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Hybridization Analysis (hybridization + analysis)
Kinds of Hybridization Analysis Selected AbstractsIdentification of Three Strains of a Virus Associated with Cassava Plants Affected by Frogskin DiseaseJOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2008L. A. Calvert Abstract Cassava Frogskin Disease (CFSD) can cause severe damage to cassava roots and is one of the most important diseases of cassava in Latin America. The principal objective of this study was to identify the causal agent of CFSD. Electron microscopy, viral purifications, double-stranded RNA (dsRNA) analysis, cloning, sequencing, rtPCR and hybridizations were carried out to characterize and associate a novel virus with the disease. Virus-like particles of 70 and 45 nm in diameter were found in affected cassava plants and partially purified preparations respectively. Nine species of dsRNA were associated with this disease and cDNA clones to six genomic segments were synthesized from the purified dsRNAs. The putative proteins predicted from the sequence of the cassava virus cDNA clones have similarity with the P1, P2, P3, P4, P5 and P10 proteins of Rice ragged stunt virus (RRSV). Phylogenic analysis confirmed that this virus is a member of the family Reoviridae and is most closed related to RRSV. Hybridization analyses of dsRNA identified S1, S2, S3, S4, S5 and S10 genomic segments in the CFSD-affected plants, but not in healthy controls. Additionally, 26 isolates were compared using a portion of the putative polymerase gene. The virus was detected in all 26 isolates, and they were classified into three distinct races. The association of this virus with CFSD was strengthened by the detection of this virus in diseased plants collected from different locations throughout Colombia. [source] Mutation in the abcb7 gene causes abnormal iron and fatty acid metabolism in developing medaka fishDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 9 2008Akimitsu Miyake The medaka fish (Oryzias latipes) is an emerging model organism for which a variety of unique developmental mutants have now been generated. Our recent mutagenesis screening of the medaka isolated a unique mutant that develops a fatty liver at larval stages. Positional cloning identified the responsible gene as medaka abcb7. Abcb7, a mitochondrial ABC (ATP binding cassette) half-transporter, has been implicated in iron metabolism. Recently, human Abcb7 was found to be mutated in X-linked sideroblastic anemia with cerebellar ataxia (XLSA/A). The homozygous medaka mutant exhibits abnormal iron metabolism in erythrocytes and accumulation of lipid in the liver. Microarray and in situ hybridization analyses demonstrated that the expression of genes involved in iron and lipid metabolisms are both affected in the mutant liver, suggesting novel roles of Abcb7 in the development of physiologically functional liver. The medaka abcb7 mutant thus could provide insights into the pathogenesis of XLSA/A as well as the normal function of the gene. [source] Analysis of Sir2E in the cellular slime mold Dictyostelium discoideum: Cellular localization, spatial expression and overexpressionDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 8 2008Takahiro Katayama It has been reported that Dictyostelium discoideum encodes four silent information regulator 2 (Sir2) proteins (Sir2A,D) showing sequence similarity to human homologues of Sir2 (SIRT1,3). Further screening in a database revealed that D. discoideum encodes an additional Sir2 homologue (Sir2E). The amino acid sequence of Sir2E is not similar to those of SIRTs but is similar to those of proteins encoded by Giardia lamblia, Cryptosporidium hominis and Cryptosporidium parvum. Fluorescence of Sir2E-green fluorescent protein fusion protein was detected in the D. discoideum nucleus, indicating that Sir2E is a nuclear localizing protein. Reverse transcription,polymerase chain reaction and whole-mount in situ hybridization analyses showed that D. discoideum expressed sir2E in amoebae in the growth phase and in prestalk cells in the developmental phase. D. discoideum overexpressing sir2E grew faster than the wild type. These results indicate that Sir2E plays important roles both in the growth phase and developmental phase of D. discoideum. [source] Identification and characterization of novel calcium-binding proteins of Dictyostelium and their spatial expression patterns during developmentDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5-6 2003Haruyo Sakamoto Five putative Ca2+ -binding proteins, CBP5, 6, 7, 8 and 9, all having EF-hand motifs, were found by searching the Dictyostelium cDNA database (http://www.csm.biol.tsukuba.ac.jp/cDNAproject.html). 45Ca2+ -overlay experiments revealed that four of these (excluding CBP9) are real Ca2+ -binding proteins. Northern blot analysis revealed that the genes encoding CBP5, 6, 7 and 8 are all developmentally regulated. In situ hybridization analyses revealed that spatial expression of these genes was regulated in several different ways. CBP1, 2, 3, 5, 6 and 7 are expressed in prespore cells in the slug stage. Transcripts of the genes for CBP1 and 5 are enriched in prestalk subtype PstO cells. In contrast, CBP4 is expressed predominantly in PstO cells. CBP8 is evenly expressed at a very low level throughout the whole slug. Such distinct spatial expression patterns suggest that the CBP might be involved in morphogenesis and might have their own roles either in prespore or in prestalk cell differentiation of Dictyostelium. [source] Shh/BMP-4 signaling pathway is essential for intestinal epithelial development during Xenopus larval-to-adult remodelingDEVELOPMENTAL DYNAMICS, Issue 12 2006Atsuko Ishizuya-Oka Abstract During amphibian larval-to-adult intestinal remodeling, progenitor cells of the adult epithelium actively proliferate and differentiate under the control of thyroid hormone (TH) to form the intestinal absorptive epithelium, which is analogous to the mammalian counterpart. We previously found that TH,up-regulated expression of bone morphogenetic protein-4 (BMP-4) spatiotemporally correlates with adult epithelial development in the Xenopus laevis intestine. Here, we aimed to clarify the role of BMP-4 in intestinal remodeling. Our reverse transcriptase-polymerase chain reaction and in situ hybridization analyses indicated that mRNA of BMPR-IA, a type I receptor of BMP-4, is expressed in both the developing connective tissue and progenitor cells of the adult epithelium. More importantly, using organ culture and immunohistochemical procedures, we have shown that BMP-4 not only represses cell proliferation of the connective tissue but promotes differentiation of the intestinal absorptive epithelium. In addition, we found that the connective tissue-specific expression of BMP-4 mRNA is up-regulated by sonic hedgehog (Shh), whose epithelium-specific expression is directly induced by TH. These results strongly suggest that the Shh/BMP-4 signaling pathway plays key roles in the amphibian intestinal remodeling through epithelial,connective tissue interactions. Developmental Dynamics 235:3240,3249, 2006. © 2006 Wiley-Liss, Inc. [source] Overexpression of spermidine/spermine N1 -acetyltransferase in transgenic mice protects the animals from kainate-induced toxicityEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2000Kyllikki Kaasinen Abstract We recently generated a transgenic mouse line with activated polyamine catabolism through overexpression of spermidine/spermine N1 -acetyltransferase (SSAT). A detailed analysis of brain polyamine concentrations indicated that all brain regions of these animals showed distinct signs of activated polyamine catabolism, e.g. overaccumulation of putrescine (three- to 17-fold), appearance of N1 -acetylspermidine and decreases in spermidine concentrations. In situ hybridization analyses revealed a marked overexpression of SSAT-specific mRNA all over the brain tissue of the transgenic animals. The transgenic animals appeared to tolerate subcutaneous injections of high-dose kainate substantially better as their overall mortality was less than 50% of that of their syngenic littermates. We used the expression of glial fibrillary acidic protein (GFAP) as a marker of brain injury in response to kainate. In situ hybridization analysis with GFAP oligonucleotide up to 7 days after the administration of sublethal kainate doses showed reduced GFAP expression in transgenic animals in comparison with their non-transgenic littermates. This difference was especially striking in the cerebral cortex of the transgenic mice where the exposure to kainate hardly induced GFAP expression. The treatment with kainate likewise resulted in loss of the hippocampal (CA3) neurons in non-transgenic but not transgenic animals. These results support our earlier findings indicating that elevated concentrations of brain putrescine, irrespective whether derived from an overexpression of ornithine decarboxylase, or as shown here, from an overexpression of SSAT, play in all likelihood a neuroprotective role in brain injury. [source] Genetic characterization of the dibenzofuran-degrading Actinobacteria carrying the dbfA1A2 gene homologues isolated from activated sludgeFEMS MICROBIOLOGY LETTERS, Issue 1 2004Takashi Noumura Abstract Thirteen dibenzofuran (DF)-utilizing bacteria carrying the DF terminal dioxygenase genes homologous to those of Terrabacter sp. strain DBF63 (dbfA1A2) were newly isolated from activated sludge samples. The amplified ribosomal DNA restriction analysis and the hybridization analyses showed that these strains were grouped into five genetically different types of bacteria. The sequence analyses of the 16S rRNA genes and the dbfA1A2 homologues from these five selected isolates revealed that the isolates belonged to the genus Rhodococcus, Terrabacter or Janibacter and that they shared 99,100% conserved dbfA1A2 homologues. We investigated the genetic organizations flanking the dbfA1A2 homologues and showed that the minimal conserved DNA region present in all five selected isolates consisted of an ,9.0-kb region and that their outer regions became abruptly non-homologous. Among them, Rhodococcus sp. strain DFA3 possessed not only the 9.0-kb region but also the 6.2-kb region containing dbfA1A2 homologues. Sequencing of their border regions suggested that some genetic rearrangement might have occurred with insertion sequence-like elements. Also, within their conserved regions, some insertions or deletions were observed. [source] MLL/GRAF fusion in an infant acute monocytic leukemia (AML M5b) with a cytogenetically cryptic ins(5;11)(q31;q23q23)GENES, CHROMOSOMES AND CANCER, Issue 4 2004Ioannis Panagopoulos More than 30 fusions involving the MLL gene at 11q23 have been reported in acute myeloid leukemia (AML). Some of these chimeras are rather common, such as MLL/MLLT3(AF9), but many are quite rare, with some, for example, MLL/GRAF, described only in a single case. The MLL/GRAF fusion, in which the reciprocal hybrid was not expressed, suggesting that the former transcript was the leukemogenic one, was detected in a juvenile myelomonocytic leukemia with a t(5;11)(q31;q23). Here, we report a second case,an infant acute monocytic leukemia (AML M5b),with an MLL/GRAF fusion. By conventional G-banding, the karyotype was normal. However, Southern blot and fluorescence in situ hybridization analyses revealed that MLL was rearranged and that the 5, part of the MLL gene was inserted into 5q in the vicinity of 5q31, which harbors GRAF. Reverse-transcriptase polymerase chain reaction (PCR) showed that exon 9 of MLL was fused in-frame with exon 19 of GRAF. Extralong genomic PCR with subsequent sequence analysis demonstrated that the breakpoints occurred in intron 9 of MLL, nine base pairs (bp) downstream from exon 9, and in intron 18 of GRAF, 117 bp downstream from exon 18. A 6-bp insertion (ACACTC) of unknown origin was present at the junction. The putative MLL/GRAF fusion protein would retain the AT-hook DNA-binding domain, the DNA methyl transferase motif, the transcription repression domain of MLL, and the SH3 domain of GRAF. As expected, the reciprocal GRAF/MLL was neither expressed nor generated at the genomic level as a consequence of the ins(5;11)(q31;q23q23). On the basis of the now-reported two cases with MLL/GRAF, we conclude that this transcript,but not the reciprocal one,characterizes a rare genetic subgroup of infant AML. © 2004 Wiley-Liss, Inc. [source] Prevalence of Duodenal Ulcer-Promoting Gene (dupA) of Helicobacter pylori in Patients with Duodenal Ulcer in North Indian PopulationHELICOBACTER, Issue 6 2007H. S. Jayasinghe Arachchi Abstract Background: , The duodenal ulcer (DU)-promoting gene (dupA) of Helicobacter pylori has been identified as a novel virulent marker associated with an increased risk for DU. The presence or absence of dupA gene of H. pylori present in patients with DU and functional dyspepsia in North Indian population was studied by polymerase chain reaction (PCR) and hybridization analysis. Materials and Methods: , One hundred and sixty-six patients (96 DU and 70 functional dyspepsia) were included in this study. In addition, sequence diversity of dupA gene of H. pylori found in these patients was analyzed by sequencing the PCR products jhp0917 and jhp0918 on both strands with appropriate primers. Results: , PCR and hybridization analyses indicated that dupA gene was present in 37.5% (36/96) of H. pylori strains isolated from DU patients and 22.86% (16/70) of functional dyspepsia patients (p .05). Of these, 35 patients with DU (97.2%) and 14 patients with functional dyspepsia (81.25%) were infected by H. pylori positive for cagA genotype. Furthermore, the presence of dupA was significantly associated with the cagA -positive genotype (p .02). Conclusion: , Results of our study have shown that significant association of dupA gene with DU in this population. The dupA gene can be considered as a novel virulent marker for DU in this population. [source] Nutritional channels in breast cancerJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 9b 2009Alejandro Godoy Abstract Breast cancers increase glucose uptake by increasing expression of the facilitative glucose transporters (GLUTs), mainly GLUT1. However, little is known about the relationship between GLUT1 expression and malignant potential in breast cancer. In this study, expression and subcellular localization of GLUT1 was analysed in vivo in breast cancer tissue specimens with differing malignant potential, based on the Scarff-Bloom-Richardson (SBRI, II, III) histological grading system, and in vitro in the breast cancer cell lines, MDA-MB-468 and MCF-7, and in MDA-MB-468 cells grown as xenografts in nude athymic BALB/c male mice. In situ hybridization analyses demonstrated similar levels of GLUT1 mRNA expression in tissue sections from breast cancers of all histological grades. However, GLUT1 protein was expressed at higher levels in grade SBRII cancer, compared with SBRI and SBRIII, and associated with the expression of the proliferation marker PCNA. Immunolocalization analyses in SBRII cancers demonstrated a preferential localization of GLUT1 to the portions of the cellular membrane that faced neighbouring cells and formed ,canaliculi-like structures', that we hypothesize could have a potential role as ,nutritional channels'. A similar pattern of GLUT1 localization was observed in confluent cultures of MDA-MB-468 and MCF-7, and in MDA-MB-468 cells grown as xenografts, but not in the normal breast epithelial cell line HMEC. However, no relationship between GLUT1 expression and malignant potential of human breast cancer was observed. Preferential subcellular localization of GLUT1 could represent a physiological adaptation of a subset of breast cancer cells that form infiltrative tumours with a nodular growth pattern and that therefore need a major diffusion of glucose from blood vessels. [source] Expression of SLURP-1, an endogenous ,7 nicotinic acetylcholine receptor allosteric ligand, in murine bronchial epithelial cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2009Kazuhide Horiguchi Abstract Mammalian secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) is a positive allosteric ligand for ,7 nicotinic acetylcholine (ACh) receptors (,7 nAChRs) that potentiates responses to ACh and elicits proapoptotic activity in human keratinocytes. Mutations in the gene encoding SLURP-1 have been detected in patients with Mal de Meleda, a rare autosomal recessive skin disorder characterized by transgressive palmoplantar keratoderma. On the basis of these findings, SLURP-1 is postulated to be involved in regulating tumor necrosis factor-, (TNF-,) release from keratinocytes and macrophages via ,7 nAChR-mediated pathways. In the present study, we assessed SLURP-1 expression in lung tissue from C57BL/6J mice to investigate the functions of SLURP-1 in pulmonary physiology and pathology. Immunohistochemical and in situ hybridization analyses revealed expression of SLURP-1 protein and mRNA, respectively, exclusively in ciliated bronchial epithelial cells. This was supported by Western blotting showing the presence of the 9.5-kDa SLURP-1 protein in whole-lung tissue and trachea. In addition, high-affinity choline transporter (CHT1) was detected in apical regions of bronchial epithelial cells and in neurons located in the lamina propria of the bronchus, suggesting that bronchial epithelial cells are able to synthesize both SLURP-1 and ACh. We also observed direct contact between F4/80-positive macrophages and bronchial epithelial cells and the presence of invading macrophages in close proximity to CHT1-positive nerve elements. Collectively, these results suggest that SLURP-1 contributes to the maintenance of bronchial epithelial cell homeostasis and to the regulation of TNF-, release from macrophages in bronchial tissue. © 2009 Wiley-Liss, Inc. [source] Identification of a protein, YneA, responsible for cell division suppression during the SOS response in Bacillus subtilisMOLECULAR MICROBIOLOGY, Issue 4 2003Yoshikazu Kawai Summary A knock-out mutant of the dinR gene that encodes the SOS regulon repressor in Bacillus subtilis was constructed. The yneA, yneB and ynzC genes transcribed divergently from the dinR gene were strongly induced in mutant cells. Northern hybridization analyses revealed that these genes collectively form an operon and belong to the SOS regulon. The simultaneous deletion of dinR and yneA suppressed the filamentous phenotype of the dinR mutant. Furthermore, although yneA is suppressed in the wild-type cell in the absence of SOS induction, artificial expression of the YneA protein using an IPTG-inducible promoter resulted in cell elongation. Disruption of yneA significantly reduced cell elongation after the induction of the SOS response by mitomycin C in dinR+ cells. These results indicate that the YneA protein is responsible for cell division suppression during the SOS response in B. subtilis. Localization of the FtsZ protein to the cell division site was reduced in dinR -disrupted or yneA -expressing cells, further suggesting that the YneA protein suppresses cell division through the suppression of FtsZ ring formation. Interestingly, the B. subtilis YneA protein is structurally and phylogenetically unrelated to its functional counterpart in Escherichia coli, SulA. [source] Induction of centrosome amplification and chromosome instability in p53 -deficient lung cancer cells exposed to benzo[a]pyrene diol epoxide (B[a]PDE),THE JOURNAL OF PATHOLOGY, Issue 3 2008K Shinmura Abstract Benzo[a]pyrene diol epoxide (B[a]PDE), the ultimate carcinogenic metabolite of benzo[a] pyrene, has been implicated in the mutagenesis of the p53 gene involved in smoking-associated lung cancer. To further understand the role of B[a]PDE in lung tumour progression, we investigated its effect on the numerical integrity of centrosomes and chromosome stability in lung cancer cells lacking p53. Exposure of p53 -deficient H1299 lung cancer cells to B[a]PDE resulted in S-phase arrest, leading to abnormal centrosome amplification. Analysis of H1299 cells stably expressing fluorescence-tagged centrin (a known centriolar marker) revealed that the centrosome amplification was primarily attributable to excessive centrosome duplication rather than to centriole splitting. Forced expression of POLK DNA polymerase, which has the ability to bypass B[a]PDE,guanine lesions in an error-free manner, suppressed the B[a]PDE-induced centrosome amplification. Fluorescence in situ hybridization analyses with probes specific for chromosomes 2, 3, and 16 revealed that B[a]PDE exposure also led to chromosome instability, which was likely to have resulted from centrosome amplification. We extended these findings to primary lung carcinomas containing non-functional p53, and found a strong association between centrosome amplification and a high level of B[a]PDE,DNA accumulation. Therefore B[a]PDE contributes to neoplasia by inducing centrosome amplification and consequent chromosome destabilization as well as its mutagenic activity. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Ternary complex formation between HvMYBS3 and other factors involved in transcriptional control in barley seedsTHE PLANT JOURNAL, Issue 2 2006I. Rubio-Somoza Summary The SHAQKYF R1MYB transcription factor (TF) HvMYBS3 from barley is an activator of gene expression both during endosperm development and in aleurone cells upon seed germination. Its mRNA was detected as early as 10 days after flowering in developing barley endosperm, with a peak at 18 days, and in aleurone cells at 8 h after water imbibition, as shown by Northern blot and in situ hybridization analyses. The HvMYBS3 protein expressed in bacteria binds to oligonucleotides containing a GATA core derived from the promoters of: (i) the developing endosperm gene Itr1 (5,- GATAAGATA -3,) encoding trypsin inhibitor BTI-CMe, and (ii) the post-germinating aleurone gene Amy6.4 (5,-TATCCAC-3,/5,-GTGGATA -3,) encoding a high-pI , -amylase. Transient expression experiments in co-bombarded developing endosperms and in barley aleurone layers demonstrated that HvMYBS3 trans -activated transcription both from Itr1 and Amy6.4 promoters, in contrast with a previously reported seed-expressed R1MYB, HvMCB1, which was an activator of Itr1 and a transcriptional repressor of the Amy6.4 gene. In the yeast three-hybrid system, the HvMYBS3 protein formed a ternary complex with BPBF and BLZ2, two important seed TFs. However, no binary interactions could be detected between HvMYBS3 and BLZ2 or between HvMYBS3 and BPBF. [source] HvMCB1, a R1MYB transcription factor from barley with antagonistic regulatory functions during seed development and germinationTHE PLANT JOURNAL, Issue 1 2006Ignacio Rubio-Somoza Summary The functional analysis of hydrolase gene promoters induced by gibberellin (GA) in barley aleurone cells upon germination has identified a tripartite GA-response complex (GARC) containing a 5,- TATCCAC-3, box as well as the GA-responsive element (GARE) recognized by GAMYB and the pyrimidine box interacting with the DOF transcription factors BPBF and SAD. We show here that the MCB1 gene encoding a R1MYB protein binds to the 5,- TATCCAC-3, (GATA core) box in vitro and is a transcriptional repressor of a GA-induced amylase (Amy6.4) promoter in bombarded aleurone layers. Northern blot and mRNA in situ hybridization analyses showed that the MCB1 transcripts accumulate in the aleurone cells upon germination, as well as in endosperm tissues during seed development. The HvMCB1 protein expressed in bacteria binds in a specific manner to a 27-mer oligonucleotide containing the 5,-TATCCAC-3, sequence, derived from the promoter region of the Amy6.4 gene. Accumulation of the MCB1 transcript diminished in response to external GA incubation in aleurone cells, and in transient expression experiments HvMCB1 repressed transcription of the Amy6.4 promoter in GA-treated aleurone layers and reversed the GAMYB-mediated activation of this amylase promoter. In contrast, during endosperm maturation HvMCB1 acted as a transcription activator of the seed-specific Itr1 gene promoter through binding to a 5,-GATAAGATA-3, box. [source] Gene expression analyses on embryonic external genitalia: identification of regulatory genes possibly involved in masculinization processesCONGENITAL ANOMALIES, Issue 2 2008Hisayo Nishida ABSTRACT Androgen plays a crucial role in initiating and maintaining the expression of male sexual characteristics in mammals. In humans and mice, any defects along the pathway of androgen functions result in congenital urogenital abnormalities. The genital tubercle (GT), an anlage of the external genitalia, differentiates into a penis in males and a clitoris in females. Although masculinization of the external genitalia is androgen-dependent, the molecular pathway of its potential downstream genes is largely unclear. To identify the genes involved in mouse GT masculinization, we performed gene expression analyses, such as real-time quantitative polymerase chain reaction and section in situ hybridization analysis. From our studies we have identified candidate genes, Cyp1b1, Fkbp51 and MafB as potential androgen targets during mouse GT masculinization. [source] The potential contribution of fluorescent in situ hybridization analysis to the cytopathological diagnosis of Merkel cell carcinomaCYTOPATHOLOGY, Issue 1 2008V. Suciu We report the cases of two patients with head and neck Merkel cell carcinoma (MCC) who developed local recurrences confirmed by cytopathology. Interphase fluorescent in situ hybridization (FISH) analysis was performed for research purposes using centromeric probes of chromosomes 6 and 8, on cytological slides. Trisomy of chromosome 6 was found in 85% of tumour cells in the first case of MCC and case 2 exhibited trisomy 8 in 77% of tumour cells. In the absence of specific molecular markers, detection of trisomy 6 and/or trisomy 8 could help in identifying MCC. FISH analysis is easily and quickly performed on interphase nuclei obtained through fine needle aspiration and may be extended to the study of other relevant genetic abnormalities. [source] Identification of asymmetrically localized transcripts along the animal,vegetal axis of the Xenopus eggDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 8 2005Kensuke Kataoka In many organisms, proper embryo development depends on the asymmetrical distribution of mRNA in the cytoplasm of the egg. Here we report comprehensive screening of RNA localized in the animal or vegetal hemisphere of the Xenopus egg. Macroarrays including over 40 000 independent embryonic cDNA clones, representing at least 17 000 unigenes, were differentially hybridized with labeled probes synthesized from the mRNA of animal or vegetal blastomeres. After two rounds of screening, we identified 33 clones of transcripts that may be preferentially distributed in the vegetal region of the early stage embryo, but transcripts localized in the animal region were not found. To assess the array results, we performed northern blot and quantitative real-time reverse transcription,polymerase chain reaction analysis. As a result, 21 transcripts of the 33 were confirmed to be localized in the vegetal region of the early stage embryo. Whole-mount in situ hybridization analysis revealed that 11 transcripts, including 7 previously reported genes, were localized in the vegetal hemisphere of the egg. These 11 transcripts were categorized into three groups according to their expression patterns in the egg. The first group, which contained four transcripts, showed uniform expression in the vegetal hemisphere, similar to VegT. The second group, which contained three transcripts, showed gradual expression from the vegetal pole to the equator, similar to Vg1. The last group, which contained three transcripts, was expressed at the germ plasm, similar to Xdazl. One transcript, Xwnt11, showed both the second and the third expression patterns. [source] Expression patterns of the opsin 5,related genes in the developing chicken retinaDEVELOPMENTAL DYNAMICS, Issue 7 2008Sayuri Tomonari Abstract The opsin gene family encodes G protein,coupled seven-transmembrane proteins that bind to a retinaldehyde chromophore for photoreception. It has been reported that opsin 5 is expressed in mammalian neural tissue, but its function has been elusive. As a first step to understand the function for opsin 5 in the developing eye, we searched for chicken opsin 5 -related genes in the genome by a bioinformatic approach and isolated opsin 5 cDNA fragments from the embryonic retina by RT-PCR. We found that there are three opsin 5,related genes, designated cOpn5m (chicken opsin 5, mammalian type), cOpn5L1 (chicken opsin 5 - like 1), and cOpn5L2 (chicken opsin 5 - like 2), in the chicken genome. Quantitative PCR analysis has revealed that cOpn5m is the most abundant in the developing and early posthatching neural retina. In situ hybridization analysis has shown that cOpn5m is specifically expressed in subsets of differentiating ganglion cells and amacrine cells. These results suggest that the mammalian type opsin 5 may contribute to the development of these retinal cells in the chicken. Developmental Dynamics 237:1910,1922, 2008. © 2008 Wiley-Liss, Inc. [source] Dynamic expression patterns of RhoV/Chp and RhoU/Wrch during chicken embryonic developmentDEVELOPMENTAL DYNAMICS, Issue 4 2008Cécile Notarnicola Abstract Rho GTPases play central roles in the control of cell adhesion and migration, cell cycle progression, growth, and differentiation. However, although most of our knowledge of Rho GTPase function comes from the study of the three classic Rho GTPases RhoA, Rac1, and Cdc42, recent studies have begun to explore the expression, regulation, and function of some of the lesser-known members of the Rho GTPase family. In the present study, we cloned the avian orthologues of RhoV (or Chp for Cdc42 homologous protein) and RhoU (or Wrch - 1 for Wnt-regulated Cdc42 homolog-1) and examined their expression patterns by in situ hybridization analysis both during early chick embryogenesis and later on, during gastrointestinal tract development. Our data show that both GTPases are detected in the primitive streak, the somites, the neural crest cells, and the gastrointestinal tract with distinct territories and/or temporal expression windows. Although both proteins are 90% identical, our results indicate that cRhoV and cRhoU are distinctly expressed during chicken embryonic development. Developmental Dynamics 237:1165,1171, 2008. © 2008 Wiley-Liss, Inc. [source] Changes in gravitational force cause changes in gene expression in the lens of developing zebrafishDEVELOPMENTAL DYNAMICS, Issue 10 2006Naoko Shimada Abstract Gravity has been a constant physical factor during the evolution and development of life on Earth. We have been studying effects of simulated microgravity on gene expression in transgenic zebrafish embryos expressing gfp under the influence of gene-specific promoters. In this study, we assessed the effect of microgravity on the expression of the heat shock protein 70 (hsp70) gene in lens during development using transgenic zebrafish embryos expressing gfp under the control of hsp70 promoter/enhancer. Hsp70:gfp expression was up-regulated (45%) compared with controls during the developmental period that included the lens differentiation stage. This increase was lens specific, because the entire embryo showed only a 4% increase in gfp expression. Northern blot and in situ hybridization analysis indicated that the hsp70:gfp expression recapitulated endogenous hsp70 mRNA expression. Hypergravity exposure also increased hsp70 expression during the same period. In situ hybridization analysis for two lens-specific crystallin genes revealed that neither micro- nor hypergravity affected the expression level of ,B1 - crystallin, a non-hsp gene used as a marker for lens differentiation. However, hypergravity changed the expression level of ,A - crystallin, a member of the small hsp gene family. Terminal deoxynucleotidyl transferase,mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) assay analysis showed that altered-gravity (,g) decreased apoptosis in lens during the same period and the decrease correlated with the up-regulation of hsp70 expression, suggesting that elimination of nuclei from differentiating lens fiber cells was suppressed probably through hsp70 up-regulation. These results support the idea that ,g influences hsp70 expression and differentiation in lens-specific and developmental period specific manners and that hsp family genes play a specific role in the response to ,g. Developmental Dynamics 235:2686,2694, 2006. © 2006 Wiley-Liss, Inc. [source] Regulation of the Neurofibromatosis 2 gene promoter expression during embryonic developmentDEVELOPMENTAL DYNAMICS, Issue 10 2006Elena M. Akhmametyeva Abstract Mutations in the Neurofibromatosis 2 (NF2) gene are associated with predisposition to vestibular schwannomas, spinal schwannomas, meningiomas, and ependymomas. Presently, how NF2 is expressed during embryonic development and in the tissues affected by neurofibromatosis type 2 (NF2) has not been well defined. To examine NF2 expression in vivo, we generated transgenic mice carrying a 2.4-kb NF2 promoter driving ,-galactosidase (,-gal) with a nuclear localization signal. Whole-mount embryo staining revealed that the NF2 promoter directed ,-gal expression as early as embryonic day E5.5. Strong expression was detected at E6.5 in the embryonic ectoderm containing many mitotic cells. ,-gal staining was also found in parts of embryonic endoderm and mesoderm. The ,-gal staining pattern in the embryonic tissues was corroborated by in situ hybridization analysis of endogenous Nf2 RNA expression. Importantly, we observed strong NF2 promoter activity in the developing brain and in sites containing migrating cells including the neural tube closure, branchial arches, dorsal aorta, and paraaortic splanchnopleura. Furthermore, we noted a transient change of NF2 promoter activity during neural crest cell migration. While little ,-gal activity was detected in premigratory neural crest cells at the dorsal ridge region of the neural fold, significant activity was seen in the neural crest cells already migrating away from the dorsal neural tube. In addition, we detected considerable NF2 promoter activity in various NF2-affected tissues such as acoustic ganglion, trigeminal ganglion, spinal ganglia, optic chiasma, the ependymal cell-containing tela choroidea, and the pigmented epithelium of the retina. The NF2 promoter expression pattern during embryogenesis suggests a specific regulation of the NF2 gene during neural crest cell migration and further supports the role of merlin in cell adhesion, motility, and proliferation during development. Developmental Dynamics 235:2771,2785, 2006. © 2006 Wiley-Liss, Inc. [source] Pod1 is required in stromal cells for glomerulogenesisDEVELOPMENTAL DYNAMICS, Issue 3 2003Shiying Cui Abstract Pod1 (capsulin/epicardin/Tcf21) is a basic-helix-loop-helix transcription factor that is highly expressed in the mesenchyme of developing organs that include the kidney, lung, gut, and heart. Null Pod1 mice are born but die shortly after birth due to a lack of alveoli in the lungs and cardiac defects. In addition, the kidneys are hypoplastic and demonstrate disrupted branching morphogenesis of the ureteric bud epithelium, a marked reduction in the number of nephrons, a delay in glomerulogenesis, and blood vessel abnormalities. To further dissect the cellular function of Pod1 during kidney development, chimeric mice were generated through aggregations of null Pod1 embryonic stem cells and murine embryos ubiquitously expressing enhanced green fluorescent protein (GFP). Histologic, immunohistochemical, and in situ hybridization analysis of the resulting chimeric offspring demonstrated both cell autonomous and non,cell autonomous roles for Pod1 in the differentiation of specific renal cell lineages that include peritubular interstitial cells and pericytes. Most strikingly, the glomerulogenesis defect was rescued by the presence of wild-type stromal cells, suggesting a non,cell autonomous role for Pod1 in this cell population. © 2003 Wiley-Liss, Inc. [source] An SNF2 factor involved in mammalian development and cellular proliferationDEVELOPMENTAL DYNAMICS, Issue 1 2001Eric H. Raabe Abstract Members of the SNF2 (Sucrose Non-Fermenter) family of chromatin-remodeling proteins function in processes ranging from DNA repair to transcription to methylation. Using differential display, we recently identified a novel member of the SNF2 family that is highly expressed at the mRNA level in proliferating cells and is down-regulated during apoptosis. We have named this gene PASG (Proliferation-Associated SNF2-like Gene). Northern blot analysis of adult mouse tissues shows PASG to be highly expressed in proliferating organs such as thymus, bone marrow, and testis and absent from nonproliferative tissues such as brain and heart. In situ hybridization analysis of mouse embryos shows that PASG is differentially expressed during development, with highest expression in developing face, limbs, skeletal muscle, heart, and tail. In vitro, PASG expression correlates with a shift from a quiescent to a proliferative state. Mice null for PASG (also known as LSH or Hells) are reported to die perinatally, although the mechanism for lethality is unclear (Geiman and Muegge, 2000). To test the hypothesis that PASG functions in cell proliferation, we compared 5-bromodeoxyuridine (BrdU) incorporation in C33A cells transiently transfected with PASG versus empty vector and found that PASG transfected cells showed a significant decrease in the amount of BrdU incorporation. These findings suggest that PASG plays a role in cell proliferation and may function in the development of multiple cell lineages during murine embryogenesis. © 2001 Wiley-Liss, Inc. [source] DNA Hybridization at Magnetic Nanoparticles with Electrochemical Stripping DetectionELECTROANALYSIS, Issue 23 2004Ningning Zhu Abstract A simple and practical method for electrochemical DNA hybridization assay has been developed to take advantage of magnetic nanoparticles for ssDNA immobilization and zinc sulfide nanoparticle as oligonucleotide label. Magnetic nanoparticles were prepared by coprecipitation of Fe2+ and Fe3+ with NH4OH, and then amino silane was coated onto the surface of magnetite nanoparticles. The magnetic nanoparticles have the advantages of easy preparation, easy surface modification and low cost. The target ssDNA with the phosphate group at the 5, end was then covalently immobilized to the amino group of magnetite nanoparticles by forming a phosphoramidate bond in the presence of 1-ethyl-3-(3-dimeth-ylaminopropyl)carbodiimide (EDAC). The zinc sulfide (ZnS) nanoparticle-labeled oligonucleotides probe was used to identify the target ssDNA immobilized on the magnetic nanoparticles based on a specific hybridization reaction. The hybridization events were assessed by the dissolution of the zinc sulfide nanoparticles anchored on the hybrids and the indirect determination of the dissolved zinc ions by anodic stripping voltammetry (ASV) at a mercury film glassy carbon electrode (GCE). The proposed method couples the high sensitivity of anodic stripping analysis for zinc ions with effective magnetic separation for eliminating nonspecific adsorption effects and offers great promise for DNA hybridization analysis. [source] Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitisENVIRONMENTAL MICROBIOLOGY, Issue 10 2008Gabriel J. Milinovich No abstract is available for this article. [source] Fungal rDNA signatures in coronary atherosclerotic plaquesENVIRONMENTAL MICROBIOLOGY, Issue 12 2007Stephan J. Ott Summary Bacterial DNA has been found in coronary plaques and it has therefore been concluded that bacteria may play a role as trigger factors in the chronic inflammatory process underlying coronary atherosclerosis. However, the microbial spectrum is complex and it is not known whether microorganisms other than bacteria are involved in coronary disease. Fungal 18S rDNA signatures were systematically investigated in atherosclerotic tissue obtained through catheter-based atherectomy of 38 patients and controls (unaffected coronary arteries) using clone libraries, denaturating gradient gel analysis (DGGE), in situ hybridization and fluorescence in situ hybridization (FISH). Fungal DNA was found in 35 of 38 (92.11%) coronary heart disease patients by either polymerase chain reaction (PCR) with universal primers or in situ hybridization analysis (n = 5), but not in any control sample. In a clone library with more than 350 sequenced clones from pooled patient DNA, an overall richness of 19 different fungal phylotypes could be observed. Fungal profiles of coronary heart disease patients obtained by DGGE analysis showed a median richness of fungal species of 5 (range from 2 to 9) with a high interindividual variability (mean similarity 18.83%). For the first time, the presence of fungal components in atherosclerotic plaques has been demonstrated. Coronary atheromatous plaques harbour diverse and variable fungal communities suggesting a polymicrobial contribution to the chronic inflammatory aetiology. [source] Reduced ethanol response in the alcohol-preferring RHA rats and neuropeptide mRNAs in relevant structuresEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2006Marc Guitart-Masip Abstract Roman rat strains, genetically selected for high (RHA) or low (RLA) active avoidance acquisition in the two-way shuttle box, differ in dopaminergic activity. These two strains appear to be a valid laboratory model of divergent sensation/novelty and substance-seeking profiles. RHA rats show higher ethanol intake and preference than do RLA rats, and it was suggested that RHA rats are more tolerant than RLA to the effects of alcohol. In the hole-board test, we found that the non-alcohol-preferring RLA rats showed enhanced responsiveness to the stimulatory effects of intraperitoneal administration of 0.25 g/kg ethanol when compared with RHA rats. In situ hybridization analysis showed higher levels of preprodynorphin in the accumbens shell and higher levels of preproenkephalin in the cingulate cortex in RHA rats. RLA rats showed higher levels of enkephalin gene transcripts in restricted areas of the dorsal striatum. Finally, differences in cholecystokinin gene transcript, suggestive of a different arrangement of certain interneurons, were found in different cortical areas. The differences in peptide gene expression found between the two strains might reflect the differences in alcohol preference and sensitivity. RHA rats may have more predictive value than other rodent alcoholism models, as high initial tolerance to ethanol is a risk factor for alcoholism in humans. [source] Spatio-temporal distribution of cellular retinoid binding protein gene transcripts in the developing and the adult cochlea.EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2000CRBPI-null mutant mice, Morphological, functional consequences in CRABP- Abstract The expression patterns of the mouse cellular retinoid binding protein genes were investigated by in situ hybridization analysis in the inner ear from 10.5 days post coďtum (dpc) up to the adult stage. The cellular retinoic acid binding protein II (CRABPII) and cellular retinol binding protein I (CRBPI) were present in a widespread and abundant pattern in cochlear structures during embryogenesis. Expression of the cellular retinoic acid binding protein I (CRABPI) is restricted during development in Kölliker's organ whilst cellular retinol binding protein II (CRBPII) is only visible after birth with a ubiquitous distribution in most regions of the cochlea including nervous components. No CRABP or CRBP transcripts were observed in the auditory receptors. Morphological observations of CRBPI- and CRABPI/CRABPII-null mutant fetus at 18.5 dpc do not show any structural modification at the level of the organ of Corti. Furthermore, electrophysiological tests performed by measuring distorsion-product otoacoustic emissions and auditory brainstem evoked responses did not present significant alteration of the auditory function for the different types of mutants. The expression of retinoid binding proteins in cochlear structures during embryogenesis could suggest important roles for these proteins during ontogenesis and morphogenesis of the inner ear. Despite these observations, morphological and functional data from mutant mice did not present obvious modifications of the cochlear structures and auditory thresholds. It is therefore unlikely that CRABPs and CRBPI are directly involved in development of the cochlea and hair cell differentiation. [source] Overexpression of spermidine/spermine N1 -acetyltransferase in transgenic mice protects the animals from kainate-induced toxicityEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2000Kyllikki Kaasinen Abstract We recently generated a transgenic mouse line with activated polyamine catabolism through overexpression of spermidine/spermine N1 -acetyltransferase (SSAT). A detailed analysis of brain polyamine concentrations indicated that all brain regions of these animals showed distinct signs of activated polyamine catabolism, e.g. overaccumulation of putrescine (three- to 17-fold), appearance of N1 -acetylspermidine and decreases in spermidine concentrations. In situ hybridization analyses revealed a marked overexpression of SSAT-specific mRNA all over the brain tissue of the transgenic animals. The transgenic animals appeared to tolerate subcutaneous injections of high-dose kainate substantially better as their overall mortality was less than 50% of that of their syngenic littermates. We used the expression of glial fibrillary acidic protein (GFAP) as a marker of brain injury in response to kainate. In situ hybridization analysis with GFAP oligonucleotide up to 7 days after the administration of sublethal kainate doses showed reduced GFAP expression in transgenic animals in comparison with their non-transgenic littermates. This difference was especially striking in the cerebral cortex of the transgenic mice where the exposure to kainate hardly induced GFAP expression. The treatment with kainate likewise resulted in loss of the hippocampal (CA3) neurons in non-transgenic but not transgenic animals. These results support our earlier findings indicating that elevated concentrations of brain putrescine, irrespective whether derived from an overexpression of ornithine decarboxylase, or as shown here, from an overexpression of SSAT, play in all likelihood a neuroprotective role in brain injury. [source] | |||||||||||||||||||||||||||||||