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Hybridization Techniques (hybridization + techniques)
Kinds of Hybridization Techniques Selected AbstractsEpidemiology of tomato yellow leaf curl begomovirus in the Fayium area, EgyptEPPO BULLETIN, Issue 2 2000A. E. Aboul-Ata Tomato yellow leaf curl begomovirus (TYLCV) severely invaded tomato plantations in Egypt (Lower and Middle Egypt) in 1989. This study aimed to discover the relationship between TYLCV and other epidemic-associated factors in the Fayium area. The rate of TYLCV infection was inspected visually for three successive years (1994/1996) in the Fayium area. During the same period, whiteflies were collected for virus detection using bait-plant and DNA hybridization techniques. DAS-ELISA was used to detect mixed virus infections in tomato plants. TYLCV infection was prevalent (60,68%) and severe (2.1,3.0) in the Fayium fields. Cucumber mosaic cucumovirus (CMV) was found in some fields (5,28%) with moderate severity (1.0,20). Potato Y potyvirus (PVY) and potato leaf roll polerovirus (PLRV) were found in few fields (5,19% and 5% respectively) at very low severity. There was a negative correlation between TYLCV occurrence and distance from the source of infection, and a positive correlation (98%) between TYLCV intensity and percentage of viruliferous whiteflies in 1994 and 1995. There was no positive correlation between TYLCV and the total population of whiteflies caught during the same period. Five percent of viruliferous whiteflies, as proved by cDNA hybridization, led to 46% TYLCV infection. The same percentage of whiteflies, as shown by bioassay, led to 68% TYLCV infection. Monitoring of viruliferous whiteflies could be used for early prediction of TYLCV infection. [source] Simultaneous detection of the establishment of seed-inoculated Pseudomonas fluorescens strain DR54 and native soil bacteria on sugar beet root surfaces using fluorescence antibody and in situ hybridization techniquesFEMS MICROBIOLOGY ECOLOGY, Issue 1 2000Peter Stephensen Lübeck Abstract Colonization at sugar beet root surfaces by seedling-inoculated biocontrol strain Pseudomonas fluorescens DR54 and native soil bacteria was followed over a period of 3 weeks using a combination of immunofluorescence (DR54-targeting specific antibody) and fluorescence in situ hybridization (rRNA-targeting Eubacteria EUB338 probe) techniques with confocal laser scanning microscopy. The dual staining protocol allowed cellular activity (ribosomal number) to be recorded in both single cells and microcolonies of strain DR54 during establishment on the root. After 2 days, the population density of strain DR54 reached a constant level at the root basis. From this time, however, high cellular activity was only found in few bacteria located as single cells, whereas all microcolony-forming cells occurring in aggregates were still active. In contrast, a low density of strain DR54 was observed at the root tip, but here many of the bacteria located as single cells were active. The native population of soil bacteria, comprising a diverse assembly of morphologically different forms and size classes, initiated colonization at the root basis only after 2 days of incubation. Hence the dual staining protocol allowed direct microscopic studies of early root colonization by both inoculant and native soil bacteria, including their differentiation into active and non-active cells and into single or microcolony-forming cells. [source] Mechanisms of gene amplification and evidence of coamplification in drug-resistant human osteosarcoma cell linesGENES, CHROMOSOMES AND CANCER, Issue 4 2009Claudia M. Hattinger Gene amplification and copy number changes play a pivotal role in malignant transformation and progression of human tumor cells by mediating the activation of genes and oncogenes, which are involved in many different cellular processes including development of drug resistance. Since doxorubicin (DX) and methotrexate (MTX) are the two most important drugs for high-grade osteosarcoma (OS) treatment, the aim of this study was to identify genes gained or amplified in six DX- and eight MTX-resistant variants of the human OS cell lines U-2OS and Saos-2, and to get insights into the mechanisms underlying the amplification processes. Comparative genomic hybridization techniques identified amplification of MDR1 in all six DX-resistant and of DHFR in three MTX-resistant U-2OS variants. In addition, progressive gain of MLL was detected in the four U-2OS variants with higher resistance levels either to DX or MTX, whereas gain of MYC was found in all Saos-2 MTX-resistant variants and the U-2OS variant with the highest resistance level to DX. Fluorescent in situ hybridization revealed that MDR1 was amplified in U-2OS and Saos-2/DX-resistant variants manifested as homogeneously staining regions and double minutes, respectively. In U-2OS/MTX-resistant variants, DHFR was amplified in homogeneously staining regions, and was coamplified with MLL in relation to the increase of resistance to MTX. Gene amplification was associated with gene overexpression, whereas gene gain resulted in up-regulated gene expression. These results indicate that resistance to DX and MTX in human OS cell lines is a multigenic process involving gene copy number and expression changes. © 2008 Wiley-Liss, Inc. [source] Genetics of dermatofibrosarcoma protuberans family of tumors: From ring chromosomes to tyrosine kinase inhibitor treatmentGENES, CHROMOSOMES AND CANCER, Issue 1 2003Nicolas Sirvent Dermatofibrosarcoma protuberans (DP) is a rare, slow-growing, infiltrating dermal neoplasm of intermediate malignancy, made up of spindle-shaped tumor cells often positive for CD34. The preferred treatment is wide surgical excision with pathologically negative margins. At the cytogenetic level, DP cells are characterized by either supernumerary ring chromosomes, which have been shown by using fluorescence in situ hybridization techniques to be derived from chromosome 22 and to contain low-level amplified sequences from 17q22-qter and 22q10,q13.1, or t(17;22), that are most often unbalanced. Both the rings and linear der(22) contain a specific fusion of COL1A1 with PDGFB. Similar to other tumors, the COL1A1-PDGFB fusion is occasionally cryptic, associated with complex chromosomal rearrangements. Although rings have been mainly observed in adults, translocations have been reported in all pediatric cases. DP is therefore a unique example of a tumor in which (i) the same molecular event occurs either on rings or linear translocation derivatives, (ii) the chromosomal abnormalities display an age-related pattern, and (iii) the presence of the specific fusion gene is associated with the gain of chromosomal segments, probably taking advantage of gene dosage effects. In all DP cases that underwent molecular investigations, the breakpoint localization in PDGFB was found to be remarkably constant, placing exon 2 under the control of the COL1A1 promoter. In contrast, the COL1A1 breakpoint was found to be variably located within the exons of the ,-helical coding region (exons 6,49). No preferential COL1A1 breakpoint and no correlation between the breakpoint location and the age of the patient or any clinical or histological particularity have been described. The COL1A1-PDGFB fusion is detectable by multiplex RT-PCR with a combination of forward primers designed from a variety of COL1A1 exons and one reverse primer from PDGFB exon 2. Recent studies have determined the molecular identity of "classical" DP, giant cell fibroblastoma, Bednar tumor, adult superficial fibrosarcoma, and the granular cell variant of DP. In approximately 8% of DP cases, the COL1A1-PDGFB fusion is not found, suggesting that genes other than COL1A1 or PDGFB might be involved in a subset of cases. It has been proposed that PDGFB acts as a mitogen in DP cells by autocrine stimulation of the PDGF receptor. It is encouraging that inhibitory effects of the PDGF receptor tyrosine kinase antagonist imatinib mesylate have been demonstrated in vivo; such targeted therapies might be warranted in the near future for treatment of the few DP cases not manageable by surgery. © 2003 Wiley-Liss, Inc. [source] Identification of Novel Target Genes of the Bone-Specific Transcription Factor Runx2,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2004Michael Stock Abstract Fifteen putative transcriptional target genes regulated by the osteogenic transcription factor Runx2 were identified by cDNA microarray and differential hybridization techniques. Expression pattern and regulation of one gene, Pttg1ip, was analyzed in detail. Introduction: The transcription factor Runx2 is a key regulator of osteoblast development and plays a role in chondrocyte maturation. The identification of transcriptional target genes of Runx2 may yield insight into how osteoblastic differentiation is achieved on a molecular level. Materials and Methods: Using a differential hybridization technique (selective amplification through biotin and restriction-mediated enrichment [SABRE]) and cDNA microarray analysis, 15 differentially expressed genes were identified using mRNA from C3H 10T1/2 cells with constitutive and inducible overexpression of Runx2. Results and Conclusions: Among the 15 genes identified, 4 encode the extracellular matrix proteins Ecm1, Mgp, Fbn5, and Osf-2, three represent the transcription factors Esx1, Osr1, and Sox9, whereas others were Ptn, Npdc-1, Hig1, and Tem1. The gene for Pttg1ip was upregulated in Runx2-expressing cells. Pttg1ip is widely expressed during development, but at highest levels in limbs and gonads. The Pttg1ip promoter binds Runx2 in a sequence specific manner, and Runx2 is able to transactivate the Pttg1ip promoter in MC3T3-E1 cells. Therefore, Pttg1ip is likely to be a novel direct transcriptional target gene of Runx2. In conclusion, the genes identified in this study are important candidates for mediating Runx2 induced cellular differentiation. [source] Collagen Metabolism Is Markedly Altered in the Hypertrophic Cartilage of Growth Plates from Rats with Growth Impairment Secondary to Chronic Renal FailureJOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2001Jesús Álvarez Abstract Skeletal growth depends on growth plate cartilage activity, in which matrix synthesis by chondrocytes is one of the major processes contributing to the final length of a bone. On this basis, the present work was undertaken to ascertain if growth impairment secondary to chronic renal insufficiency is associated with disturbances of the extracellular matrix (ECM) of the growth plate. By combining stereological and in situ hybridization techniques, we examined the expression patterns of types II and X collagens and collagenase-3 in tibial growth plates of rats made uremic by subtotal nephrectomy (NX) in comparison with those of sham-operated rats fed ad libitum (SAL) and sham-operated rats pair-fed with NX (SPF). NX rats were severely uremic, as shown by markedly elevated serum concentrations of urea nitrogen, and growth retarded, as shown by significantly decreased longitudinal bone growth rates. NX rats showed disturbances in the normal pattern of chondrocyte differentiation and in the rates and degree of substitution of hypertrophic cartilage with bone, which resulted in accumulation of cartilage at the hypertrophic zone. These changes were associated with an overall decrease in the expression of types II and X collagens, which was especially marked in the abnormally extended zone of the hypertrophic cartilage. Unlike collagen, the expression of collagenase-3 was not disturbed severely. Electron microscopic analysis proved that changes in gene expression were coupled to alterations in the mineralization as well as in the collagen fibril architecture at the hypertrophic cartilage. Because the composition and structure of the ECM have a critical role in regulating the behavior of the growth plate chondrocytes, results obtained are consistent with the hypothesis that alteration of collagen metabolism in these cells could be a key process underlying growth retardation in uremia. [source] Regional and Developmental Expression of the Npc1 mRNA in the Mouse BrainJOURNAL OF NEUROCHEMISTRY, Issue 3 2000A. Prasad Abstract: Niemann-Pick type C (NP-C) disease is a fatal, autosomal recessive disorder of cholesterol metabolism that results in progressive central nervous system deterioration and premature death. Recently, a gene mutated in NP-C disease (NPC1) was identified in both human patients and in the npcnih mouse model. Although the function of the NPC1 gene is at present unknown, determining the pattern of its expression in the brain may facilitate identification of the mechanisms underlying the neuropathology of this disease and in identifying relevant targets for any potential therapeutic intervention. We have used in situ hybridization techniques to characterize the pattern of Npc1 mRNA expression in both the wild-type and the npcnih mutant mouse brain. In adult animals of both genotypes, the Npc1 mRNA was detected in the majority of neurons in nearly all regions, but at significantly higher levels in the cerebellum and in specific pontine nuclei. Analysis of Npc1 mRNA levels during development in the wild-type mouse indicated that this transcript was expressed in neurons as early as embryonic day 15 and that a significant region-specific pattern of expression was established by postnatal day 7. Our data suggest that whereas the NPC1 gene is widely expressed in neurons of the brain, the higher levels of expression in the cerebellum and pontine structures established by early postnatal ages may make these regions more susceptible to neuronal dysfunction in NP-C disease. [source] Polymorphic microsatellite markers for paternity assessment in southern calamari Sepioteuthis australis (Cephalopoda: Loliginidae)MOLECULAR ECOLOGY RESOURCES, Issue 4 2003L. M. Van Camp Abstract Recent decades have seen the fast growth of cephalopod fisheries but their management is compromised by the critical gaps in our knowledge of cephalopod life histories. Molecular markers are invaluable tools for studying the evolutionary significance and management implications of variation in mating systems. We have developed seven polymorphic microsatellite loci for mating system analysis in the southern calamari Sepioteuthis australis Quoy & Gaimard 1833 using magnetic enrichment and colony hybridization techniques. Observed heterozygosities range from 32% to 100% and will have sufficient power to examine the relative success of alternate mating strategies in S. australis. [source] The reaction of glial progenitor cells in remyelination following ethidium bromide-induced demyelination in the mouse spinal cordNEUROPATHOLOGY, Issue 4 2002Shigeko Fushimi The present study investigated how glial progenitor cells participated in the process of remyelination following ethidium bromide (EBr)-induced demyelination in the adult mouse spinal cord. In situ hybridization techniques for detecting mRNA for platelet-derived growth factor , receptor (PDGF,R) and proteolipid protein (PLP) were employed to identify glial progenitor cells and mature oligodendrocytes, respectively. During the demyelination stage and early stage of remyelination, large cells strongly expressing PDGF,R mRNA were observed in the border of the demyelinating lesion, and with immunohistochemistry they exhibited positive labeling of the astrocytic marker glial fibrillary acidic protein (GFAP). Other glial progenitor cells expressing PDGF,R mRNA proliferated around the lesion during the demyelination stage. During the remyelination stage, some PDGF,R mRNA-positive cells partly expressed mRNA for PLP in the periphery of the demyelinating lesion. These results suggest that PDGF,R mRNA-positive glial progenitor cells may give rise to both astrocytes and oligodendrocytes, which participate in remyelination following demyelination. [source] Craniofacial cephalometric morphology in children with CATCH 22 syndromeORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 4 2006A Heliövaara Structured abstract Authors ,, Heliövaara A, Hurmerinta K Objectives ,, To evaluate cephalometrically the craniofacial, pharyngeal and cervical morphology in children with CATCH 22, and to compare and quantify the findings with age- and sex-matched controls. Design ,, A retrospective case,control study. Setting and Sample Population , Forty-one children (20 girls) with CATCH 22 were compared with age- and sex-matched controls from lateral cephalograms taken at the mean age of 8.5 years (range 5.8,12.9). The deletion of 22q11 was verified by fluorescence in situ hybridization techniques. Thirteen of the children with CATCH 22 had palatal clefts. Outcome measure ,, Linear and angular measurements were obtained from lateral cephalograms. A Student's t -test and a paired Student's t -test were used in the statistical analysis. Standard deviation scores (SDS) were calculated to quantify the variation. Results ,, Children with CATCH 22 had obtuse cranial base angles and long anterior cranial bases. Their faces were long with increased facial convexity. The maxillae were long but both jaws were retrognathic and the lower jaws posteriorly diverged. The pharynges were wide in the nasopharyngeal area and narrow in the hypopharyngeal area. The development of the hyoid bones was delayed, and hyoid bone and atlas lengths were reduced. The morphology of the children with CATCH 22 with and without a palatal cleft was similar. Despite several statistically significant differences between the children with CATCH 22 and the controls, the SDS did not exceed ±2 for any of the measurements. Conclusion ,, Children with CATCH 22 have several minor distinctive morphological features in the craniofacial, pharyngeal, and cervical areas. [source] | |||||||||||||