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Hybridization Method (hybridization + method)
Kinds of Hybridization Method Selected AbstractsA Microarray Based Genomic Hybridization Method for Identification of New Genes in Plants: Case Analyses of Arabidopsis and OryzaJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 6 2007Chuanzhu Fan Abstract To systematically estimate the gene duplication events in closely related species, we have to use comparative genomic approaches, either through genomic sequence comparison or comparative genomic hybridization (CGH). Given the scarcity of complete genomic sequences of plant species, in the present study we adopted an array based CGH to investigate gene duplications in the genus Arabidopsis. Fragment genomic DNA from four species, namely Arabidopsis thaliana, A. lyrata subsp. lyrata, A. lyrata subsp. petraea, and A. halleri, was hybridized to Affymetrix (Santa Clara, CA, USA) tiling arrays that are designed from the genomic sequences of A. thaliana. Pairwise comparisons of signal intensity were made to infer the potential duplicated candidates along each phylo-genetic branch. Ninety-four potential candidates of gene duplication along the genus were identified. Among them, the majority (69 of 94) were A. thaliana lineage specific. This result indicates that the array based CGH approach may be used to identify candidates of duplication in other plant genera containing closely related species, such as Oryza, particularly for the AA genome species. We compared the degree of gene duplication through retrotransposon between O. sativa and A. thaliana and found a strikingly higher number of chimera retroposed genes in rice. The higher rate of gene duplication through retroposition and other mechanisms may indicate that the grass species is able to adapt to more diverse environments. [source] Phylogenetic analyses of ribosomal DNA-containing bacterioplankton genome fragments from a 4000 m vertical profile in the North Pacific Subtropical GyreENVIRONMENTAL MICROBIOLOGY, Issue 9 2008Vinh D. Pham Summary High-throughput identification of rRNA gene-containing clones in large insert metagenomic libraries is difficult, because of the high background of host ribosomal RNA (rRNA) and rRNA genes. To address this challenge, a membrane hybridization method was developed to identify all bacterial small subunit rRNA-containing fosmid clones of microbial community DNA from seven different depths in the North Pacific Subtropical Gyre. Out of 101,376 clones screened, 751 rDNA-containing clones were identified that grouped in ,60 different clades. Several rare sequences only remotely related to known groups were detected, including a Wolbachia -related sequence containing a putative intron or intervening sequence, as well as seven sequences from Order Myxococcales not previously detected in pelagic habitats. Stratified, depth-specific population structure was evident within both cultured and uncultured lineages. Conversely, some eurybathyal members of the genera Alcanivorax and Rhizobium shared identical small subunit ribosomal DNA sequences that were distributed from surface waters to the 4000 m depth. Comparison with similar analyses in Monterey Bay microbial communities revealed previously recognized, as well as some distinctive, depth-stratified partitioning that distinguished coastal from open ocean bacterioplankton populations. While some bias was evident in fosmid clone recovery in a few particular lineages, the overall phylogenetic group recovery and distributions were consistent with previous studies, as well as with direct shotgun sequence data from the same source DNA. [source] Molecular identification and expression study of differentially regulated genes in the Pacific oyster Crassostrea gigas in response to pesticide exposureFEBS JOURNAL, Issue 2 2005Arnaud Tanguy The effects of pesticide contamination on the metabolism of marine molluscs are poorly documented. We investigated the response of a marine bivalve, the Pacific oyster, Crassostrea gigas, using a suppression subtractive hybridization method to identify up- and down-regulated genes after a 30-day exposure period to herbicides (a cocktail of atrazine, diuron and isoproturon, and to the single herbicide glyphosate). A total of 137 unique differentially expressed gene sequences was identified, as well as their associated physiological process. The expression of 18 of these genes was analyzed by RT-PCR under laboratory experimental conditions. The metabolic functions they are associated with include xenobiotic detoxification, energy production, immune system response and transcription. This study provides a preliminary basis for studying the response of marine bivalves to long-term herbicide exposure in terms of regulated gene expression and characterizes new potential genetic markers of herbicide contamination. [source] Human leukocyte antigen DR status and clinical features in Japanese patients with type 1 autoimmune hepatitisHEPATOLOGY RESEARCH, Issue 1 2008Yasuhiro Miyake Aim:, Human leukocyte antigen (HLA) DR status affects the clinical features of autoimmune hepatitis. In Caucasians, patients with DR3 have poorer outcomes. In Japan, the relationship between HLA DR status and clinical features has yet to be fully examined. Methods:, We investigated 79 patients with type 1 autoimmune hepatitis who underwent liver biopsy and were screened for HLA DR status by the polymerase chain reaction sequence specific oligonucleotide hybridization method. Results:, Fifty-five patients had DR4 and 23 had DR2. Thirteen patients had both DR2 and DR4. None had DR3. Of patients aged <30 years, 70% did not have DR4. A tendency toward higher serum levels of immunoglobulin G was seen in patients with DR4 compared to those without, while patients with neither DR2 nor DR4 had lower serum levels of immunoglobulin G than those with only DR2 and those with only DR4. Patients with DR2 had a lower frequency of concurrentautoimmune disease. Concurrence of thyroid disease was seen only in patients with DR4. The cumulative incidental rate of the normalization of serum alanine aminotransferase levels within six months after the introduction of corticosteroid treatment was not associated with HLA DR status. Conclusion:, HLA DR status is considered to affect the clinical features of Japanese patients with type 1 autoimmune hepatitis. Japanese patients with DR2 may have different clinical features from others. In addition, diagnoses of type 1 autoimmune hepatitis should be made carefully in Japanese patients with neither DR2 nor DR4 and in those aged <30 years. [source] Wounding-mediated gene expression and accelerated viviparous reproduction of the pea aphid Acyrthosiphon pisumINSECT MOLECULAR BIOLOGY, Issue 6 2008B. Altincicek Abstract Most insects mount a potent antimicrobial defence upon contact with microbes or microbe-associated pattern molecules. Using a combined set of methods for analysis of insect innate immunity, we report here that piercing of the pea aphid Acyrthosiphon pisum with a bacteria-contaminated needle elicits lysozyme-like activity in the haemolymph but no detectable activities against live bacteria. Confirming these results, we found no homologues of known antimicrobial peptides in our cDNA library generated by using the suppression subtractive hybridization method or in over 90 000 public expressed sequence tag (EST) sequences, but lysozyme genes have recently been described in A. pisum. Interestingly, we discovered that production of viviparous offspring was significantly accelerated upon wounding. Therefore, we postulate that aphids may increase terminal reproductive investment and limit antibacterial defence in response to a threat to their survival. [source] Influence of HLA-DR2 on perforin-positive cells in pulmonary tuberculosisINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 5 2007D. N. Rajeswari Summary Perforin is one of the key effector molecules of cytotoxic T cells and natural killer cells. The influence of HLA-DRB1 alleles on peripheral blood perforin-positive CD4, CD8, CD16 and CD56 cells was studied by flow cytometry. HLA-DRB1 typing was done in normal healthy subjects (NHS: n = 156) and patients with pulmonary tuberculosis (PTB: n = 102) by polymerase chain reaction-based sequence-specific oligonucleotide hybridization method. We observed a significantly decreased percentage of total perforin-positive cells (per+) (P = 0.0004); CD8+/Per+ (P = 0.0005); CD16+/Per+ (P = 0.05) and CD56+/Per+ cells (P = 0.001) in HLA-DR2-positive PTB patients compared to non-DR2 patients. Subtyping of HLA-DR2-positive subjects at the allelic level revealed that the percentage of CD8+/Per+ cells did not differ among DRB1*1501 and DRB1*1502 patients while a trend towards a decreased percentage of CD16+/Per+ and CD56+/Per+ cells was noticed in DRB1*1501 patients compared to DRB1*1502 patients. The present study suggests that HLA-DR2 may be associated with down-regulation of perforin-positive cytotoxic lymphocytes and natural killer cells in pulmonary tuberculosis. [source] Noxp20 and Noxp70, two new markers of early neuronal differentiation, detected in teratocarcinoma-derived neuroectodermic precursor cellsJOURNAL OF NEUROCHEMISTRY, Issue 2 2006M. Boucquey Abstract The murine 1C11 cell line, derived from F9 pluripotent teratocarcinoma cells, exhibits features of a bipotential neuronal precursor as it converts into serotonergic or catecholaminergic neurons under appropriate induction. In order to point out molecular markers expressed in this early neuroectodermic commitment, we used a cDNA subtractive hybridization method. The 105 different isolated cDNAs represented 75 known genes, expressed sequence tags (EST) or genomic fragments. A majority of known proteins encoded by these sequences are involved in cellular mobility or migration. We characterized two sequences showing identities with ESTs and we called them Noxp20 and Noxp70. The Noxp20 transcript encodes a putative protein with a predicted caspase recruitment domain and the Noxp70 transcript encodes a putative protein displaying a Zn-finger domain. Consistent with their roles in neuronal cell development, in situ hybridization showed that Noxp20 and Noxp70 are over-expressed in brain. At embryonic days 12 and 15, Noxp20 is strongly expressed in the ventricular and intermediate zones of the brain and of the spinal cord. At embryonic day 15, Noxp70 was found to be strongly expressed in the ventricular zone around the telencephalic ventricle, and to a lower extent in the thalamus and hypothalamus. At post-natal day 10, Noxp20 mRNA was detected in the dentate gyrus, the hippocampus, the cerebellum and the olfactory bulb. [source] Bacterial Culture and DNA Checkerboard for the Detection of Internal Contamination in Dental ImplantsJOURNAL OF PROSTHODONTICS, Issue 5 2009Rodrigo Edson Santos Barbosa DDS Abstract Purpose: The aim of this in vitro study was to evaluate the bacterial leakage along the implant,abutment interface by the conventional bacterial culture and DNA Checkerboard hybridization method. Materials and Methods: Twenty Branemark-compatible implants with a 3.75-mm diameter and external hexagonal platform were randomly placed in two groups of ten implant,abutment assemblies each. One group was used to analyze bacterial counts by DNA Checkerboard hybridization and the other by a conventional bacterial culture. Suspensions of Fusobacterium nucleatum (3 ,l) were injected into the grooved internal cylinders of each implant assembly, and the abutment was connected by a 32 Ncm torque. The combined implant,abutments were individually placed in tubes containing the CaSaB culture medium and incubated in a bacteriological constant temperature oven for 14 days. The samples were observed daily as to the presence of turbidity, and after the designated time the microorganisms were collected from the implant interiors and analyzed by the two methods. Results: After 14 days, six implant,abutment assemblies showed turbidity. Both methods indicated reduced microorganism counts in samples from the interior of the implant,abutment assemblies after incubation in the culture medium; however, the number of counts of F. nucleatum was higher by the DNA Checkerboard method when compared to the group analyzed by conventional bacterial cultures (p < 0.05). Conclusion: The DNA Checkerboard method was shown to be more sensitive than conventional cultures in the detection of microorganisms. [source] A new checkerboard panel for testing bacterial markers in periodontal diseaseMOLECULAR ORAL MICROBIOLOGY, Issue 1 2006G. Dahlén Background/aims:, Various microbiological methods have been used for testing bacterial markers for periodontitis and periodontal disease progression. Most studies have used only a limited number of well recognized bacterial species. The purpose of the present study was to evaluate the association of 13 more recently identified bacterial species in a new panel in comparison with 12 previously more recognized periodontotopathogens (,old panel') using the ,checkerboard' DNA,DNA hybridization method. Methods:, Fifty individuals were chosen who showed at least one site with a probing pocket depth of 6 mm or more (disease) and bleeding on probing and at least one site with a probing pocket depth of 3 mm and without bleeding on probing (health). One diseased and one healthy site on each individual were sampled with the paperpoint technique and the samples were processed in the checkerboard technique against deoxigenin-labeled whole genomic probes to 25 subgingival species representing 12 well recognized and 13 newly identified periodontitis associated species. Results:, Twenty-four (out of 25) species were detected more frequently in the subgingival plaque of diseased than healthy sites both at score 1 (> 104) and score 3 (> 105). A significant difference at the higher score (score 3) was noticed for all species of the old panel except for three (Streptococcus intermedius, Selenomonas noxia, and Eikenella corrodens). Of the species in the new panel only Prevotella tannerae, Filifactor alocis, and Porphyromonas endodontalis showed a statistical significant difference between diseased and healthy sites. Conclusion:, It was concluded that P. tannerae, F. alocis, and P. endodontalis should be added to the 12 species used for routine diagnostics of periodontitis-associated bacterial flora. [source] Chromogenic in situ hybridization analysis of HER-2/neu status in breast carcinoma: Application in screening of patients for trastuzumab (Herceptin®) therapyPATHOLOGY INTERNATIONAL, Issue 8 2001Hiroyuki Kumamoto Evaluation of HER-2/neu status is important in the management of patients with breast carcinoma, especially in determining the possible application of trastuzumab, a humanized anti-HER-2/neu monoclonal antibody. Chromogenic in situ hybridization (CISH) detection of the HER-2/neu oncogene is a newly developed in situ hybridization method that utilizes a robust and unique-sequence DNA probe labeled with digoxygenin, and sequential incubations with antidigoxygenin fluorescein, antifluorescein peroxidase, and diaminobenzidine. In this study, we examined 20 archival specimens of human breast carcinoma using CISH, and we correlated findings with immunohistochemical findings for HER-2/neu. HER-2/neu immunohistochemistry was carried out with HercepTestTM, a standardized immunohistochemical examination system for HER-2/neu overexpression in surgical pathology specimens. CISH analysis could be done in 18 out of 20 cases examined. Gene copy signals for HER-2/neu were recognized as intranuclear brown dots in both neoplastic and non-neoplastic cells. Seven carcinomas showed an increased number or size of signals and were interpreted as being positive for HER-2/neu amplification. Eight cases were positive with the HercepTestTM. Seven out of eight carcinoma cases found to overexpress immunoreactive HER-2/neu also demonstrated HER-2/neu gene amplification following CISH analysis. There was a significant correlation between immunohistochemical and CISH analyses (P< 0.001). We found that CISH was a specific, sensitive and easily applicable method for the detection of HER-2/neu gene amplification, which may be used together with immunohistochemical examination for the evaluation of patients with breast carcinoma. [source] Gene Expression Analysis in Cucumber Leaves Primed by Root Colonization with Pseudomonas chlororaphis O6 upon Challenge-Inoculation with Corynespora cassiicolaPLANT BIOLOGY, Issue 2 2004M. S. Kim Abstract: Root colonization by Pseudomonas chlororaphis O6, a non-pathogenic rhizobacterium, induced systemic resistance in cucumber against target leaf spot caused by Corynespora cassiicola. A cDNA library was constructed using mRNA extracted from cucumber leaves 12 h after inoculation with C. cassiicola, using plants colonized by O6. To identify genes involved in O6-mediated induced systemic resistance (ISR), we employed a subtractive hybridization method using mRNAs extracted from pathogen-challenged cucumber leaves of plants lacking colonization. Differential screening of the cDNA library led to the isolation of six distinct genes encoding a GTP binding protein, a 60S ribosomal protein, a hypersensitive-induced reaction protein, a ubiquitin extension protein, a pyridine nucleotide-disulfide oxidoreductase, and a signal recognition particle receptor. Expression of these genes was not induced by O6 colonization alone. Rather, transcript accumulation of these genes increased significantly faster and stronger in the O6 colonized than in non-colonized plants after challenge infection. Therefore, O6-mediated ISR may be associated with an enhanced capacity for the rapid and effective activation of cellular defence responses after challenge inoculation. [source] Clinical and microbiological analysis of subjects treated with Brånemark or AstraTech implants: a 7-year follow-up studyCLINICAL ORAL IMPLANTS RESEARCH, Issue 4 2008S. Renvert Abstract Aims: To assess the impact of different implant systems on the clinical conditions and the microbiota at implants, and whether the presence of bacteria at tooth sites was predictive of the presence at implant sites. Materials and methods: Subjects with either AstraTech or Brånemark in function for 7 years were enrolled. Sub-gingival bacterial samples at tooth and implant sites were collected with sterile endodontic paper points, and analyzed by the checkerboard DNA,DNA hybridization method (40 species). Results: Fifty-four subjects, 27 supplied with AstraTech (n=132 implants) and 27 with Brånemark (n=102) implants, were studied. Test tooth sites had significantly less evidence of bleeding on probing (P<0.001) and presence of plaque (P<0.001) than implant test sites. Implant sites presented with deeper probing pocket depth than tooth sites (mean difference: 1.1 mm, standard error of differences: 0.08, 95% confidence intervals (CI): 0.9,1.3, P<0.001). Tannerella forsythia (P<0.05), Capnocytophaga sputigena (P<0.05), Actinomyces israelii (P<0.05) and Lactobacillus acidophilus (P<0.05) were found at higher levels at tooth surfaces. No differences in bacterial load for any species were found between the two implant systems. The odds of being present/absent at tooth and implants sites were only significant for Staphylococcus aureus [odds ratio (OR): 5.2 : 1, 95% CI: 1.4,18.9, P<0.01]. Conclusions: After 7 years in function, implants presented with deeper probing depths than teeth. S. aureus was commonly present at both teeth and implants sites. S. aureus at tooth sites was predictive of also being present at implant sites. [source] Comparison of bacterial plaque samples from titanium implant and tooth surfaces by different methodsCLINICAL ORAL IMPLANTS RESEARCH, Issue 1 2006Jeanne Gerber Abstract: Studies have shown similarities in the microflora between titanium implants or tooth sites when samples are taken by gingival crevicular fluid (GCF) sampling methods. The purpose of the present study was to study the microflora from curette and GCF samples using the checkerboard DNA,DNA hybridization method to assess the microflora of patients who had at least one oral osseo-integrated implant and who were otherwise dentate. Plaque samples were taken from tooth/implant surfaces and from sulcular gingival surfaces with curettes, and from gingival fluid using filter papers. A total of 28 subjects (11 females) were enrolled in the study. The mean age of the subjects was 64.1 years (SD±4.7). On average, the implants studied had been in function for 3.7 years (SD±2.9). The proportion of Streptococcus oralis (P<0.02) and Fusobacterium periodonticum (P<0.02) was significantly higher at tooth sites (curette samples). The GCF samples yielded higher proportions for 28/40 species studies (P -values varying between 0.05 and 0.001). The proportions of Tannerella forsythia (T. forsythensis), and Treponema denticola were both higher in GCF samples (P<0.02 and P<0.05, respectively) than in curette samples (implant sites). The microbial composition in gingival fluid from samples taken at implant sites differed partly from that of curette samples taken from implant surfaces or from sulcular soft tissues, providing higher counts for most bacteria studied at implant surfaces, but with the exception of Porphyromonas gingivalis. A combination of GCF and curette sampling methods might be the most representative sample method. [source] | |||||||||||||