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Results for “Expression analysis”

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PubMed2000

Transcription mapping and expression analysis of candidate genes in the vicinity of the mouse Loop-tail mutation.

Loop-tail (Lp) is a semidominant mutation that maps to the distal portion of mouse Chromosome (Chr) 1 and is an established model for neural tube defects (NTDs). Homozygous embryos exhibit an open neural tube from the caudal midbrain to the tip of the tail that results from over-differentiation of the floor plate. To facilitate the positional cloning of the Lp gene, both cDNA selection and assignment of sequence-tagged-sites from the human transcript map have been used to identify genes within the Lp interval. Together with previous physical mapping, this has allowed the placement of 13 transcription units within an approximately 1-Mb region that spans the Lp genetic interval, and eight of these genes map to the nonrecombinant interval. This map includes genes that encode proteins involved in protein sorting and targeting (Tim23 and Copa), ion transport (Atp1a2, Atp1a4, and Girk3), transcription (Nhlh1), immune regulation (Cd48 and Fcer1alpha), cell adhesion (R88252), apoptosis (Pea15), and several of unknown function (H326, Kiaa0253, and Estm34). Expression analysis by Northern blotting indicated that a subset of these genes are expressed preferentially in the developing nervous system. Finally, this region of mouse Chr 1 represents a conserved linkage group with genes on human chromosome 1q21, a region that is frequently altered in human cancers and that harbors loci for several genetic conditions. Consequently, analysis of the Lp interval may provide important tools to understand how the corresponding region of human Chr 1 contributes to disease, in addition to defining a key gene product required for neurulation.

Animals↗

PubMed1994

Nucleotide sequence and expression analysis of the Acetobacter xylinum phosphoglucomutase gene.

The Acetobacter xylinum gene (celB) encoding phosphoglucomutase (EC 5.4.2.2) has previously been cloned by complementation of cellulose-negative mutants. In the present report the nucleotide sequence of a 2.0 kb DNA fragment containing celB is described. Expression analysis using the bacteriophage T7 RNA polymerase promoter phi 10 resulted in identification of a probable translational start codon of celB, and this conclusion was confirmed by N-terminal amino acid sequencing of the recombinant protein. From the nucleotide sequence data it was deduced that celB encodes a protein with a calculated molecular mass of 59.6 kDa. A protein of similar size was visualized after in vitro transcription and translation, using the cloned 2.0 kb fragment as template. The results of an amino acid sequence comparison and a biochemical analysis indicated that the CelB protein is structurally and functionally related to the previously characterized human and rabbit phosphoglucomutases.

Amino Acid Sequence↗

PubMed1991

Nucleotide sequence and expression analysis of the Acetobacter xylinum uridine diphosphoglucose pyrophosphorylase gene.

The nucleotide sequence of the Acetobacter xylinum uridine diphosphoglucose pyrophosphorylase gene was determined; this is the first procaryotic uridine diphosphoglucose pyrophosphorylase gene sequence reported. The sequence data indicated that the gene product consists of 284 amino acids. This finding was consistent with the results obtained by expression analysis in vivo and in vitro in Escherichia coli.

Amino Acid Sequence↗

PubMed2004

Expression analysis of chick Wnt and frizzled genes and selected inhibitors in early chick patterning.

Wnt signaling is an important component in patterning the early embryo and specifically the neural plate. Studies in Xenopus, mouse, and zebrafish have shown that signaling by members of the Wnt family of secreted signaling factors, their Frizzled receptors and several inhibitors (sFRP1, sFRP2, sFRP3/Frzb1, Crescent/Frzb2, Dkk1, and Cerberus) are involved. However, very little is known about the expression of genes in the Wnt signaling pathway during early anterior neural patterning in chick. We have performed an expression analysis at neural plate stages of several Wnts, Frizzled genes, and Wnt signaling pathway inhibitors using in situ hybridization. The gene expression patterns of these markers are extremely dynamic. We have identified two candidate molecules for anterior patterning of the neural plate, Wnt1 and Wnt8b, which are expressed in the rostral ectoderm at these stages. Further functional studies on the roles of these markers are underway.

Animals↗

PubMed2006

From gene expression analysis to tissue microarrays: a rational approach to identify therapeutic and diagnostic targets in lymphoid malignancies.

Mantle cell lymphoma (MCL) is an aggressive lymphoid malignancy for which better treatment strategies are needed. To identify potential diagnostic and therapeutic targets, a signature consisting of MCL-associated genes was selected based on a comprehensive gene expression analysis of malignant and normal B cells. The corresponding protein epitope signature tags were identified and used to raise monospecific, polyclonal antibodies, which were subsequently analyzed on paraffin-embedded sections of malignant and normal tissue. In this study, we demonstrate that the initial selection strategy of MCL-associated genes successfully allows identification of protein antigens either uniquely expressed or overexpressed in MCL compared with normal lymphoid tissues. We propose that genome-based, affinity proteomics, using protein epitope signature tag-induced antibodies, is an efficient way to rapidly identify a number of disease-associated protein candidates of both previously known and unknown identities.

Antibodies, Neoplasm↗

PubMed2003

Chemical discovery and global gene expression analysis in zebrafish.

The zebrafish (Danio rerio) provides an excellent model for studying vertebrate development and human disease because of its ex utero, optically transparent embryogenesis and amenability to in vivo manipulation. The rapid embryonic developmental cycle, large clutch sizes and ease of maintenance at large numbers also add to the appeal of this species. Considerable genomic data has recently become publicly available that is aiding the construction of zebrafish microarrays, thus permitting global gene expression analysis. The zebrafish is also suitable for chemical genomics, in part as a result of the permeability of its embryos to small molecules and consequent avoidance of external confounding maternal effects. Finally, there is increasing characterization and analysis of zebrafish models of human disease. Thus, the zebrafish offers a high-quality, high-throughput bioassay tool for determining the biological effect of small molecules as well as for dissecting biological pathways.

Animals↗

PubMed2001

Impact of CD133 (AC133) and CD90 expression analysis for acute leukemia immunophenotyping.

BACKGROUND AND OBJECTIVES: AC133 is a novel monoclonal antibody (Moab) reacting with a population of immature/primitive or granulo-monocytic committed CD34+ve cells. Up to now, only few studies with small numbers of cases have examined AC133 (recently designated CD133) expression in acute leukemia. To determine the value of this Moab for acute leukemia immunophenotyping, we investigated a large series of leukemic cell samples for their reactivity with Moab AC133. DESIGN AND METHODS. A total of 298 cell samples from patients with de novo acute myeloid leukemia (AML) (n=142), acute lymphoblastic leukemia (ALL) (n=119), CD34+ve biphenotypic acute leukemia (n=13), and CD34+ve CML blast crisis (=BC; 21 myeloid BC/3 lymphoid BC) were investigated by flow cytometry for Moab AC133 reactivity.CD133 expression was compared with CD90(Thy-1) expression, another CD34-associated antigen. RESULTS: Fifteen (5%) samples expressed CD90, whereas 114 (38%) samples were positive for Moab AC133 (20% cut-off level). No significant differences in CD133 and CD90 expression levels between AML and ALL were observed. In AML, but not ALL, CD133 was more often expressed in CD34+ve cases than in CD34-ve ones (p<0.00001). However, CD133 expression was not restricted to CD34+ve leukemic cells in individual cell samples. All 8 pro-B-ALL cell samples with 11q23-anomalies and MLL (mixed lineage leukemia) gene translocations were positive for CD133, whereas only 2 of 9 pro-B-ALL without MLL gene translocations expressed CD133 (p<0.002). In contrast, none of the 5 AML cell samples with a t(9;11) and MLL gene translocation reacted with Moab AC133. CD34+ve CML cells in myeloid BC were less often positive for CD133 than CD34+ve de novo AML cells (p<0.0001). INTERPRETATION AND CONCLUSIONS: CD133 and CD90 expression analysis is not helpful for lineage determination in acute leukemia immunophenotyping. However, MoabAC133 may be an informative marker for the detection and further characterization of immature AML cells, as well as pro-B-ALL cells with MLL gene translocations, by flow cytometry.

AC133 Antigen↗

PubMed2004

Gene expression analysis in platelets from a single donor: evaluation of a PCR-based amplification technique.

BACKGROUND: Genetic analysis of platelet mRNA may facilitate the diagnosis of disorders affecting the megakaryocytic-platelet lineage. Its use, however, is limited by the exceptionally small yield of platelet mRNA and the risk of leukocyte contamination during platelet preparation. METHODS: We depleted platelet suspensions of leukocytes by filtration and used a PCR-based RNA amplification step [switching mechanism at the 5' end of RNA templates (SMART)]. We tested the reliability and precision of the RNA amplification procedure by use of real-time PCR to measure quantities of specific transcripts: von Willebrand factor (vWF), A-subunit of coagulation factor XIII (F13A), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Microarray analysis was performed on platelet RNA with and without amplification. RESULTS: Microgram quantities of platelet-specific cDNAs were produced from as little as 50 ng of total platelet RNA or 40 mL of whole blood. At cycle numbers <16, amplification of all transcripts tested was exponential with slightly more efficient amplification of low-abundance transcripts. Expression profiling of 9850 genes gave identical results for 9815 genes (1576 positive/8239 negative). Eight transcripts failed to be amplified by the SMART procedure. Expression of vWF, F13A, and GAPDH transcripts showed only minor day-to-day variations in three healthy individuals. CONCLUSION: The proposed protocol makes extremely small amounts of platelet RNA available for gene expression analysis in single patients.

Blood Platelets↗

PubMed2006

Molecular cloning, chromosomal location, and expression analysis of porcine CD14.

CD14 is a membrane-associated glycosylphosphatidylinositol (GPI)-anchored protein that binds lipopolysaccharide (LPS) of Gram-negative bacteria and enables LPS-dependent responses in a variety of cells. In this study a cDNA containing the porcine CD14 coding sequence has been cloned and its complete sequence determined. The amino acid sequence deduced from pig CD14 cDNA encodes a 373 amino acid polypeptide that exhibits 75%, 72%, 69%, 66%, 57% and 56% similarity to CD14 from cow, horse, human, rabbit, mouse and rat, respectively. Structural analysis showed that the porcine CD14 is a membrane glycoprotein with a GPI-anchor site and an extracellular domain containing 11 leucine-rich repeats. In addition, the LPS-binding regions identified in the human CD14 are highly conserved in the N-terminal domain of the porcine sequence. Fluorescence in situ hybridization was used to locate the CD14 gene on the pig chromosome 2, band q28. Expression analysis revealed that porcine CD14 transcripts were detected in all tissues and cells examined, suggesting that the expression of porcine CD14 gene is not restricted to myeloid cell lineage. Finally, we report that LPS stimulation significantly up-regulated CD14 gene expression in porcine alveolar macrophages.

Amino Acid Sequence↗

PubMed2006

Expression analysis of genes for callose synthases and Rho-type small GTP-binding proteins that are related to callose synthesis in rice anther.

The most chilling-sensitive stage of rice has been found to be at the onset of microspore release. The microsporocytes produce a wall of callose between the primary cell wall and the plasma membrane, and it has been shown that precise regulation of callose synthesis and degradation in anther is essential for fertile pollen formation. In this study, genes for 10 callose synthases in the rice genome were fully annotated and phylogenetically analyzed. Expression analysis of these genes showed that OsGSL5, an ortholog of microsporogenesis-related AtGSL2, was specifically expressed in anthers, and was notably downregulated by cooling treatment. Gene expression profiles of Rho-type small GTP-binding proteins in rice anther were also analyzed. The mechanisms of callose synthesis in rice pollen formation and its relationships with cool tolerance are discussed.

Cell Membrane↗

PubMed2005

Searching for non-RET molecular alterations in medullary thyroid carcinoma: expression analysis by mRNA differential display.

Some 25%-70% of sporadic medullary thyroid carcinomas (MTCs) are associated with somatic mutations within the RET proto-oncogene. In a significant number of MTCs, however, no such genetic variations can be detected, which implies alternative pathogenic molecular alterations. To assess altered RET mutation-specific gene expression, and to identify yet unknown gene transcripts involved in the tumorigenesis of MTC, we performed an expression analysis by mRNA differential display (RT-DD). Snap-frozen tumor tissues and corresponding normal thyroid tissues of 8 patients suffering from MTC (6 sporadic, 2 hereditary tumors) were included in the study; 5/8 MTCs harbored RET point mutations (codons 618, 634, 918). The RT-DD method was refined by use of fluorescence-labeled arbitrary oligonucleotides, electrophoresis on an automated sequencer, and a novel fragment-recovery technique utilizing a high-performance fluorescence scanner. More than 400 differentially expressed mRNA transcripts--representing upregulated or downregulated genes in the compared tissues--were detected. In all, 28 selected fragments were recovered, cloned, sequenced, and identified. Differential expression of gene transcripts with known association to cell proliferation or tumor progression--such as annexin A2, Rab11a, trefoil proteins, superoxide dismutase (SOD1), mitochondrial displacement loop (D-loop), and G protein subunit gamma11--as well as of the neuroendocrine marker chromogranin was observed. Furthermore, several mRNA transcripts of yet unknown genes displayed mutation-specific upregulation or downregulation in MTC. Illumination of the molecular basis especially of C-cell carcinomas without detectable alterations of the RET receptor tyrosine kinase will be required for the development of therapeutic strategies for advanced tumors that cannot be bridled or cured by surgical interventions alone.

Adult↗

PubMed2004

Gene expression analysis of the pro-oestrous-stage rat uterus reveals neuroligin 2 as a novel steroid-regulated gene.

In the present study, differential gene expression in the uteri of ovariectomised (OVX) and pro-oestrous rats (OVX v. pro-oestrus pair) was investigated using cDNA expression array analysis. Differential uterine gene expression in OVX rats and progesterone (P(4))-injected OVX rats (OVX v. OVX + P(4) pair) was also examined. The uterine gene expression profiles of these two sets of animals were also compared for the effects of P(4) treatment. RNA samples were extracted from uterine tissues and reverse transcribed in the presence of [alpha(32)P]-dATP. Membrane sets of rat arrays were hybridised with cDNA probe sets. Northern blot analysis was used to validate the relative gene expression patterns obtained from the cDNA array. Of the 1176 cDNAs examined, 23 genes showed significant (>two-fold) changes in expression in the OVX v. pro-oestrus pair. Twenty of these genes were upregulated during pro-oestrus compared with their expression in the OVX rat uterus. In the OVX v. OVX + P(4) pair, 22 genes showed significant (>two-fold) changes in gene expression. Twenty of these genes were upregulated in the OVX + P(4) animals. The genes for nuclear factor I-XI, afadin, neuroligin 2, semaphorin Z, calpain 4, cyclase-associated protein homologue, thymosin beta-4X and p8 were significantly upregulated in the uteri of the pro-oestrus and OVX + P(4) rats of both experimental pairs compared with the OVX rat uteri. These genes appear to be under the control of P(4). One of the most interesting findings of the present study is the unexpected and marked expression of the neuroligin 2 gene in the rat uterus. This gene is expressed at high levels in the central nervous system and acts as a nerve cell adhesion factor. According to Northern blot analysis, neuroligin 2 gene expression was higher during the pro-oestrus and metoestrus stages than during the oestrus and dioestrus stages of the oestrous cycle. In addition, neuroligin 2 mRNA levels were increased by both 17beta-oestradiol (E(2)) and P(4), although P(4) administration upregulated gene expression to a greater extent than injection of E(2). These results indicate that neuroligin 2 gene expression in the rat uterus is under the control of both E(2) and P(4), which are secreted periodically during the oestrous cycle.

Animals↗

PubMed2003

Cloning, physical mapping and expression analysis of the human 5-HT3 serotonin receptor-like genes HTR3C, HTR3D and HTR3E.

For more than 50 years the serotonin system has been the subject of intense research. This has provided an exciting insight and led to the discovery of multiple drugs targeting serotonin receptors, metabolising enzymes and re-uptake sites. During the past few years researchers focussed especially on elucidating the complexity of different physiological actions in the serotonergic network. We have identified two novel human serotonin 5-hydroxytryptamine type 3 receptor-like genes, HTR3D and HTR3E, by performing homology searches using the public human sequence databases and subsequently cloned the full length cDNAs by 5' and 3' rapid amplification of complementary DNA ends. Mapping of HTR3D and HTR3E by hybridisation, polymerase chain reaction and fluorescence in situ hybridisation revealed that both genes together with HTR3C are clustered in a subinterval of less than 100 kb on chromosome 3q27. Comparative expression analysis of all HTR3 genes, namely HTR3A, B, C, D and E showed HTR3D expression to be restricted to kidney, colon and liver and HTR3E expression to colon and intestine, whereas all other genes are widely expressed in many tissues including brain.

Amino Acid Sequence↗

PubMed2002

GeneChip expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes.

Upon iron restriction, the opportunistic pathogen Pseudomonas aeruginosa produces various virulence factors, including siderophores, exotoxin, proteases and haemolysin. The ferric uptake regulator (Fur) plays a central role in this response and also controls other regulatory genes, such as pvdS, which encodes an alternative sigma factor. This circuit leads to a hierarchical cascade of direct and indirect iron regulation. We used the GeneChip to analyse the global gene expression profiles in response to iron. In iron-starved cells,the expression of 118 genes was increased at least fivefold compared with that in iron-replete cells, whereas the expression of 87 genes was decreased at least fivefold. The GeneChip data correlated well with results obtained using individual lacZ gene fusions. Strong iron regulation was observed for previously identified genes involved in biosynthesis or uptake of the siderophores pyoverdine and pyochelin, utilization of heterologous siderophores and haem and ferrous iron transport. A low-iron milieu led to increased expression of the genes encoding TonB, alkaline protease,PrpL protease, exotoxin A, as well as fumarase C, Mn-dependent superoxide dismutase SodA, a ferredoxin and ferredoxin reductase and several oxidoreductases and dehydrogenases. Iron-controlled regulatory genes included seven alternative sigma factors and five other transcriptional regulators. Roughly 20% of the iron-regulated genes encoded proteins of unknown function and lacked any conclusive homologies. Under low-iron conditions, expression of 26 genes or operons was reduced in a DeltapvdS mutant compared with wild type, including numerous novel pyoverdine biosynthetic genes. The GeneChip proved to be a very useful tool for rapid gene expression analysis and identification of novel genes controlled by Fur or PvdS.

Bacterial Proteins↗

PubMed2002

Gene expression analysis from nuclear Poly(A) RNA.

Quantitation of the level of specific mRNA involves the isolation of total RNA or poly(A)+ RNA as a starting materiaL Thus, this result is the sum of the transcription and degradation of mRNA. Here we report a rapid, sensitive, and high-throughput methodology for gene expression analysis from nuclear poly(A)+ RNA via the reduction of the cytosolic components. The cells were first trapped on the glass fiber membranes of 96-well filter plates and subsequently exposed to non-ionic detergent to achieve cell membrane permeation. The cytosolic components, which contain preexisting mRNA, were removed by washing with the appropriate buffer, while nuclei remained in the filter plates. Lysis buffer was then used to release nuclear mRNA, which was collected on oligo(dT)-immobilized PCR plates for the capture of poly(A)+ RNA, on which RT-PCR was performed. The reduction of the cytosolic components and the preservation of the nuclear components were confirmed by electron microscopy, agarose gel electrophoresis, PCR of mtDNA, and RT-PCR of pre-splicing immature beta-actin poly(A)+ RNA. Using this method, we clearly identified UVC-induced p21 gene expression that is not detectable with conventional whole cell methods.

Cell Nucleus↗

PubMedDate not supplied

Comparative expression analysis of Adh3 during arthropod, urochordate, cephalochordate, and vertebrate development challenges its predicted housekeeping role.

Gene and genome duplications in the vertebrate lineage explain the complexity of extant gene families. Among these, the medium-chain alcohol dehydrogenase (ADH), which expanded by tandem duplications after the cephalochordate-vertebrate split, is a good model with which to analyze the evolution of gene function. Although the ancestral member of this family, ADH3, has been strictly conserved throughout animal evolution, its physiological role is still controversial. Previous evidence indicates that it contributes to formaldehyde cytoprotection, retinoic acid metabolism, and nitric oxide homeostasis. We performed in situ hybridization during Drosophila, ascidian (Ciona intestinalis), and zebrafish (Danio rerio) development. We showed that Adh3 expression was restricted to the fat body in Drosophila embryos at stage 17 and to the anterior endoderm in C. intestinalis tail bud, whereas in the zebrafish 2.5-day larvae the signal appeared widespread. A more comprehensive expression analysis including amphioxus and mice revealed that ancestral Adh3 was tissue specific, whereas a widespread expression was later attained in vertebrates. These variations occurred concomitantly with the expansion of the ADH family and the acquisition of new functions but were unlinked to the genomic changes that led to the transition from fractional to global methylation in vertebrates. Our data challenge the housekeeping role of ADH3 and question its involvement in the prevertebrate retinoic acid pathway.

Alcohol Dehydrogenase↗

PubMed2006

Allelic expression analysis suggests that cis-acting polymorphism of FRZB expression does not contribute to osteoarthritis susceptibility.

OBJECTIVE: The FRZB gene codes for secreted frizzled-related protein 3 (sFRP3), a soluble antagonist of wnt signalling that is required for maintaining cartilage integrity. Two common single nucleotide polymorphisms (SNPs) that code for the substitution of conserved arginine residues have previously been identified in FRZB and found to be associated with osteoarthritis (OA). Functional studies revealed that the arginine substitutions lead to a loss-of-function of sFRP3 activity. We set out to assess whether cis-acting polymorphism in the regulation of FRZB expression exists, as this may be an additional mechanism through which sFRP3 activity could be modulated. METHODS: RNA was extracted from the articular cartilage chondrocytes of 25 individuals who had undergone joint replacement for OA and who were heterozygous for one of the two FRZB SNPs. Allelic output was measured by single base extension in to the SNPs and deviations from the expected 1:1 pattern were assessed using the Mann-Whitney U test. RESULTS: Differential allelic expression was observed in six of the 25 individuals. However, the average fold difference in allelic expression in the six was only 1.19. CONCLUSIONS: The presence of a small degree of differential allelic expression in a low proportion (24%) of the individuals studied suggests that polymorphism in FRZB cis-acting regulatory elements can be discounted as a major factor that could influence the development of OA.

Aged↗

PubMed2004

Expression analysis of endogenous retroviral elements belonging to the HERV-F family from human tissues and cancer cells.

Based on the primer binding site for Phe-tRNA, human endogenous retroviruses (HERVs) have been classified as HERV-F family members, HERV-F, HERV-FXA, HERV-Fb, and HERV-Fc. The HERV-F family was expressed in human placenta preferentially. We investigated pol fragments of HERV-F family in various human tissues and cancer cells. The pol gene was expressed in human placenta only among tissues (brain, prostate, testis, heart, kidney, liver, lung, placenta, skeletal muscle, spleen, thymus, uterus), but detected in various cancer cell lines (RT4, BT-474, HCT-116, TE-1, UO-31, Jurkat, A549, MCF7, OVCAR-3, MIA-PaCa-2, PC3, LOX-IMVI, AZ521, 2F7, U-937, and C-33A) except for PFSK-1 and HepG2 by RT-PCR analysis. Expression and sequencing data of the 37 clones imply that the pol gene of HERV-F family is active in cancer cells, which may have potential role related to cancer development.

Amino Acid Sequence↗