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CDO is a cell-surface protein of the immunoglobulin/fibronectin type III repeat family that positively regulates myogenic differentiation in vitro. To gain a better understanding of the role of cdo during vertebrate development, we carried out an extensive in situ hybridization study to characterize its expression pattern from postimplantation to late stages of mouse embryogenesis and in rat brain from E13 to adult. Our results show a broad pattern of cdo expression that is spatially and temporally restricted during embryogenesis. In the central nervous system (CNS), cdo expression is detected as early as E7.5 and maintained in the dorsal ventricular zones of the brain and spinal cord, becoming increasingly restricted in the adult. High levels of cdo are detected in developing sensory organs, such as the eye and ear. Outside the CNS, cdo is expressed mainly in neural crest and mesodermal derivatives, including skeletal muscle precursors. Overall, the highest levels of cdo expression are seen from E9.0 to E15.5. The temporal onset and restricted expression of cdo suggest that cdo plays a role in the determination and/or differentiation of a number of cell types during embryogenesis. 相似文献
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Arjan C Houweling Semir Somi Maurice J B Van Den Hoff Antoon F M Moorman Vincent M Christoffels 《The Anatomical record》2002,266(2):93-102
In mouse, atrial natriuretic factor (ANF) gene expression was shown to be a marker for chamber formation within the embryonic heart. To gain insight into the process of chamber formation in the chicken embryonic heart, we analyzed the expression pattern of cANF during development. We found cANF to be specifically expressed in the myocardium of the morphologically distinguishable atrial and ventricular chambers, similar to ANF in mouse. cANF expression was never detected in the myocardium of the atrioventricular canal (AVC), inner curvature, and outflow tract (OFT), which is lined by endocardial cushions. Expression was strictly excluded from the interventricular myocardium and most proximal part of the bundle branches, as identified by the expression of Msx-2, whereas the rest of the bundle branches, trabeculae, and surrounding working myocardium did express cANF. The myocardium that forms de novo within the cushions after looping did not express cANF. At HH9 cANF expression was first observed in a subset of cardiomyocytes, which was localized ventrally in the fused heart tube and laterally in the unfused cardiac sheets. Together, these results show that cANF expression can be used to distinguish differentiated chamber (working) myocardium, including the peripheral ventricular conduction system, from embryonic myocardium. We conclude that differentiation of chamber myocardium takes place already at HH9 at the ventral side of the linear heart tube, possibly preceded by latero-medial signals in the unfused cardiac sheets. 相似文献
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Summary A monoclonal antibody that reacts with all known isoforms of troponin I detected a single isoform of cardiac troponin I in both a trial and ventricular chambers of adult chicken and rat hearts in an immunoblotting analysis. Another isoform of troponin I in addition to the adult cardiac form, however, was present in all chambers of the heart during early development in both species. This developmental isoform appeared to have the same electrophoretic mobility on SDS tris glycine polyacrylamide gels as that observed for the adult slow skeletal muscle isoform. In the rat, only the developmental isoform of troponin I was present in the early foetal heart and small amounts of the adult cardiac isoform were not apparent until late in gestation, whereas the developmental and adult isoforms were expressed in approximately equal amounts throughout embryonic development in the chicken. The level of developmental isoform of troponin I in both the chicken and the rat hearts gradually decreased so that only small amounts of this variant were detectable two weeks after birth or post hatching. 相似文献
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Sigmar Stricker Nicole Verhey van Wijk Florian Witte Norbert Brieske Kathrin Seidel Stefan Mundlos 《Developmental dynamics》2006,235(12):3456-3465
Ror2 is a receptor tyrosine kinase mutated in the human syndromes Brachydactyly type B (BDB) and recessive Robinow syndrome (RS). In this study, we used the chick as a model to investigate the role of Ror2 in skeletogenesis and to elucidate the functional consequences of Ror2 mutations. For this purpose, we cloned chicken Ror2 and analyzed its expression pattern at various embryonic stages by in situ hybridization and immunolabeling. We document expression of cRor2 in several organs, including mesonephros, heart, nervous system, intestine and cartilage. The high conservation of expression when compared with the mouse underlines the validity of the chick as a model system. Using replication-competent retroviral vector-mediated overexpression, we analyzed the functional consequences of truncating BDB and RS mutations in the developing chick limb. Overexpression of Ror2 mutants led to a disturbance of growth plate architecture and a severe block of chondrocyte differentiation, demonstrating the functional importance of Ror2 in skeletogenesis. 相似文献
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Identification and characterization of the Treponema pallidum tpn50 gene, an ompA homolog. 总被引:11,自引:3,他引:8
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Treponema pallidum is a pathogenic spirochete that has no known genetic exchange mechanisms. In order to identify treponemal genes encoding surface and secreted proteins, we carried out TnphoA mutagenesis of a T. pallidum genomic DNA library in Escherichia coli. Several of the resulting clones expressed enzymatically active T. pallidum-alkaline phosphatase fusion proteins. The DNA sequence of the 5' portion of a number of the treponemal genes was obtained and analyzed. A recombinant clone harboring plasmid p4A2 that encoded a treponemal protein with an approximate molecular mass of 50,000 Da was identified. Plasmid p4A2 contained an open reading frame of 1,251 nucleotides that resulted in a predicted protein of 417 amino acids with a calculated molecular mass of 47,582 Da. We have named this gene tpn50 in accordance with the current nomenclature for T. pallidum genes. A 1.9-kb HincII-ClaI fragment from p4A2 that contained the tpn50 gene was subcloned to produce p4A2HC2. Comparison of the predicted amino acid sequence of TpN50 with protein sequences in the National Center for Biotechnology Information data base indicated statistically significant homology to the Pseudomonas sp. OprF, E. coli OmpA, Bordetella avium OmpA, Neisseria meningitidis RmpM, Neisseria gonorrhoeae PIII, Haemophilus influenzae P6, E. coli PAL, and Legionella pneumophila PAL proteins. These proteins are all members of a family of outer membrane proteins that are present in gram-negative bacteria. The tpn50 gene complemented E. coli ompA mutations on the basis of two separate criteria. First, morphometry and electron microscopy data showed that E. coli C386 (ompA lpp) cells harboring plasmid vector pEBH21 were rounded while cells of the same strain harboring p4A2HC2 (TpN50+), pWW2200 (OprF+), or pRD87 (OmpA+) were rod shaped. Second, E. coli BRE51 (MC4100 delta sulA-ompA) cells harboring pEBH21 grew poorly at 42 degrees C in minimal medium, while the growth of BRE51 cells harboring p4A2HC2 was similar to that of the parental MC4100 cells. These results demonstrate that the TpN50 protein is functionally equivalent to the E. coli OmpA protein. If TpN50 functions in a similar fashion in T. pallidum, then it may be localized to the treponemal outer membrane. 相似文献
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Arjan C. Houweling Semir Somi Maurice J.B. Van Den Hoff Antoon F.M. Moorman Vincent M. Christoffels 《Anatomical record (Hoboken, N.J. : 2007)》2002,266(2):93-102
In mouse, atrial natriuretic factor (ANF) gene expression was shown to be a marker for chamber formation within the embryonic heart. To gain insight into the process of chamber formation in the chicken embryonic heart, we analyzed the expression pattern of cANF during development. We found cANF to be specifically expressed in the myocardium of the morphologically distinguishable atrial and ventricular chambers, similar to ANF in mouse. cANF expression was never detected in the myocardium of the atrioventricular canal (AVC), inner curvature, and outflow tract (OFT), which is lined by endocardial cushions. Expression was strictly excluded from the interventricular myocardium and most proximal part of the bundle branches, as identified by the expression of Msx‐2, whereas the rest of the bundle branches, trabeculae, and surrounding working myocardium did express cANF. The myocardium that forms de novo within the cushions after looping did not express cANF. At HH9 cANF expression was first observed in a subset of cardiomyocytes, which was localized ventrally in the fused heart tube and laterally in the unfused cardiac sheets. Together, these results show that cANF expression can be used to distinguish differentiated chamber (working) myocardium, including the peripheral ventricular conduction system, from embryonic myocardium. We conclude that differentiation of chamber myocardium takes place already at HH9 at the ventral side of the linear heart tube, possibly preceded by latero‐medial signals in the unfused cardiac sheets. Anat Rec 266:93–102, 2002. © 2002 Wiley‐Liss, Inc. 相似文献
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Cloning, expression, and molecular characterization of the dermonecrotic toxin gene of Bordetella spp. 总被引:3,自引:0,他引:3
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A cosmid library of random fragments of Bordetella bronchiseptica genomic DNA was prepared and screened with oligonucleotides designed from the sequence of the B. pertussis dermonecrotic toxin (DNT) gene. Two cosmid clones which apparently contained the complete B. bronchiseptica DNT gene were identified, but they did not express the toxin. A 5-kb fragment containing the DNT gene was subcloned from one of the cosmid clones onto a high-copy-number plasmid, and this resulted in low-level expression of the toxin. The expression level was increased by deletion of a small region upstream of the coding sequence. Assays for biological activity, including the infant mouse dermonecrosis assay, confirmed that the product of the cloned gene was DNT. The complete sequence of the B. bronchiseptica DNT gene was determined and was more than 99% homologous to the DNT gene of B. pertussis. A putative purine nucleotide-binding motif was shown to be important for toxic activity. Extracts containing the recombinant or the native toxin induced DNA synthesis in Swiss 3T3 cells but inhibited cell division leading to binucleation. 相似文献
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Identification, characterization, and expression of the BiP endoplasmic reticulum resident chaperonins in Pneumocystis carinii. 总被引:1,自引:0,他引:1
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We have isolated, characterized, and examined the expression of the genes encoding BiP endoplasmic reticulum (ER) resident chaperonins responsible for transport, maturation, and proper folding of membrane and secreted proteins from two divergent strains of Pneumocystis carinii. The BiP genes, Pcbip and Prbip, from the P. c. carinii (prototype) strain and the P. c. rattus (variant) strain, respectively, are single-copy genes that reside on chromosomes of approximately 330 and approximately 350 kbp. Both genes encode approximately 72.5-kDa proteins that are most homologous to BiP genes from other organisms and exhibit the amino-terminal signal peptides and carboxyl-terminal ER retention sequences that are hallmarks of BiP proteins. We established short-term P. carinii cultures to examine expression and induction of Pcbip in response to heat shock, glucose starvation, inhibition of protein transport or N-linked glycosylation, and other conditions known to affect proper transport, glycosylation, and maturation of membrane and secreted proteins. These studies indicated that Pcbip mRNA is constitutively expressed but induced under conditions known to induce BiP expression in other organisms. In contrast to mammalian BiP genes but like other fungal BiP genes, P. carinii BiP mRNA levels are induced by heat shock. Finally, the Prbip and Pcbip coding sequences surprisingly exhibit only approximately 83% DNA and approximately 90% amino acid sequence identity and show only limited conservation in noncoding flanking and intron sequences. Analyses of the P. carinii BiP gene sequences support inclusion of P. carinii among the fungi but suggest a large divergence and possible speciation among P. carinii strains infecting a given host. 相似文献
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摘要:目的 研究红系分化过程中,红系分化特异转录因子EKLF对miR-96的表达调控。方法:氯化高铁血红素(Hemin)诱导人K562细胞(人红白血病细胞系)向红系分化,Real-time PCR检测miR-96表达。生物信息学分析miR-96转录起始点上游可能的EKLF结合位点。并经染色质免疫共沉淀(ChIP)与双荧光报告基因实验验证EKLF与这些位点结合的生物学意义。结果 Hemin诱导K562细胞向红系分化过程中,miR-96表达显著升高,且在48h表达最高(3.94156±0.63995, p<0.05)。生物信息学分析显示miR-96转录起始位点上游2.0kb内含有多个EKLF结合位点。经ChIP验证这些位点有EKLF的结合,且EKLF与这些位点结合能募集RNA聚合酶II(Pol II)结合,起始转录。进一步的双荧光报告基因实验也证明EKLF对miR-96转录起始点上游序列具有转录激活作用。结论EKLF转录激活miR-96在红系分化过程中的表达。 相似文献
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On the basis of behavioral interactions with mutations in a potassium channel gene of Drosophila--Shaker (Sh)--we have isolated mutations in a new gene called inebriated (ine). In a wildtype background, ine mutants display no observable behavioral defects. However, in a Sh mutant background, ine mutations cause downturned wings and an indented thorax. This distinctive phenotype is also exhibited by flies of other genotypes that cause extreme neuronal hyperexcitability. We utilized the potassium channel blocking drugs quinidine and dideoxy forskolin (DDF) to test the effects of ine on synaptic transmission. DDF and ine mutations each potentiated the effects of quinidine on synaptic transmission, but neither had any observable effects in the absence of quinidine. Application of DDF to ine mutants had no effects either in the presence or absence of quinidine. We conclude that ine mutations increase neuronal membrane excitability and perhaps block a DDF-sensitive potassium channel. 相似文献
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Despite the increasing quality and quantity of genomic sequence that is available to researchers, predicting gene function from sequence information remains a challenge. One method for obtaining rapid insight into potential functional roles of novel genes is through gene expression mapping. We have performed a high throughput whole-mount in situ hybridization (ISH) screen with chick embryos to identify novel, differentially expressed genes. Approximately 1,200 5' expressed sequence tags (ESTs) were generated from cDNA clones of a Hamburger and Hamilton (HH) stage 4-7 (late gastrula) chick embryo endoderm-mesoderm library. After screening to remove ubiquitously expressed cDNAs and internal clustering and after comparison to GenBank sequences, remaining cDNAs (representing both characterized and uncharacterized genes) were screened for expression in HH stage 3-14 embryos by automated high throughput ISH. Of 786 cDNAs for which ISH was successfully performed, approximately 30% showed ubiquitous expression, 40% were negative, and approximately 30% showed a restricted expression pattern. cDNAs were identified that showed restricted expression in every embryonic region, including the primitive streak, somites, developing cardiovascular system and neural tube/neural crest. A relational database was developed to hold all EST sequences, ISH images, and corresponding BLAST report information, and to enable browsing and querying of data. A user interface is freely accessible at http://geisha.biosci.arizona.edu. Results show that high throughput whole-mount ISH provides an effective approach for identifying novel genes that are differentially expressed in the developing chicken embryo. 相似文献
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Identification, cloning, expression, and characterization of the gene for Plasmodium knowlesi surface protein containing an altered thrombospondin repeat domain
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Mahajan B Jani D Chattopadhyay R Nagarkatti R Zheng H Majam V Weiss W Kumar S Rathore D 《Infection and immunity》2005,73(9):5402-5409
Proteins present on the surface of malaria parasites that participate in the process of invasion and adhesion to host cells are considered attractive vaccine targets. Aided by the availability of the partially completed genome sequence of the simian malaria parasite Plasmodium knowlesi, we have identified a 786-bp DNA sequence that encodes a 262-amino-acid-long protein, containing an altered version of the thrombospondin type I repeat domain (SPATR). Thrombospondin type 1 repeat domains participate in biologically diverse functions, such as cell attachment, mobility, proliferation, and extracellular protease activities. The SPATR from P. knowlesi (PkSPATR) shares 61% and 58% sequence identity with its Plasmodium falciparum and Plasmodium yoelii orthologs, respectively. By immunofluorescence analysis, we determined that PkSPATR is a multistage antigen that is expressed on the surface of P. knowlesi sporozoite and erythrocytic stage parasites. Recombinant PkSPATR produced in Escherichia coli binds to a human hepatoma cell line, HepG2, suggesting that PkSPATR is a parasite ligand that could be involved in sporozoite invasion of liver cells. Furthermore, recombinant PkSPATR reacted with pooled sera from P. knowlesi-infected rhesus monkeys, indicating that native PkSPATR is immunogenic during infection. Further efficacy evaluation studies in the P. knowlesi-rhesus monkey sporozoite challenge model will help to decide whether the SPATR molecule should be developed as a vaccine against human malarias. 相似文献
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《Journal of chemical neuroanatomy》2010,39(4):282-291
Here we report a functional autoradiographic study of [35S]GTPγS binding induced by α2-adrenoceptor activation in chicken brain tissue sections using both 10−4 M UK 14304 (bromoxidine or brimonidine) and 10−6 M epinephrine as α2-adrenoceptor agonists. Assays were performed using two different incubation buffers: glycylglycine or Tris–HCl. Changes in the [35S]GTPγS basal binding values were detected, and different [35S]GTPγS specific binding values were also obtained depending on the buffer used for each drug. The best results were obtained with epinephrine in Tris–HCl, with slightly higher stimulation values than the observed with UK 14304 in glycylglycine buffer. The effect of the addition of adenosine deaminase to the incubation buffer was also tested. This effect decreasing basal binding in chicken was very small when compared to mammals, according with differences found in adenosine 1 receptor expression levels. Structures presenting α2-adrenoceptor-mediated Gi/o protein stimulation fitted with areas previously described as enriched in α2-adrenoceptors in chicken brain, and their homologous areas in mammals. These data confirm the specificity of the results and reinforce the implication of the α2-adrenoceptors in the function of these brain nuclei. On the other hand, the expression level of the different α2-adrenoceptor subtypes was tested with real-time PCR. Contrasting with the α2-adrenoceptor subtype distribution previously described with radioligand competition assays, where α2A was the predominant α2-adrenoceptor subtype (≥75%); in the present work, the ratio of α2A:α2B/C gene expression was lower than expected both in telencephalon, tectum opticum, and cerebellum. 相似文献