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Situ Hybridization Experiments (situ + hybridization_experiment)
Selected AbstractsLineage-independent mosaic expression and regulation of the Ciona multidom gene in the ancestral notochordDEVELOPMENTAL DYNAMICS, Issue 7 2007Izumi Oda-Ishii Abstract The transcription factor Ciona Brachyury (Ci-Bra) plays an essential role in notochord development in the ascidian Ciona intestinalis. We characterized a putative Ci-Bra target gene, which we named Ci - multidom, and analyzed in detail its expression pattern in normal embryos and in embryos where Ci - Bra was misexpressed. Ci - multidom encodes a novel protein, which contains eight CCP domains and a partial VWFA domain. We show that an EGFP-multidom fusion protein localizes preferentially to the endoplasmic reticulum (ER), and is excluded from the nucleus. In situ hybridization experiments demonstrate that Ci - multidom is expressed in the notochord and in the anterior neural boundary (ANB). We found that the expression in the ANB is fully recapitulated by an enhancer element located upstream of Ci - multidom. By means of misexpression experiments, we provide evidence that Ci-Bra controls transcription of Ci - multidom in the notochord; however, while Ci-Bra is homogeneously expressed throughout this structure, Ci - multidom is transcribed at detectable levels only in a random subset of notochord cells. The number of notochord cells expressing Ci - multidom varies among different embryos and is independent of developmental stage, lineage, and position along the anterior,posterior axis. These results suggest that despite its morphological simplicity and invariant cell-lineage, the ancestral notochord is a mosaic of cells in which the gene cascade downstream of Brachyury is differentially modulated. Developmental Dynamics 236:1806,1819, 2007. © 2007 Wiley-Liss, Inc. [source] Localization of nAChR subunit mRNAs in the brain of Macaca mulattaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2000Zhi-Yan Han Abstract We present here a systematic mapping of nAChR subunit mRNAs in Macaca mulatta brain. A fragment, from the transmembrane segments MIII to MIV of Macaca neuronal nAChR subunits was cloned, and shown to exhibit high identity (around 95%) to the corresponding human subunits. Then, specific oligodeoxynucleotides were synthesized for in situ hybridization experiments. Both ,4 and ,2 mRNA signals were widely distributed in the brain, being stronger in the thalamus and in the dopaminergic cells of the mesencephalon. Most brain nuclei displayed both ,4 and ,2 signals with the exception of some basal ganglia regions and the reticular thalamic nucleus which were devoid of ,4 signal. ,6 and ,3 mRNA signals were selectively concentrated in the substantia nigra and the medial habenula. The strongest signals for ,3 or ,4 mRNAs were found in the epithalamus (medial habenula and pineal gland), whereas there were no specific ,3 or ,4 signals in mesencephalic dopaminergic nuclei. ,5 and ,7 mRNA signals were found in several brain areas, including cerebral cortex, thalamus and substantia nigra, although at a lower level than ,4 and ,2. The distribution of ,3, ,4, ,5, ,6, ,7, ,2, ,3 and ,4 subunit mRNAs in the monkey is substantially similar to that observed in rodent brain. Surprisingly, ,2 mRNA signal was largely distributed in the Macaca brain, at levels comparable with those of ,4 and ,2. This observation represents the main difference between rodent and Macaca subunit mRNA distribution and suggests that, besides ,4,2*, ,2,2* nAChRs constitute a main nAChR isoform in primate brain. [source] Altered expression of transcripts for ,-tubulin and an unidentified gene in the spinal cord of phenyl saligenin phosphate treated hens (Gallus gallus)JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 5 2003Jonathan H. Fox Abstract Phenyl saligenin phosphate (PSP) induces a central-peripheral distal axonopathy in domestic fowl that develops 7,21 days after a single exposure. Neurotoxic esterase (NTE) is the initial molecular target for this neurotoxicity. PSP has to covalently bind to NTE and chemically "age" for induction of axonopathy. It was hypothesized that exposure to PSP results in early changes in spinal cord gene expression that do not occur with phenylmethylsulfonyl fluoride, a non-neuropathic compound that also inhibits NTE, or DMSO controls. Targeted display was used to screen ,15,000 gel bands. Three candidate genes were identified, but only the transcript designated P1 showed decreased expression following PSP exposure (2 mg/kg i.m.) in subsequent Northern blot and in situ hybridization experiments in samples taken <48 h after exposure. Additional experiments revealed that a ,2.5 kb ,-tubulin transcript had decreased expression at 12,48 h after PSP exposure, with maximum change at 48 h (33%, p = 0.0479). A ,4.5 kb ,-tubulin transcript had increased expression at 12 h (38%, p = 0.0125) and decreased expression at 48 h (28%, p = 0.0576). In situ hybridization on spinal cord revealed neuronal expression of P1 and ,-tubulin transcripts. Decreased expression of transcripts for P1 and ,-tubulin was present at 12 and 48 h, respectively. This decrease occurred in all neurons, not just those whose axons degenerate. Results suggest that (1) in PSP-induced OPIDN (organophosphorus-induced delayed neurotoxicity) some gene transcript expression changes are associated with initiation of axonopathy, and (2) PSP modulates spinal cord gene expression in neuronal types that do not undergo axonal degeneration. © 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:263,271, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10088 [source] Modular changes of cis-regulatory elements from two functional Pit1 genes in the duplicated genome of Cyprinus carpioJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2006G. Kausel Abstract The pituitary-specific transcription factor Pit1 is involved in its own regulation and in a network of transcriptional regulation of hypothalamo-hypophyseal factors including prolactin (PRL) and growth hormone (GH). In the ectotherm teleost Cyprinus carpio, Pit1 plays an important role in regulation of the adaptive response to seasonal environmental changes. Two Pit1 genes exist in carp, a tetraploid vertebrate and transcripts of both genes were detected by RT-PCR analysis. Powerful comparative analyses of the 5,-flanking regions revealed copy specific changes comprising modular functional units in the naturally evolved promoters. These include the precise replacement of four nucleotides around the transcription start site embedded in completely conserved regions extending upstream of the TATA-box, an additional transcription factor binding site in the 5,-UTR of gene-I and, instead, duplication of a 9 bp element in gene-II. Binding of nuclear factors was assessed by electro mobility shift assays using extracts from rat pituitary cells and carp pituitary. Binding was confirmed at one conserved Pit1, one conserved CREB and one consensus MTF1. Interestingly, two functional Pit1 sites and one putative MTF1 binding site are unique to the Pit1 gene-I. In situ hybridization experiments revealed that the expression of gene-I in winter carp was significantly stronger than that of gene-II. Our data suggest that the specific control elements identified in the proximal regulatory region are physiologically relevant for the function of the duplicated Pit1 genes in carp and highlight modular changes in the architecture of two Pit1 genes that evolved for at least 12 MYA in the same organism. J. Cell. Biochem. 99: 905,921, 2006. © 2006 Wiley-Liss, Inc. [source] A simple and rapid technique for the detection of Epstein-Barr virus DNA in HIV-associated oral hairy leukoplakia biopsiesJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 3 2000M. J. E. M. F. Mabruk Abstract: A method of generating nucleic acid probes by polymerase chain reaction (PCR) for the detection of Epstein,Barr virus (EBV)-DNA by in situ hybridization in oral hairy leukoplakia (OHL) lesions is described. This method has the advantage over older methods of being cheaper, quicker and retaining sensitivity and specificity. Purified PCR products of Epstein-Barr virus DNA of 110 bp and 328 bp were labelled with biotin by nick translation or random primer labelling and were compared in in situ hybridization experiments with probes prepared by incorporation of biotin-labelled nucleotides in the PCR reaction mixture, with EBV viral DNA as a template. These probes were applied to 18 OHL tongue biopsies known to be positive for EBV-DNA, using a commercially available biotin-labelled BamHI "V" fragment EBV-DNA probe. To determine the specificity of the probes, we applied them to 20 normal tongue tissue samples and to 12 biopsies taken from keratotic tongue lesions from patients without risk factors for HIV infection and known to be negative for EBV-DNA. Clear positive signals for EBV-DNA were detected in all 18 cases of OHL biopsies using the amplimer of 328 bp labelled by PCR and random primer labelling. However, nick translation labelling was less efficient and sensitive. All control specimens were negative for EBV-DNA. [source] Affinity of corpora amylacea for oligonucleotides: Sequence dependency and proteinaceous binding motifNEUROPATHOLOGY, Issue 4 2006Ioan A. Balea Corpora amylacea (CA) have an affinity to nucleic acids as shown by in situ hybridization experiments. However, little is known about the specificity of this interaction, as well as the mechanism involved. We investigated the ability of different probes of digoxigenin-labeled oligonucleotides corresponding to some specific neuronal receptors, both sense and antisense, to bind to CA from human autopsy brain tissue. The bound nucleotides were detected with antidigoxigenin antibody and the signal was further amplified using the tyramide signal amplification system. The affinity of binding varies with the sequence of nucleotides. The most intense signal is produced by the adenosine-2A receptor antisense probe and the least intense signal is produced by the N-methyl-D-aspartate receptor sense probe. The affinity of binding for the same probe does not depend on the localization of CA in the central nervous system. Complete staining loss by proteinase K pretreatment in higher concentrations shows that the binding motif is partially proteinaceous. The circumferential but not the punctate internal staining is diminished by mild amylglucosidase pretreatment, suggesting a process of progressive apposition and condensation. [source] Association of ANA, a Member of the Antiproliferative Tob Family Proteins, with a Cafl Component of the CCR4 Transcriptional Regulatory ComplexCANCER SCIENCE, Issue 6 2001Yutaka Yoshida A 35-kDa protein, ANA, belongs to an emerging family of antiproliferative proteins consisting of Tob, Tob2, ANA/BTG3, PC3B, PC3/TIS21/BTG2, and BTG1. All of these, except ANA and PC3B, have been shown to interact with the CCR4 transcription factor-associated protein Cafl. Here we show that ANA also associates with Cafl, ANA being the preferred partner of Cafl among the Tob family proteins. Although ANA is likely to interact with Cafl at its amino-terminal hall', which is conserved among the family members, our data suggest that the carboxyl-terminal half of ANA plays a role in the interaction. Finally, in situ hybridization experiments revealed that expression of Caf1 overlaps at least in part with that of ANA. Thus, ANA could function through its interaction with Caf1. [source] | |||||||||||||