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The phylogenetic diversity of dissimilatory sulfite reductase (DSR, EC 1.8.99.3) -subunit genes from
a deep-sea cold seep was analyzed. Bulk genomic DNA was extracted from the cold seep sediment and used for
amplification by polymerase chain reaction (PCR) of DSR -subunit gene. Two sizes of PCR products, 1.4 kb
(expected) and 1.3 kb (unexpected), were amplified. Sixteen clones of the 1.4-kb amplicons and 16 clones of
1.3-kb amplicons, a total of 32 clones, were obtained and grouped into operational DSR units (ODUs) based
on restriction fragment length polymorphism (RFLP) by digestion with HaeIII and MboI. A total of 14 ODUs,
i.e., 5 ODUs from 1.4-kb amplicon clones and 9 ODUs from 1.3-kb amplicon clones, were recovered. About
400 bp of the 5 ends of all the clones was sequenced and validated the RFLP-based ODU grouping. All the
5-end 400-bp sequences of ODUs, even from the 1.3-kb amplicons, showed the characteristic DSR amino acid
sequence motifs. The ODUs from 1.4-kb amplicons were closely related to the -Proteobacterial lineage with
the DSR genes from -Proteobacterial epibionts of the hot vent worm Alvinella pompejana. The ODUs from
1.3-kb amplicons were mostly related to the unknown but possibly archaeal lineage. The diversity of the DSR
genes may indicate the diversity of sulfate reducers in the seep sediment as well as the complexity of electron
donors including methane. 相似文献
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DNA was extracted from water and sediment samples taken from soda lakes of the Kenyan-Tanzanian Rift Valley. DNA was also extracted from microbial enrichment cultures of sediment samples. 16S rRNA genes were amplified by the polymerase chain reaction and microbial diversity was studied using denaturing gradient gel electrophoresis (DGGE) of 16S rDNA amplicons. Cloning and sequencing of single DGGE bands showed that they usually contained mixed amplicons. Several of the amplicon sequences had high identities, up to 99%, with 16S rRNA genes of organisms previously isolated from soda lakes, while others were only distantly related, with identities as low as 82%. Phylogenetic analysis of the sequenced amplicons indicated that sequences were related to the haloarchaeal, Bacillus/Clostridium, Rhodobacterium/Thioalcalovibrio/ Methylobacter, and Cytophaga/Flavobacterium/Bacteroides (CFB) groups and the enterobacteria/Aeromonas/Vibrio part of the 3 subdivision of the Proteobacteria.Communicated by K. Horikoshi 相似文献
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BACKGROUND:
Cornelia de Lange syndrome (CdLS) is a multisystem developmental disorder in children. The disorder is caused mainly due to mutations in Nipped-B-like protein. The molecular data for CdLS is available from developed countries, but not available in developing countries like India. In the present study, the hotspot region of NIPBL gene was screened by Polymerase Chain Reaction which includes exon 2, 22, 42, and a biggest exon 10, in six CdLS patients and ten controls.MATERIALS AND METHODS:
The method adopted in present study was amplification of the target exon by using polymerase chain reaction, qualitative confirmation of amplicons by Agarose Gel Electrophoresis and use of amplicons for Conformation Sensitive Gel Electrophoresis to find heteroduplex formation followed by sequencing.RESULTS:
We report two polymorphisms in the studied region of gene NIPBL. The polymorphisms are in the region of intron 1 and in exon 10. The polymorphism C/A is present in intron 1 region and polymorphism T/G in exon 10.CONCLUSION:
The intronic region polymorphism may have a role in intron splicing whereas the polymorphism in exon 10 results in amino acid change (Val to Gly). These polymorphisms are disease associated as these are found in CdLS patients only and not in controls. 相似文献4.
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K. Ostrowski J. V. Watson P. J. Barnard E. A. Barnard K. Thomas L. Freedman B. de Stavola 《Histochemistry and cell biology》1986,85(5):423-429
Summary A common approach to the study of cell substrate interactions is the measurement of the attachment of cells to different substrates or to cultured cell layers. The evaluation of attachment is made either by scintillation counting of previously labelled adhering cells, or by light microscopy using the criterion of cell shape, sometimes refined by automatic image analysis. These methods have many drawbacks. This paper suggests the use of fluorescence-activated flow cytophotometry, (FC) which yields direct counts of the non-adhering cells. These free cells are removed after completion of the adhesion experiment from the microtitre plate wells. An internal standard, in the form of fluorescent polystyrene beads is added, allowing evaluation of the percentage of cells adhering to the well walls. Flow cytophotometry then produces data based on the analysis of large populations of cells. Unequivocal discrimination is obtained between the counted cells and counted fluorescent beads eliminating counting errors. The results can be processed on line by computer.A suspension of mouse splenocytes was used for the evaluation of the overall error of the method arising from maccuracies in pipetting, interference of glutaraldehyde with ethidium bromide (EB) staining and instrumental error. Each adhesion experiment was terminated by staining and post-fixation and it was established that this introduces no change in cell counting, in comparison with the original unfixed cells. Prefixation, however, quenches the EB staining and would interfere with the counting procedure. The overall standard error of the technique was found to be 5%–10%.A comparison was made of this technique with conventional cell counting by light microscopy, in a study of mouse splenocytes and of myoblasts isolated from 12 day old chicken embryos, Suspensions of these cells and of myoblasts were applied to microtitre plate wells precoated with fibronectin or Concanavalin A (Con A). The specific phase of cell contact was blocked by preincubation of the cells in media containing Con A or -methyl-d-mannoside, and the kinetics of cell attachment to Con A coated surfaces were compared. Claims in the literature for a difference between the fibronectin-type and lectin-type curves were not supported by our results. It was found that the curves for the attachment of both cell types to fibronectin or Con A coated surfaces are similar in shape.The results obtained by the FC technique were evaluated statistically and compared with cell counts obtained by microscopy. The definition of adhering cells is different in flow-cytophotometry (i.e. cells which cannot be removed from wells) from that used in microscopic counting (i.e. cells which have lost their round shape). Nevertheless, the results are found to be parallel which allows us to suggest the use of flowcytophotometry as a routine technique for evaluation of cell adhesion. 相似文献
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Comparison of different approaches for the incorporation of non-radioactive labels into polymerase chain reaction products 总被引:1,自引:0,他引:1
Different methods for labelling polymerase chain reaction (PCR) products with non-radioactive labels for their detection by hybridization with immobilized DNA probes were compared. The use of digoxigenin (DIG) as a label provided greater sensitivity than biotin in a PCR system targeting the invA gene from Salmonella typhimurium. Incorporation of digoxigenin into amplicons in the form of 5-DIG-labelled oligonucleotide primers resulted in better assay signals and was more economical than DIG-labelled dUTP. 相似文献
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S. Lebonvallet T. Mennesson N. Bonnet S. Girod C. Plotkowski J. Hinnrasky E. Puchelle 《Histochemistry and cell biology》1991,96(3):245-250
Summary The quantitation of electron dense labelling is very tedious when it is done by hand. Accordingly we developed software allowing, at electron microscopic level, a semi-automatic counting of dense markers in biological specimens. It includes the digitization of images and extraction of dense particles from the grey level of the background. The definition of the areas of interest was carried out by the observer but all quantitative calculations were done automatically. This method was applied to different biological materials (phospholipid and lysozyme labelling in secretory granules of human submucosal bronchial gland cells). The results obtained by this semi-automatic procedure were in good agreement with those obtained by manual counting of colloïdal gold labelling (r=0.97). 相似文献
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Vassiliki Kotoula Aggeliki Lyberopoulou Kyriaki Papadopoulou Elpida Charalambous Zoi Alexopoulou Chryssa Gakou Sotiris Lakis Eleftheria Tsolaki Konstantinos Lilakos George Fountzilas 《PloS one》2015,10(6)