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At least 811 records · Page 45Linked to original sources
PubMed2006

Gene expression analysis of primary normal human hepatocytes infected with human hepatitis B virus.

AIM: To find the relationship between hepatitis B virus (HBV) and hepatocytes during the initial state of infection by cDNA microarray. METHODS: Primary normal human hepatocytes (PNHHs) were isolated and infected with HBV. From the PNHHs, RNA was isolated and inverted into complement DNA (cDNA) with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-kappaB luciferase reporter assay. RESULTS: From the cDNA microarray, we obtained 98 differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes: LT-alpha, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, Huh7, and Chang liver cells, which were transfected with pHBV1.2x, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBV-mediated NF-kappaB activation. CONCLUSION: During the initial state of HBV infection, hepatocytes facilitate the activation of NF-kappaB through up regulation of LT-alpha, TRAF2, and NIK.

DNA, Complementary↗
PubMed1999

Expression analysis of glycogen synthase kinase-3 in human tissues.

Human glycogen synthase kinase-3 (GSK-3) is a multisubstrate, proline-directed kinase that phosphorylates tau, beta-amyloid and neurofilaments. In this study, the expression levels of the two GSK-3 isoforms, alpha and beta, RNA and proteins in different human tissues were examined. Northern analysis demonstrated that GSK-3alpha is encoded by a 2.6-kb mRNA and GSK-3beta by 8.3- and 2.8-kb mRNAs. The two GSK-3beta mRNA species were variably expressed in different tissues. Northern and quantitative polymerase chain reaction demonstrated that both GSK-3alpha and GSK-3beta mRNA were prominently expressed in testis, thymus, prostate and ovary but were low in adult lung and kidney. Western blot analysis showed that the 51-kDa GSK-3alpha protein was highly expressed in lung, ovary, kidney and testis, whereas the 46-kDa GSK-3beta protein was highly expressed in lung, kidney and brain. The differential expression of GSK-3alpha and GSK-3beta mRNA and proteins and the lack of relationship between transcription and translation in some tissues indicate that GSK-3alpha and GSK-3beta are subject to different means of regulation.

Adult↗
PubMed2004

Quantitative expression analysis of blastocyst-derived gene transcripts in preimplantation developmental stages of in vitro-produced bovine embryos using real-time polymerase chain reaction technology.

The main objective of the present study was to analyse the quantitative expression pattern of genes from a subtracted blastocyst transcriptome throughout the preimplantation developmental stages of in vitro-produced bovine oocytes and embryos. For this purpose, Day 5 morula (M) cDNAs were subtracted from Day 7 blastocyst (B) cDNAs (B-M) and used to establish a B-M subtracted cDNA library, as reported previously. From the total generated clones, 19 were analysed quantitatively. The mRNA samples isolated from pools of immature oocytes (n = 150), mature oocytes (n = 150) and two-cell (n = 80), four-cell (n = 40), eight-cell (n = 20), morula (n = 6) and blastocyst (n = 3) embryos were reverse transcribed and subjected to real-time polymerase chain reaction (PCR) using sequence-specific primers and SYBR green as the DNA dye. A relative standard curve method was used to analyse the real-time data taking the morula stage as a calibrator. Applying suppression subtractive hybridisation (SSH), a total of 71 clones, which represent 33 different expressed sequence tags, were generated and available for analysis. Most transcripts were analysed for the first time in bovine embryogenesis. The real-time PCR has validated the results of SSH positively for 84% (16/19) of transcripts, whereas 16% (3/19) showed deviation in the expression pattern from the one seen during SSH. Several transcript-specific expression patterns were observed for genes that play decisive roles in bovine embryogenesis. In addition to identification, accurately quantifying the expression profiles of transcripts during development will pave the way towards understanding the molecular mechanisms of embryogenesis and their potential role in early embryo development. Most importantly, the present study has contributed to the enrichment of bovine embryo gene collection by generating new transcripts involved in bovine embryo development.

Animals↗
PubMed2004

Comprehensive gene expression analysis of the response to straight-chain alcohols in Saccharomyces cerevisiae using cDNA microarray.

AIMS: The purpose of this study was to examine the gene expression profiles of yeast Saccharomyces cerevisiae subjected to straight-chain alcohols. METHODS AND RESULTS: Lipophilic alcohols with high log Pow values were more toxic to yeast than those with low log Pow values. Morphological changes after exposure to ethanol, 1-pentanol, 1-octanol were observed, whereas n-pentane as a model hydrocarbon affected the surface of the outer membrane, with little change in organelles. Using cDNA microarrays, quite a few up-regulated gene categories were classified into the category 'cell rescue, defence and virulence' by ethanol, and the category 'energy' and 'metabolism' by 1-pentanol. Meanwhile, the characteristic genes up-regulated by n-pentane were not observed, and the expression profile was distantly related to ethanol, 1-pentanol and 1-octanol. CONCLUSIONS: This study suggests that gene expression profiles at the whole genome level were intimately associated with the cell growth inhibition and morphological changes by straight-chain alcohols with differing log Pow values. SIGNIFICANCE AND IMPACT OF THE STUDY: The study of comprehensive gene expression profiles by cDNA microarrays elucidates the straight-chain alcohol adaptation mechanisms.

1-Octanol↗
PubMedDate not supplied

Molecular genetic and expression analysis of alpha-tocopherol transfer protein mRNA in German shepherd dogs with degenerative myelopathy.

Degenerative Myelopathy (DM) is a progressive neurological disorder of the spinal cord preferentially occurring in German shepherd dogs. The pathogenesis of the disease is unknown. However, there are indications that vitamin E deficiency may be involved in the pathogenesis of DM. Therefore, we analyzed the expression and the nucleotide sequence of the canine alpha-tocopherol transfer protein (alpha Ttp) of German shepherd dogs with DM in order to determine whether a deficiency or a defect of the alpha Ttp could be a primary factor in the pathogenesis of DM, as found in human patients with Ataxia with vitamin E deficiency (AVED). The cDNA of the coding region of the canine alpha Ttp-mRNA was generated from total liver RNA using RT-PCR and 5' RACE technique. We determined the sequence of 707 out of 834 base pairs or 84.8% of the canine alpha Ttp coding region. Sequence comparison of canine alpha Ttp between affected and control dogs revealed no differences in either nucleotide or predicted amino acid sequence. Using Northern blot analysis alpha Ttp-mRNA expression was solely found in the liver of the dogs, rats and humans, while various other organs showed no alpha Ttp-mRNA expression. No significant differences in expression levels of canine alpha Ttp mRNA were found between DM and control dogs. Our data suggest that the canine alpha Ttp gene is unlikely to be involved in the pathogenesis of DM in German shepherd dogs.

Amino Acid Sequence↗
PubMed2006

Comparative expression analysis of Pax3 and Pax7 during mouse myogenesis.

Pax3 and Pax7 are closely related transcription factors involved in the commitment of myogenic precursors in the developing trunk. However, it is not yet clear whether these genes are required for myogenic cell specification in the head and for post-somitic myogensis per se. In part, this uncertainty is due to the scarce information about their normal time course and pattern of expression. Here, we present a systematic immunohistochemical in situ analysis of spatiotemporal characteristics of Pax3 and Pax7 protein expression in comparison to that of MyoD and myogenin in the developing trunk and head muscles. The observed patterns of expression suggest that Pax3 is not involved in myogenesis in the head and its post-somitic expression in the trunk and limb muscles is mostly repressed after stage E13.5. In contrast, Pax7 expression is shared among all striated muscles and exhibits a uniform pattern. Pax7 is expressed only in mononucleated cells that either differentiate into myotubes or later form satellite cells. During development of head muscles, expression of Pax7 follows expression of MyoD and myogenin, implying that Pax7 is not required to induce the initial steps of the myogenic program in the head. In Pax7 homozygous mutants, in which muscle development proceeds normally, expression of Pax3 is indistinguishable from its wild-type pattern (i. e. absent), suggesting that after stage E13.5 myogenesis does not require Pax3 and Pax7. These data challenge the concept that Pax3 and Pax7 determine a persistent lineage of myogenic precursors in pre-natal and post-natal muscle development.

Animals↗
PubMed2004

Expression analysis suggests novel roles for the plastidic phosphate transporter Pht2;1 in auto- and heterotrophic tissues in potato and Arabidopsis.

A cDNA encoding Pht2;1 from potato, a new member of the plant Pht2 gene family of low-affinity orthophosphate (Pi) transporters, was isolated. The expression pattern of the corresponding gene as well as its ortholog from Arabidopsis was analyzed and the encoded proteins were localized in the two plants. Pht2;1 expression is strongly upregulated by light in potato and Arabidopsis leaf tissue. RNA gel blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), promoter/GUS, and protein/green fluorescent protein (GFP) fusion studies, respectively, indicate that the gene is expressed in both auto- and heterotrophic tissues and its encoded protein is localized to the plastids. The similar patterns of Pht2;1 gene regulation in potato and Arabidopsis prompted us to screen publicly available gene expression data from 228 Arabidopsis oligonucleotide microarrays covering 83 different experimental conditions. Modulation of Pht2;1 transcript levels was overall moderate, except for a limited number of experimental conditions where Pht2;1 mRNA concentrations varied between 2- and 3.7-fold. Overall, these analyses suggest involvement of the Pht2;1 protein in cell wall metabolism in young, rapidly growing tissues, independent of other Pi transporters such as the high-affinity Solanum tuberosum Pi transporter 1 (StPT1). Cluster analysis allowed identification of colinear or antiparallel expression profiles of a small set of genes involved in post-translational regulation, and photosynthetic carbon metabolism. These data give clues about the possible biological function of Pht2;1 and shed light on the complex web of interactions in which Pht2;1 could play a role.

Amino Acid Sequence↗
PubMed1998

Sequence of murine CDC10 cDNA, gene organization and expression analysis.

The sequence of the 2391 bp murine CDC10 cDNA is reported. The gene transcription unit, composed of 13 exons, encodes a potential 417/446 amino acid GTP-binding protein, highly similar to human CDC10. The ubiquitous expression of the gene, as judged by Northern analysis, is consistent with its putative promoter structure.

Amino Acid Sequence↗
PubMed2000

Human KLRF1, a novel member of the killer cell lectin-like receptor gene family: molecular characterization, genomic structure, physical mapping to the NK gene complex and expression analysis.

The human NK gene complex localized on chromosome 12p12.3 - p13.2 codes for several lectin-like receptor genes expressed by NK cells as well as by other hematopoietic cells. In this study, by using the expressed sequence tag database we identified a novel receptor gene, designated as killer cell lectin-like receptor, subfamily F, member 1 (KLRF1), encoding a putative type II transmembrane glycoprotein. The KLRF1 gene has been localized on the high-resolution physical map of chromosome 12p. The genomic structure of the KLRF1 gene and the existence of one spliced variant are also described. KLRF1 was expressed at the mRNA level in peripheral blood leukocytes, activated NK cells, monocytes and NK and myeloid cell lines. The presence of two immunoreceptor tyrosine-based inhibitory-like motifs within the cytoplasmic tail of KLRF1 suggests an inhibitory role in NK cell and monocyte activity.

Amino Acid Sequence↗
PubMed2002

Gene expression analysis during liver stage development of Plasmodium.

The complex life cycle of malaria parasites requires significant changes in gene expression as the parasites move from vector to host and back to the vector. Although recognised as an important vaccine and drug target, the liver stage parasite has remained difficult to study. One of the major impediments in identifying parasite gene expression at the liver stage has remained the large number of uninfected hepatocytes relative to the number of infected hepatocytes in the liver after sporozoite inoculation. This article describes several of the approaches that have been utilised to overcome this difficulty in rodent models of malaria. While significant progress has been made to identify genes that are expressed during liver stage parasite development, a great deal more work remains to be done.

Animals↗
PubMed2002

Cloning and developmental expression analysis of ltd-1, the Caenorhabditis elegans homologue of the mouse kyphoscoliosis (ky) gene.

We have characterized the developmental expression pattern of the Caenorhabditis elegans homologue of the mouse ky gene. The Ky protein has a putative key function in muscle development and has homologues in invertebrates, fungi and a cyanobacterium. The C. elegans Ky homologue gene has been named ltd-1 for LIM and transglutaminase domains gene. The LTD-1::GFP construct is expressed in developing hypodermal cells from the twofold stage embryo through adulthood. These data define the ltd-1 gene as a novel marker for C. elegans epithelial cell development.

Amino Acid Sequence↗
PubMed2006

Qdot nanobarcodes for multiplexed gene expression analysis.

We report a quantum dot (Qdot) nanobarcode-based microbead random array platform for accurate and reproducible gene expression profiling in a high-throughput and multiplexed format. Four different sizes of Qdots, with emissions at 525, 545, 565, and 585 nm are mixed with a polymer and coated onto the 8-mum-diameter magnetic microbeads to generate a nanobarcoded bead termed as QBeads. Twelve intensity levels for each of the four colors were used. Gene-specific oligonucleotide probes are conjugated to the surface of each spectrally nanobarcoded bead to create a multiplexed panel, and biotinylated cRNAs are generated from sample total RNA and hybridized to the gene probes on the microbeads. A fifth streptavidin Qdot (655 nm or infrared Qdot) binds to biotin on the cRNA, acting as a quantification reporter. Target identity was decoded based on spectral profile and intensity ratios of the four coding Qdots (525, 545, 565, and 585 nm). The intensity of the 655 nm Qdot reflects the level of biotinylated cRNA captured on the beads and provides the quantification for the corresponding target gene. The system shows a sensitivity of < or =10(4) target molecules detectable with T7 amplification, a level that is better than the 10(5) number achievable with a high-density microarray system, and approaching the 10(3)-10(4) level usually observed for quantitative PCR (qPCR). The QBead nanobarcode system has a dynamic range of 3.5 logs, better than the 2-3 logs observed on various microarray platforms. The hybridization reaction is performed in liquid phase and completed in 1-2 hours, at least 1 order of magnitude faster than microarray-based hybridizations. Detectable fold change is lower than 1.4-fold, showing high precision even at close to single copy per cell level. Reproducibility for this proof-of-concept study approaches that of Affymetrix GeneChip microarray, with an R(2) value between two repeats at 0.984, and interwell CV around 5%. In addition, it provides increased flexibility, convenience, and cost-effectiveness in comparison to conventional gene expression profiling methods.

Electronic Data Processing↗
PubMed2002

Gene expression analysis of cold and freeze stress in Baker's yeast.

We used mRNA differential display to assess yeast gene expression under cold or freeze shock stress conditions. We found both up- and down-regulation of genes, although repression was more common. We identified and sequenced several cold-induced genes exhibiting the largest differences. We confirmed, by Northern blotting, the specificity of the response for TPI1, which encodes triose-phosphate isomerase; ERG10, the gene for acetoacetyl coenzyme A thiolase; and IMH1, which encodes a protein implicated in protein transport. These genes also were induced under other stress conditions, suggesting that this cold response is mediated by a general stress mechanism. We determined the physiological significance of the cold-induced expression change of these genes in two baker's yeast strains with different sensitivities to freeze stress. The mRNA level of TPI1 and ERG10 genes was higher in freeze-stressed than in control samples of the tolerant strain. In contrast, both genes were repressed in frozen cells of the sensitive strain. Next, we examined the effects of ERG10 overexpression on cold and freeze-thaw tolerance. Growth of wild-type cells at 10 degrees C was not affected by high ERG10 expression. However, YEpERG10 transformant cells exhibited increased freezing tolerance. Consistent with this, cells of an erg10 mutant strain showed a clear phenotype of cold and freeze sensitivity. These results give support to the idea that a cause-and-effect relationship between differentially expressed genes and cryoresistance exists in Saccharomyces cerevisiae and open up the possibility of design strategies to improve the freeze tolerance of baker's yeast.

Blotting, Northern↗
PubMed2005

Gene expression analysis of the function of the male-specific lethal complex in Drosophila.

Dosage compensation refers to the equal expression of X-linked genes despite the difference in copy number between the two sexes. The male-specific lethal (MSL) complex is concentrated on the X chromosome in males. A gene expression assay for embryos was developed to examine the function of this complex. In mutant male embryos without either the MSL complex or MOF histone acetylase, dosage compensation is retained but autosomal expression is increased. Dosage compensation is lost in the double-mutant embryos. In embryos in which the MSL complex and MOF are targeted to the X chromosomes in females, the results are consistent with previous surveys showing that in general the X expression remains unchanged, but autosomal expression is reduced. Mutations in the ISWI chromatin-remodeling component cause increases specifically of X-linked genes in males. Thus, the function of the MSL complex in conjunction with ISWI is postulated to override the effect on gene expression of high histone acetylation on the male X. The basic determinant of dosage compensation is suggested to be an inverse dosage effect produced by an imbalance of transcription factors on the X vs. the autosomes. The sequestration of the MSL complex to the male X may have evolved to counteract a similar effect on the autosomes and to prevent an overexpression of the X chromosome in males that would otherwise occur due to the high levels of histone acetylation.

Acetyltransferases↗
PubMed2003

cDNA cloning and expression analysis of the rice (Oryza sativa L.) RNase L inhibitor.

A subtractive cDNA library was constructed using rice (Oryza sativa L.) callus cDNA as driver and differentiating callus cDNA as tester. A novel cDNA fragment encoding RNase L inhibitor (RLI) was isolated by screening the subtractive library, which had a higher expression level in differentiating callus than in callus. The full-length cDNA of rice-RLI was obtained by the method of rapid amplification of cDNA ends, which contained a 1812-bp open reading frame encoding a 604 amino acid polypeptide. Homologous analysis showed that rice-RLI contained the conserved motifs (two repeated P-loops, two ATP-binding boxes and an iron-sulfur binding motif). The fluorescence quantitative PCR analysis showed that it was a constitutive expressed gene but up-regulated in abiotic stress (low temperature and NaCl treatment) and down-regulated under the treatments of NAA and IAA.

ATP-Binding Cassette Transporters↗
PubMed2004

Genomic organization and expression analysis for hcstk, a serine/threonine protein kinase gene of Haemonchus contortus, and comparison with Caenorhabditis elegans par-1.

The organization and expression of a putative serine/threonine kinase gene (designated hcstk), proposed to relate to a conserved eukaryotic signal transduction pathway, was characterized for the socio-economically important pathogen Haemonchus contortus (Nematoda). The entire hcstk gene is approximately 26.7 kb in size, has 26 exons and is inferred to produce multiple isoforms via alternative splicing in its N-terminal header and spacer domains. Comparison of hcstk with its Caenorhabditis elegans homologue, par-1, revealed major differences in genomic organization, exon number and inferred mRNA processing. The expression of hcstk transcripts was highest in the first- and late-fourth-stage larvae of the parasite compared with other developmental stages, somewhat distinct from par-1 in C. elegans. In spite of a substantial amino acid sequence identity in the functional domains between the predicted proteins HcSTK and PAR-1, overall, the findings suggest a unique functional role for each molecule.

Alternative Splicing↗
PubMed2004

cDNA microarray gene expression analysis and toxicological phenotype for anticancer drug.

Toxicogenomics, the subdiscipline that merges genomics with toxicology, hold the promise to contributing toward the goal of elucidating mechanism by studying genomic profiling related with various drugs. The application of gene expression profiling technology to examine multiple genes and signaling pathways promises a significant advance in understanding the toxic mechanisms of various drugs and prediction of new drug candidate. Toxicogenomics is emerging field combining genomics and bioinformatics to identify and characterize mechanisms of toxicity of drug and various compounds. The principal hypothesis underlying on this field is that chemical-specific pattern of altered gene expression is related with each chemicals properties, especially toxicological property, and it will be revealed using high-density microarray analysis of sample from exposed organisms. So, in this study we compare the gene expression pattern of two anticancer drugs paclitaxel and orally absorbable paclitaxel, using the cDNA microarray. And from the result of this study, it is possible to provide the new possibility for genome-wide insight into mechanism of their anticancer activity and toxicological phenotype.

Administration, Oral↗