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Results for “Expression analysis”

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PubMed1991

Sequence and expression analysis of a cAMP-responsive gene regulated during late development of Dictyostelium discoideum.

The 7E gene is expressed late in normal development of Dictyostelium discoideum after pseudoplasmodium formation. After disaggregation of the developing cells, transcription of this gene depends entirely on exogenous 3'5' cyclic AMP (cAMP). The 5' flanking region of the 7E gene contains two TATA box-oligo (dT) promoter motifs but analysis of 7E gene expression by primer extension shows only a single primary transcript with transcription initiating immediately after the most proximal promoter motif during development or in disaggregated cells in the presence of exogenous cAMP. Four C-rich sequences lie within 350bp upstream of the cap site, analogous to the upstream elements implicated in the cAMP regulation of several other Dictyostelium genes expressed in development.

Base Composition↗

PubMed2002

Expression analysis of RSK gene family members: the RSK2 gene, mutated in Coffin-Lowry syndrome, is prominently expressed in brain structures essential for cognitive function and learning.

Coffin-Lowry syndrome (CLS) is characterized by cognitive impairment, characteristic facial and digital findings and skeletal anomalies. The gene implicated in CLS encodes RSK2, a serine/threonine kinase acting in the Ras/MAPK signalling pathway. In humans, RSK2 belongs to a family of four highly homologous proteins (RSK1-RSK4), encoded by distinct genes. RSK2 mutations in CLS patients are extremely heterogeneous. No consistent relationship between specific mutations and the severity of the disease or the expression of uncommon features has been established. Together, the data suggest an influence of environmental and/or other genetic components on the presentation of the disease. Obvious modifying genes include those encoding other RSK family members. In this study we have determined the expression of RSK1, 2 and 3 genes in various human tissues, during mouse embryogenesis and in mouse brain. The three RSK mRNAs were expressed in all human tissues and brain regions tested, supporting functional redundancy. However, tissue specific variations in levels suggest that they may also serve specific roles. The mouse Rsk3 gene was prominently expressed in the developing neural and sensory tissues, whereas Rsk1 gene expression was the strongest in various other tissues with high proliferative activity, suggesting distinct roles during development. In adult mouse brain, the highest levels of Rsk2 expression were observed in regions with high synaptic activity, including the neocortex, the hippocampus and Purkinje cells. These structures are essential components in cognitive function and learning. Based on the expression levels, our results suggest that in these areas, the Rsk1 and Rsk3 genes may not be able to fully compensate for a lack of Rsk2 function.

Animals↗

PubMed2005

High-affinity peptide transporter PEPT2 (SLC15A2) of the zebrafish Danio rerio: functional properties, genomic organization, and expression analysis.

Solute carrier 15 (SLC15) membrane proteins PEPT1 (SLC15A1) and PEPT2 (SLC15A2) have been described in great detail in mammals. In contrast, information in lower vertebrates is limited. We characterized the functional properties of a novel zebrafish peptide transporter orthologous to mammalian and avian PEPT2, described its gene (pept2) structure, and determined mRNA tissue distribution. An expressed sequence tag (EST) cDNA (Integrated Molecular Analysis of Gene Expression; IMAGE) corresponding to zebrafish pept2 was completed by inserting a stretch of 75 missing nucleotides in the coding sequence to obtain a 3,238-bp functional clone. The complete open reading frame (ORF) was 2,160 bp and encoded a 719-amino acid protein. Electrophysiological analysis after cRNA injection in Xenopus laevis oocytes suggested that zebrafish PEPT2 is a high-affinity/low-capacity transporter (K(0.5) for glycyl-L-glutamine approximately 18 microM at -120 mV and pH 7.5). Zebrafish pept2 gene was 19,435 kb, thus being the shortest vertebrate pept2 fully characterized so far. Also, zebrafish pept2 exhibited 23 exons and 22 introns, whereas human and rodent pept2 genes contain 22 exons and 21 introns only. Zebrafish pept2 mRNA was mainly detected in brain, kidney, gut, and, interestingly, otic vesicle, the embryonic structure that develops into the auditory/vestibular organ, homolog to the higher vertebrate inner ear, of the adult fish. Characterization of zebrafish pept2 will contribute to the investigation of peptide transporters using a well-established genetic model and will allow the elucidation of the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, it can represent a useful marker to screen mutations that affect choroid plexus and inner ear development.

Amino Acid Sequence↗

PubMed2000

Molecular cloning and expression analysis of the complementary deoxyribonucleic acid for chicken inhibin/activin beta(B) subunit.

Inhibins and activins are dimeric peptide hormones that play an integral role in the intraovarian regulation of folliculogenesis. The domestic hen, with its well-defined follicular hierarchy, provides a unique model in which to study the role of these hormones in follicular development. In the present study, the complete coding sequence and deduced amino acid sequence for the chicken inhibin/activin beta(B) subunit has been determined from cDNA clones isolated from a chicken ovarian granulosa cell library. This beta(B)-subunit cDNA predicts a precursor protein of 392 amino acids containing the mature C-terminal 115 amino acid beta(B) subunit. When compared to the beta(B) subunit isolated from a variety of species, the chicken cDNA clone showed high nucleotide identity in the full-length coding region (>70%) and in the mature coding region (>80%). In addition, the deduced amino acid sequence of chicken beta(B) subunit showed greater than 95% identity compared to other species in the mature peptide region. Expression of the beta(B)-subunit mRNA was detected by reverse transcription-polymerase chain reaction in both gonadal and extragonadal tissues. Northern blot analysis detected expression in the gonadal tissues only, specifically in the granulosa tissue from the F3-F5 follicles, small yellow follicles (SYF), large white follicles, and immature and mature rooster testes. A major transcript of approximately 4.1 kilobases (kb) and three minor transcripts of approximately 8.4 kb, 6.5 kb, and 1.7 kb were detected in the SYF granulosa samples. To examine the expression pattern of the beta(B) subunit around the stage of follicle selection, the SYF granulosa was subdivided into two groups: 6-8 mm and 9-12 mm. Quantification of RNA expression (n = 3) showed that expression of the beta(B) subunit was maximal in the 6-8 mm SYF. Activin B, as well as other intraovarian signals, may regulate early follicle selection and/or development in the chicken.

Activins↗

PubMed1996

Isolation and developmental expression analysis of Enx-1, a novel mouse Polycomb group gene.

Members of the Polycomb group (Pc-G) of genes encode transcriptional regulators that control the expression of key developmental effector genes in Drosophila melanogaster. Although multiple Pc-G genes have been identified and characterized in Drosophila, information about these important regulatory proteins in vertebrates, including their precise expression patterns, has remained scarce. We report here the cloning of Enx-1, a novel vertebrate Pc-G gene, which encodes the murine homolog of the Drosophila Enhancer of zeste (E(z)) gene. Drosophila E(z) controls the expression of several homeobox genes as well as some segmentation genes and its disruption causes multiple phenotypes in Drosophila development. Analysis of the primary structure of murine Enx-1 reveals the conservation of several regions, including the previously described SET domain and a newly defined CXC domain. In addition, we find the SET domain to be conserved in evolutionarily distant species ranging from vertebrates to plants and fungi. The expression pattern analysis of Enx-1 reveals ubiquitous expression throughout early embryogenesis, while in later embryonic development Enx-1 expression becomes restricted to specific sites within the central and peripheral nervous system and to the major sites of fetal hematopoiesis. In adult stages we also find Enx-1 expression to be restricted to specific tissues, including spleen, testis and placenta.

Amino Acid Sequence↗

PubMed1998

Molecular cloning and expression analysis of GFR alpha-3, a novel cDNA related to GDNFR alpha and NTNR alpha.

Glial-cell-line-derived neurotrophic factor (GDNF) and neurturin (NTN) are structurally related to TGF-beta and are survival factors for sympathetic, sensory, and central nervous system neurons. GDNF transmits its signal primarily through a receptor complex containing the receptor tyrosine kinase Ret and a glycosyl-phosphatidylinositol (GPI)-linked receptor, GDNFR alpha. NTN utilizes a receptor complex system that consists of Ret and another GPI-linked receptor, NTNR alpha. We have identified a mouse cDNA, termed GFR alpha-3, that encodes a putative GPI-linked receptor. At the protein level, mouse GFR alpha-3 is 35% identical to mouse GDNFR alpha and 36% identical to mouse NTNR alpha. Northern blot analysis showed that GFR alpha-3 is expressed in fetal mouse heart, brain, lung, and kidney and adult heart. These results indicate that the tissue distribution of GFR alpha-3 mRNA is different from that of GDNFR alpha or NTNR alpha mRNA, and suggest that GFR alpha-3 may function in differentiation of embryonic cells expressing its mRNA.

Amino Acid Sequence↗

PubMed2004

cDNA cloning and expression analysis of a novel human F-box domain containing gene.

F-box proteins are a large family of eukaryotic proteins that are characterized by an approximately 40 amino acid motif. Some F-box proteins are critical for the controlled degradation of cellular regulatory proteins. Here we report that a novel member of F-box proteins, FBXO35 gene, was cloned and identified during the large-scale sequencing analysis from a human fetal brain cDNA library. FBXO35 gene shares amino acid similarity with several putative mouse genes not only in F-box domain but also in the rest of the sequence, which indicates that FBXO35 might also contain some other unknown conserved domain. RT-PCR analysis indicated that FBXO35 gene had a ubiquitously low expression pattern in most human adult tissues. According to bioinformatics analysis, the FBXO35 gene was found located in chromosome 3p21.

Amino Acid Sequence↗

PubMed2005

Expression analysis of androgen-responsive genes in the prostate of veal calves treated with anabolic hormones.

In order to identify indirect molecular biomarkers of anabolic treatments in veal calves, an animal experiment was performed using two combinations of growth promoters (consisting of boldenone undecylenate and estradiol benzoate, and of testosterone enantate and estradiol benzoate). We selected a set of 12 genes that are known to be androgen responsive in other mammalian species. The expression profile of this set of genes was analysed on prostate samples of veal calves using a real-time RT-PCR approach. For each selected gene the corresponding bovine sequence was obtained and a gene specific real-time assay was optimised and validated. The amplification was shown to be highly specific, linear and efficient. High reproducibility (<1%) and low-test variability (<2.5%) were also been achieved. Messenger RNA levels were quantified in prostate samples, non-parametric analysis of variance showed significant up-regulation of three genes (MAF, ESR1 and AR) and significant down-regulation of four genes (HMGCS1, HPGD, DBI, and LIM) in treated samples when compared with untreated controls. To assess the possibility of identifying hormone-treated animals by molecular means we performed a discriminant analysis that was effective in classifying treated and non-treated samples with an accuracy of 93%. Our results indicate that identification of treatment with steroid hormones in veal calves by means of gene expression analysis is a feasible approach and could be improved increasing both the number of genes and the number of controls analysed.

Animals↗

PubMed1994

Cloning and mRNA expression analysis of a novel human protooncogene, c-mer.

A human B-lymphoblastoid lambda gt11 expression library was screened using anti-phosphotyrosine antibodies yielding complementary DNAs encoding active tyrosine kinases. The resulting clones were used to obtain the sequence of a novel 984 amino acid transmembrane tyrosine kinase. Analysis of the complementary DNA revealed extracellular immunoglobulin and fibronectin type III domains and the unusual kinase signature sequence KWIAIES; all are characteristic of the axl family of tyrosine kinases. The novel tyrosine kinase was not expressed in normal B- and T-lymphocytes but, unlike axl, was expressed in numerous neoplastic B- and T-cell lines. Transcripts for the novel receptor-like tyrosine kinase were detected in normal peripheral blood monocytes and bone marrow. One alternatively spliced transcript was detected which contained an insert in the membrane proximal region that could encode for a truncated, soluble receptor. Sequence comparison shows that the kinase may be the human protooncogene for the recently isolated chicken retroviral oncogene v-ryk (recently renamed v-eyk), a truncated tyrosine kinase whose expression by retroviral infection produced sarcomas in chickens. The intracellular domain of the human kinase shows 83% similarity and 71% identity to v-ryk. Since the ryk designation has been used to name another tyrosine kinase and an analysis of RNA expression demonstrated that this novel human kinase is expressed in monocytes and tissues of epithelial and reproductive origin, we have designated our novel protooncogene c-mer.

Alternative Splicing↗

PubMed1997

Cloning and expression analysis of cadherin-10 in the CNS of the chicken embryo.

A full-length cDNA of a novel cadherin of chicken (cad10) was cloned. The deduced amino acid sequence of the putative cytoplasmic domain of this molecule is highly homologous to a previously published cytoplasmic fragment of human cadherin-10, a type II cadherin. An in situ hybridization analysis in chicken embryos shows that cad10 expression starts at about 4 days' incubation (E4) and persists at least until the hatching stage. In the central nervous system (CNS), cad10 expression is spatially restricted at all stages of development. At early stages, expression reflects the neuromeric organization of the brain. For example, in the alar plate of the diencephalon, cad10 expression is restricted to the dorsal thalamic neuromere. A number of cad10-expressing brain nuclei are formed in this neuromeric domain during later development. Specific cad10-expressing gray matter structures are also found in all other major divisions of the brain. Many of these structures are known to be functionally connected to each other. The cad10 expression pattern is distinct from that of other cadherins. These results support the idea that cadherins provide a molecular code for the regionalization of the embryonic CNS at the different stages of development.

Animals↗

PubMed2003

Differential gene expression analysis by micro-preparative capillary gel electrophoresis.

Differential display analysis by cDNA fractionation, collection of differentially expressed fractions of interests and their downstream characterization is demonstrated. cDNA pools from two strains of Cochliobolus heterostrophus fungus were generated by specific restriction digestion and selective ligation. Micropreparative separation and isolation of differentially expressed transcript representatives were accomplished by high-performance capillary gel electrophoresis. The collected individual DNA molecules were polymerase chain reaction amplified and sequenced to create expressed sequence tags for the genes of interests. High resolving power and sensitivity of capillary gel electrophoresis enabled fast and automated processing of minute amounts of cDNA samples with high precision.

Base Sequence↗

PubMed2000

Identification of factors interacting with hMSH2 in the fetal liver utilizing the yeast two-hybrid system. In vivo interaction through the C-terminal domains of hEXO1 and hMSH2 and comparative expression analysis.

Mutations in DNA mismatch repair (MMR) genes have been shown to segregate with Hereditary Nonpolyposis Colorectal Cancer (HNPCC). However, because many HNPCC families fail to display mutations in known MMR genes, we argued that changes in other components of the MMR pathway may be responsible. The increasing number of proteins reported to interact in the MMR pathway suggests that larger complexes are formed, the composition of which may differ among cell types and tissues. In an attempt to identify tissue-specific MMR-associated factors, we employed the yeast two-hybrid system, using the human hMSH2 as bait and a human fetal liver library as prey. We demonstrate that hMSH2 interacts with a human 5'-3' exonuclease 1 (hEXO1/HEX1) and that this interaction is mediated through their C-terminal domains. The hMSH6 protein does not interact with hEXO1 in the two-hybrid system. Dot-blot analysis of multiple tissue RNA revealed that hMSH2 and hEXO1 are coexpressed at high levels in fetal liver as well as in adult testis and thymus. Northern blot analysis also revealed that hEXO1/HEX1 is highly expressed in several liver cancer cell lines as well as in colon and pancreas adenocarcinomas, but not in the corresponding non-neoplastic tissue.

14-3-3 Proteins↗

PubMed1998

Amino acid carriers of Ricinus communis expressed during seedling development: molecular cloning and expression analysis of two putative amino acid transporters, RcAAP1 and RcAAP2.

This study reports on the isolation of two putative amino acid carrier cDNAs, RcAAP1 and RcAAP2, from Ricinus communis. Northern analysis shows that RcAAP1 and RcAAP2 are expressed abundantly in the cotyledon and root tissues of developing seedlings and at lower levels in the endosperm and hypocotyl. In the mature plant low expression was observed in the source and sink leaves. We have further characterized the expression of RcAAP1 in Ricinus roots by in situ hybridization. The transcripts are localized in many cell types of the root tip region, including the epidermal and cortical cells, but the highest expression was observed in the cells of the stele situated adjacent to the xylem poles. This is the first report describing the cellular expression of an amino acid transporter in roots, and the results are discussed in relation to the physiological role of this transporter.

Amino Acid Sequence↗

PubMed2003

Mouse matriptase-2: identification, characterization and comparative mRNA expression analysis with mouse hepsin in adult and embryonic tissues.

We report the identification and characterization of mouse matriptase-2 (m-matriptase-2), an 811-amino-acid protein composed of an N-terminal cytoplasmic domain, a membrane-spanning domain, two CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains, three LDLR (low-density-lipoprotein receptor class A) domains and a C-terminal serine-protease domain. All m-matriptase-2 protein domain boundaries corresponded with intron/exon junctions of the encoding gene, which spans approx. 29 kb and comprises 18 exons. Matriptase-2 is highly conserved in human, mouse and rat, with the rat matriptase-2 gene ( r-maltriptase-2 ) predicted to encode transmembrane and soluble isoforms. Western-blot analysis indicated that m-matriptase-2 migrates close to its theoretical molecular mass of 91 kDa, and immunofluorescence analysis was consistent with the proposed surface membrane localization of this protein. Reverse-transcription PCR and in-situ -hybridization analysis indicated that m-matriptase-2 expression overlaps with the distribution of mouse hepsin (m-hepsin, a cell-surface serine protease identified in hepatoma cells) in adult tissues and during embryonic development. In adult tissues both are expressed at highest levels in liver, kidney and uterus. During embryogenesis m-matriptase-2 expression peaked between days 12.5 and 15.5. m-hepsin expression was biphasic, with peaks at day 7.5 to 8.5 and again between days 12.5 and 15.5. In situ hybridization of embryonic tissues indicated abundant expression of both m-matriptase-2 and m-hepsin in the developing liver and at lower levels in developing pharyngo-tympanic tubes. While m-hepsin was detected in the residual embryonic yolk sac and with lower intensity in lung, heart, gastrointestinal tract, developing kidney tubules and epithelium of the oral cavity, m-matriptase-2 was absent in these tissues, but strongly expressed within the nasal cavity by olfactory epithelial cells. Mechanistic insight into the potential role of this new transmembrane serine protease is provided by its novel expression profile in embryonic and adult mouse.

Amino Acid Sequence↗

PubMed2002

Immunohistochemical expression analysis of the human interferon-inducible gene IFI16, a member of the HIN200 family, not restricted to hematopoietic cells.

This is the first description of an extensive immunohistochemical analysis of interferon (IFN)-inducible gene IFI16 expression in normal tissues. Immunohistochemical detection of IFI16 in paraffin-embedded tissues is achieved by using a polyclonal antibody raised against its C-terminal fragment that recognizes its three closely migrating isoforms in Western blotting. The results clearly indicate that IFI16 expression is not restricted to the hematopoietic compartment. In normal adult human tissues, it is prominent in stratified squamous epithelia and particularly intense in parabasal cells in the proliferating compartments, but it gradually decreases in the more differentiated suprabasal layers. Understanding of IFI16 expression in vivo is essential for interpretation of the results obtained from in vitro studies and elucidation of its physiologic role. The constitutive expression and wider distribution of IFI16 in normal human tissues, not restricted to the hematopoietic compartment, strongly support the possibility of an important role in cell differentiation that can be further modulated by other stimuli, such as IFN.

Blotting, Western↗

PubMed2004

Cryptococcus neoformans methionine synthase: expression analysis and requirement for virulence.

This paper describes (i) the expression profile of the methionine synthase gene (MET6) in the human pathogenic fungus Cryptococcus neoformans and (ii) the phenotypes of a C. neoformans met6 mutant. In contrast to the MET3 gene, which showed no significant change in expression in any environmental condition tested, the MET6 gene showed a substantial induction in response to methionine and a dramatic transcriptional induction in response to homocysteine. Like a met3 mutant, the met6 mutant was a methionine auxotroph. However, relative to a met3 mutant, the met6 mutant grew very slowly and was less heat-shock resistant. In contrast to a met3 mutant, the met6 mutant lost viability when starved of methionine, and it was deficient in capsule formation. Like a met3 mutant, the met6 mutant was avirulent. In contrast to a met3 mutant, the met6 mutant was hypersensitive to fluconazole and to the calcineurin inhibitors FK506 and cyclosporin A. A synergistic fungicidal effect was also found between each of these drugs and met6. The phenotypic differences between the met3 and met6 mutants may be due to the accumulation in met6 mutants of homocysteine, a toxic metabolic intermediate that inhibits sterol biosynthesis.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed2005

Functional characterization and expression analysis of a gene, OsENT2, encoding an equilibrative nucleoside transporter in rice suggest a function in cytokinin transport.

We identified four genes for potential equilibrative nucleoside transporters (ENTs) from rice (Oryza sativa; designated OsENT1 through OsENT4). Growth analysis of budding yeast (Saccharomyces cerevisiae) cells expressing OsENTs showed that OsENT2 transported adenosine and uridine with high affinity (adenosine, K(m) = 3.0 microm; uridine, K(m) = 0.7 microm). Purine or pyrimidine nucleosides and 2'-deoxynucleosides strongly inhibited adenosine transport via OsENT2, suggesting that OsENT2 possesses broad substrate specificity. OsENT2-mediated adenosine transport was resistant to the typical inhibitors of mammalian ENTs, nitrobenzylmercaptopurine ribonucleoside, dilazep, and dipyridamole. The transport activity was maximal at pH 5.0 and decreased slightly at lower as well as higher pH. In competition experiments with various cytokinins, adenosine transport by OsENT2 was inhibited by isopentenyladenine riboside (iPR). Direct measurements with radiolabeled cytokinins demonstrated that OsENT2 mediated uptake of iPR (K(m) = 32 microm) and trans-zeatin riboside (K(m) = 660 microm), suggesting that OsENT2 participates in iPR transport in planta. In mature plants, OsENT2 was predominantly expressed in roots. The OsENT2 promoter drove the expression of the beta-glucuronidase reporter gene in the scutellum during germination and in vascular tissues in germinated plants, suggesting a participation of OsENT2 in the retrieval of endosperm-derived nucleosides by the germinating embryo and in the long-distance transport of nucleosides in growing plants, respectively.

Adenosine↗

PubMed2006

Functional gene expression analysis uncovers phenotypic switch in aggressive uveal melanomas.

Microarray gene expression profiling is a powerful tool for generating molecular cancer classifications. However, elucidating biological insights from these large data sets has been challenging. Previously, we identified a gene expression-based classification of primary uveal melanomas that accurately predicts metastatic death. Class 1 tumors have a low risk and class 2 tumors a high risk for metastatic death. Here, we used genes that discriminate these tumor classes to identify biological correlates of the aggressive class 2 signature. A search for Gene Ontology categories enriched in our class-discriminating gene list revealed a global down-regulation of neural crest and melanocyte-specific genes and an up-regulation of epithelial genes in class 2 tumors. Correspondingly, class 2 tumors exhibited epithelial features, such as polygonal cell morphology, up-regulation of the epithelial adhesion molecule E-cadherin, colocalization of E-cadherin and beta-catenin to the plasma membrane, and formation of cell-cell adhesions and acinar structures. One of our top class-discriminating genes was the helix-loop-helix inhibitor ID2, which was strongly down-regulated in class 2 tumors. The class 2 phenotype could be recapitulated by eliminating Id2 in cultured class 1 human uveal melanoma cells and in a mouse ocular melanoma model. Id2 seemed to suppress the epithelial-like class 2 phenotype by inhibiting an activator of the E-cadherin promoter. Consequently, Id2 loss triggered up-regulation of E-cadherin, which in turn promoted anchorage-independent cell growth, a likely antecedent to metastasis. These findings reveal new roles for Id2 and E-cadherin in uveal melanoma progression, and they identify potential targets for therapeutic intervention.

Animals↗