. Transgenic mice carrying the 3.0-kb region expressed the reporter gene in dorsal horn and interneurons of ventral horn, although those with the 0.5-kb, 1.5-kb or 6.3-kb region did not. No transgenic mice expressed the reporter gene in motor neurons of ventral horn. These results suggest that in spinal cord, the expression mechanisms of the alpha(1A) subunit gene are complex, involving both positive and negative cis-regulatory elements, and the 6.3-kb 5'-upstream region alone is not sufficient for the expression.
5' Untranslated Regions↗
Identification of differentially expressed genes in Con A-activated carp (Cyprinus carpio L.) leucocytes.
A cDNA library was constructed from the message RNA (mRNA) obtained from Con A-induced head kidney (HK) leucocytes of carp (Cyprinus carpio L.). Differential screening of the cDNA was carried out by hybridization against the total cDNA probes from normal, Con A-uninduced HK leucocytes or Con A-induced HK leucocytes of carp. The differential expression patterns of certain cDNA clones were confirmed by Southern-blot and Northern-blot analysis. Single-pass of the sequencing analysis and homology search in Genbank (EMBL) revealed those differentially expressed cDNA clones encode for cytochrome c oxidase sub-unit II and III (COII and COIII), elongation factor-1 beta (EF-1 beta), bleomycin hydrolase (BH), heat shock cognate protein 70 (HSC70) and 16S ribosomal RNA (16S rRNA).
Amino Acid Sequence↗
Expression pattern of Rhizobium etli ccmIEFH genes involved in c-type cytochrome maturation.
In different bacterial species, ccmIEFH genes have been suggested to code for subunits of a bacterial haem-lyase catalyzing the covalent attachment of haem to c-type apoproteins. In Rhizobium etli CE3 there are two copies of ccmIEFH: one in the chromosome and the other located in plasmid pf. However, the null phenotype of chromosomal ccmF mutant indicates that the gene locus of plasmid pf is not functional. Two ccmI chromosomal mutants, previously isolated, produced detectable levels of c-type cytochromes under certain culture conditions in contrast with the ccmF mutant, suggesting that ccmF could be transcribed independently. The transcriptional organization of ccmIEFH operon was established. Two promoters from the chromosomal locus were mapped by primer extension, one located upstream of ccmI and the second located upstream of ccmF. The regulation of the expression of both promoters was studied using appropriate lacZ gene fusions (ccmI-lacZ and ccmEF-lacZ). The ccmI-lacZ gene fusion was expressed in complex medium, during exponential growth, under microaerobic conditions and in a R. etli mutant that accumulates reducing power, conditions where a higher respiration rate could be limited by c-type cytochrome content. The ccmEF-lacZ fusion was also primarily expressed in complex medium and under microaerophilic conditions. The finding of two independent promoters in this gene locus could suggest that the step catalyzed by CcmFH could be a rate-limiting step for c-type cytochrome assembly under certain culture conditions.
Amino Acid Sequence↗
Mycobacterium tuberculosis H37Rv comparative gene-expression analysis in synthetic medium and human macrophage.
Mycobacteria are intracellular pathogens that survive and grow in host macrophages. Following phagocytosis, sustained intracellular bacterial growth depends on its ability to avoid destruction by macrophage-mediated host defences such as lysosomal enzymes, reactive oxygen and the reactive nitrogen intermediates. This suggests that the interaction between host cell and microbe is delicately balanced, and can be tipped in favour of either organism. The identification of Mycobacterium tuberculosis H37Rv (MTB) genes expressed within host cells would contribute greatly to the development of new strategies to fight tuberculosis. In the present study, we compared MTB gene expression in the course of intra- (human macrophages) and extracellular growth (Sauton's medium) to ascertain whether differences might occur between gene-expression patterns in the two habitats of replication. Using reverse-transcriptase polymerase chain reaction (RT-PCR) on a group of 14 MTB-Complex-specific genes, we found that MT10Sa (a small stable RNA), 35 kDa (unknown), ahpC (alkyl hydroperoxide reductase, AhpC), sigF (alternative RNA Polymerase sigma factor), and katG (catalase-peroxidase, HPI) genes are expressed in both the environments, while Ag85B, Ag85C (members of the Antigen 85 Complex), rpoV (RNA Polymerase sigma factor) and ESAT6 (early secretory antigen, 6 kDa) are expressed only in the in vitro culture; on the other hand, Ag85A (Antigen 85 Complex), rpoB (RNA Polymerase beta sub-unit), pab (Protein antigen b), invA and invB genes (encoding proteins that show homologies with p60 of Listeria monocytogenes) are expressed only inside the macrophage. Positive RT-PCR products on cDNAs for these genomic regions were not obtained from approximately 1000-fold more bacteria grown in Laboratory Broth. Identification of M. tuberculosis genes expressed in response to phagocytosis by human macrophages increases our basic understanding of the host-pathogen interaction, and helps to identify bacterial factors necessary for in vivo survival and growth.
Colony Count, Microbial↗
Glutamate transporters in neonatal cerebellar subarachnoid hemorrhage.
We have previously described the immunoreactivities of glutamate transporters, EAAT4 and GLAST, in the developing human cerebellum. In the present report, we demonstrate the different expression of EAAT4 and GLAST in the pathologic condition, neonatal subarachnoid hemorrhage. EAAT4 and GLAST were characteristically disturbed in the cerebellar cortices beneath the subarachnoid hemorrhage. In preterm infants with subarachnoid hemorrhage the decrease in EAAT4 immunoreactivity was more prominent than in term infants, and GLAST immunoreactivity in the inner granular cell layer decreased and reappeared later than in term infants with subarachnoid hemorrhage. Although Bergmann's glia removes glutamate from the extracellular space surrounding Purkinje cells in the early stage of hypoxic-ischemic brain damage, the reaction of EAAT4 and GLAST in the cerebellar cortex under the subarachnoid hemorrhage was decreased, and immature glia had a delayed reaction. These characteristics of glutamate transporters in immature cells may lead to cell death and olivocerebellar degeneration.
ATP-Binding Cassette Transporters↗
Recent advances towards understanding redox mechanisms in the activation of nuclear factor kappaB.
The transcription factor, nuclear factor-kappaB (NF-kappaB) has been studied extensively due to its prominent role in the regulation of immune and inflammatory genes, apoptosis, and cell proliferation. It has been known for more that a decade that NF-kappaB is a redox-sensitive transcription factor. The contribution of redox regulation and the location of potential redox-sensitive sites within the NF-kappaB activation pathway are subject to intense debate due to many conflicting reports. Redox regulation of NF-kappaB has been extensively addressed in this journal and the reader is referred to two comprehensive reviews on the subject [1,2]. With the identification of signaling intermediates proximal to the degradation of the inhibitor, IkappaB, the number of potential redox-sensitive sites is rapidly increasing. The purpose of this review is to address recent insights into the NF-kappaB signaling cascades that are triggered by proinflammatory cytokines such as TNF-alpha and IL-1beta. In addition, the role of nitrogen monoxide (.NO) in the regulation of NF-kappaB will be reviewed. Opportunities for redox regulation that occur upstream of IkappaB-alpha degradation, as well as the potential for redox control of phosphorylation of NF-kappaB subunits, will be discussed. Redox-sensitive steps are likely to depend on the nature of the NF-kappaB activator, the type of reactive oxygen or nitrogen species involved, the selectivity of signaling pathways activated, as well as the cell type under investigation. Lastly, it is discussed how redox regulation of NF-kappaB activation is likely to involve multiple subcellular compartments.
Animals↗
Detection of 8-oxoG DNA glycosylase activity and OGG1 transcripts in the rat CNS.
The oxoguanine DNA glycosylase (Ogg1) is a DNA repair enzyme that excises 7,8-dihydro-8-oxoguanine present in DNA damaged by oxidative stress. We have investigated the expression of the OGG1 gene in different regions of the rat CNS. Biochemical studies on brain homogenates of adult rats have shown that Ogg1 nicking activity is present at relatively similar levels in the cerebral cortex, the hypothalamus, the pons and the cerebellum. Following in situ hybridization with radiolabeled OGG1 cDNA or specific antisense oligonucleotides, OGG1 transcripts showed a widespread but heterogeneous distribution pattern among distinct brain regions of adult rats: high levels of this transcript were detected in the CA1-CA3 layers and the gyrus dentate of the hippocampal formation, the piriform cortex, the supraoptic nuclei, the olivary complex as well as in the pyramidal cells of layer V of the cortex and the Purkinje cells of the cerebellum. In peripheral organs such as the lungs, the stomach and the spleen, OGG1 transcript is however expressed in specific subpopulations of cells. Using a semi-quantitative reverse transcription - polymerase chain reaction assay on total mRNA from the frontal cortex, OGG1 mRNA was determined to be expressed with relatively the same levels in 1-day-old and 7-day-old rats as well as in adult rats. These results provide evidence for the widespread expression of the OGG1 gene in developing and adult brains.
Animals↗
Left-right asymmetry in BMP4 signalling pathway during chick gastrulation.
Determination of the left-right (L-R) axis was shown recently to implicate several genes, among which TGFbeta-related molecules such as Activin betaB, lefty1 and 2 and Nodal. We show here that Bmp4 and its signal transduction pathway partners BMPR IA and Smad1 are transiently expressed on the right side of Hensen's node, when L-R polarity is being established. Moreover, Smad1 is expressed asymmetrically in the nascent notochord. These observations suggest a role for a BMP4-dependent autocrine or paracrine mechanism during early L-R determination.
Amino Acid Sequence↗
Mouse submandibular gland mucin: embryo-specific mRNA and protein species.
Mouse submandibular salivary gland (SMG) mucin is the primary histodifferentiation product of submandibular epithelia. We demonstrate marked differences between embryonic, neonatal, and adult SMG mucin mRNA and protein by Northern and Western blot analyses: E17 and 1-day-old neonates exhibit two unique mucin transcripts (1.20 and 0.85 kb) which are approximately 19% greater or smaller in size than the single (1.01 kb) adult transcript. Two embryonic protein isoforms (Mr approximately 110 and 152 kDa) are immunodetected compared to a single adult protein (Mr approximately 136 kDa), with the larger (approximately 152 kDa) embryonic isoform persisting in neonatal glands. Mucin transcripts are localized to the branching epithelia in E14 and older SMGs, with increased hybridization signal being seen in terminal bud and proacinar epithelial cells with age; a significant 26% increase in transcript levels is detected by RNase protection assay between E14 and E19. By contrast, submandibular mucin protein is not immunodetected until E17, being primarily immunolocalized to terminal bud and proacinar epithelial cell membranes. Our data clearly shows that substantial qualitative differences exist between embryonic and adult SMG mucin mRNA and protein.
Animals↗
MDM2 expression during mouse embryogenesis and the requirement of p53.
We compared mouse embryonic expression of the MDM2 proto-oncogene, p21WAF1/CIP1 and their transcriptional regulator, p53. MDM2 expression is ubiquitous from 7.5 to 11.5 days post coitum (dpc) and more restricted from 12.5 dpc, with the highest levels in the testes and neural tube. From 14.5 to 18.5 dpc, the nasal respiratory epithelium expresses high levels of MDM2 RNA and protein and p21WAF1/CIP1 RNA, in both wild type and p53 null embryos. MDM2 expression during development is tissue-specific and, like p21WAF1/CIP1, is independent of p53. MDM2 may have a developmental role after 6.5 dpc, when MDM2 null mice die (Jones, S.N., Roe, A.E., Donehower, L.A., Bradley, A., 1995. Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206-208; Montes de Oca Luna, R., Wagner, D.S., Lozano, G., 1995. Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378, 203-206).
Animals↗
Whn and mHa3 are components of the genetic hierarchy controlling hair follicle differentiation.
The molecular basis of the characteristic hair growth disorder in nude mice that carry a defective Whn transcription factor gene is unknown. A comparison of mRNA populations from wild-type and nude mice back skin by representational difference analysis revealed the absence of acidic hair keratin gene 3 (mHa3) mRNA in mutant mice. Whn and acidic hair keratin genes are co-expressed in hair follicles, nail forming regions and filiform papillae of the tongue: expression of the mHa3 gene is generally detectable about 1 day after Whn mRNA and rapidly ceases in its absence. Whn is strongly expressed during the anagen (growth) phase of the hair cycle in matrix, cortex and outer root sheath; its expression rapidly declines during catagen and is undetectable in telogen phases. In nude mice, low levels of mHa3 expression are maintained in nails and whisker follicles, whereas expression is completely absent in pelage hair follicles and filiform papillae. Thus, the nude phenotype represents the first example of an inherited skin disorder that is associated with the loss of expression rather than structural mutation of keratin genes. The distinct molecular difference between pelage and whisker follicles correlates with the improved mechanical stability of vibrissae in nude mice, implicating mHa3 as an important structural component of the hair shaft.
Animals↗
Analysis of large-scale gene expression data.
The advent of cDNA and oligonucleotide microarray technologies has led to a paradigm shift in biological investigation, such that the bottleneck in research is shifting from data generation to data analysis. Hierarchical clustering, divisive clustering, self-organizing maps and k-means clustering have all been recently used to make sense of this mass of data.
Algorithms↗
Genomic-scale analysis of gene expression in resting and activated T cells.
Recent advances in gene array technology and isolation of lymphocytes now allow comprehensive analysis of gene expression in many different types of T cells. So far only a few sets of results have been published. However it is already clear that these analyses provide accurate measurements of gene expression in T cells. This technology offers the first opportunity to examine global and subtle changes in gene expression in response to specific stimuli.
Animals↗
Genomic-scale gene expression analysis of lymphocyte growth, tolerance and malignancy.
Immunologists are already comfortable with the need for monitoring many different gene products simultaneously. It is a common challenge to remember what CD-one-hundred-and-something is, and an ever-increasing number of colours are required for identification on the flow cytometer. Gene expression arrays now offer the possibility of extending this approach beyond the cell surface and expanding it dramatically to survey the entire catalogue of gene transcripts in a lymphoid cell.
Algorithms↗
How the host 'sees' pathogens: global gene expression responses to infection.
Innate immune responses to pathogens are believed to be patterned and stereotyped. Adaptive responses display variety but in relatively few types of products and with limited numbers of mechanisms. Is this apparent disparity between microbial pathogen diversity and a restricted set of host responses an accurate picture of infection or is it the result of a limited collection of analytic tools? DNA microarray technology permits one to address simple descriptive questions about global gene expression inside cells. In particular, it offers an opportunity to examine the relationship between host and pathogen in much greater detail than has been possible previously. One can now ask, firstly, how a host cell or organism 'sees' a microbial pathogen from the viewpoint of gene expression responses and, secondly, at what level it is able to discriminate between different agents. Other potential insights to be reaped include the identification of microbial determinants of the host response, the temporal features of the 'conversation' between host and pathogen, novel strategies for therapeutic and prophylactic intervention and prognostic markers of outcome.
Animals↗
Observing the living genome.
Dynamic pictures of living genomes are now beginning to emerge from systematic studies of gene expression patterns using DNA microarrays. The rich information represented in the variation in each gene's expression provides the basis for a new kind of genomic map.
Animals↗
Microarray analysis of replicative senescence.
BACKGROUND: Limited replicative capacity is a defining characteristic of most normal human cells and culminates in senescence, an arrested state in which cells remain viable but display an altered pattern of gene and protein expression. To survey widely the alterations in gene expression, we have developed a DNA microarray analysis system that contains genes previously reported to be involved in aging, as well as those involved in many of the major biochemical signaling pathways. RESULTS: Senescence-associated gene expression was assessed in three cell types: dermal fibroblasts, retinal pigment epithelial cells, and vascular endothelial cells. Fibroblasts demonstrated a strong inflammatory-type response, but shared limited overlap in senescent gene expression patterns with the other two cell types. The characteristics of the senescence response were highly cell-type specific. A comparison of early- and late-passage cells stimulated with serum showed specific deficits in the early and mid G1 response of senescent cells. Several genes that are constitutively overexpressed in senescent fibroblasts are regulated during the cell cycle in early-passage cells, suggesting that senescent cells are locked in an activated state that mimics the early remodeling phase of wound repair. CONCLUSIONS: Replicative senescence triggers mRNA expression patterns that vary widely and cell lineage strongly influences these patterns. In fibroblasts, the senescent state mimics inflammatory wound repair processes and, as such, senescent cells may contribute to chronic wound pathologies.
Blood Physiological Phenomena↗