The Experts below are selected from a list of 3333 Experts worldwide ranked by ideXlab platform

John M Graham - One of the best experts on this subject based on the ideXlab platform.

Tomoyuki Hori - One of the best experts on this subject based on the ideXlab platform.

  • Ultra-high-sensitivity stable-isotope probing of rRNA by high-throughput sequencing of Isopycnic Centrifugation gradients.
    Environmental microbiology reports, 2015
    Co-Authors: Tomo Aoyagi, Satoshi Hanada, Hideomi Itoh, Yuya Sato, Atsushi Ogata, Michael W. Friedrich, Yoshitomo Kikuchi, Tomoyuki Hori
    Abstract:

    Stable isotope probing (SIP) of rRNA directly identifies microorganisms assimilating an isotopically labelled substrate. High-throughput DNA sequencing is available for label screening at high resolution and high sensitivity, yet its effectiveness and validity remain to be clarified. Here, we investigated whether the detection sensitivity of rRNA-SIP could be improved by using Illumina sequencing in place of terminal restriction fragment length polymorphism (T-RFLP) analysis. A dilution series of (13) C-labelled RNA from Escherichia coli (1-0.0001%) and unlabelled RNA from Bacillus subtilis was density separated and fractionated. Illumina sequencing of Isopycnic Centrifugation gradients was able to detect (13) C-labelled RNA in the heaviest fraction with a buoyant density of 1.798 g ml(-1) even at the mixing ratio of 0.001%, whereas the detection ability of T-RFLP was not lower than 0.5%. Quantitative reverse transcription polymerase chain reaction of the density-separated RNAs showed that (13) C-labelled RNAs at mixing ratios of 0.05-0.001% had definitely accumulated in the heaviest fraction. Consequently, high-throughput sequencing provided up to 500-fold higher sensitivity for screening of (13) C-labelled RNA than T-RFLP. Ultra-high-sensitivity rRNA-SIP represents a clear advance towards a more complete understanding of microbial ecosystem function, including the ecophysiology of rare microorganisms in various natural environments.

Tomo Aoyagi - One of the best experts on this subject based on the ideXlab platform.

  • Ultra-high-sensitivity stable-isotope probing of rRNA by high-throughput sequencing of Isopycnic Centrifugation gradients.
    Environmental microbiology reports, 2015
    Co-Authors: Tomo Aoyagi, Satoshi Hanada, Hideomi Itoh, Yuya Sato, Atsushi Ogata, Michael W. Friedrich, Yoshitomo Kikuchi, Tomoyuki Hori
    Abstract:

    Stable isotope probing (SIP) of rRNA directly identifies microorganisms assimilating an isotopically labelled substrate. High-throughput DNA sequencing is available for label screening at high resolution and high sensitivity, yet its effectiveness and validity remain to be clarified. Here, we investigated whether the detection sensitivity of rRNA-SIP could be improved by using Illumina sequencing in place of terminal restriction fragment length polymorphism (T-RFLP) analysis. A dilution series of (13) C-labelled RNA from Escherichia coli (1-0.0001%) and unlabelled RNA from Bacillus subtilis was density separated and fractionated. Illumina sequencing of Isopycnic Centrifugation gradients was able to detect (13) C-labelled RNA in the heaviest fraction with a buoyant density of 1.798 g ml(-1) even at the mixing ratio of 0.001%, whereas the detection ability of T-RFLP was not lower than 0.5%. Quantitative reverse transcription polymerase chain reaction of the density-separated RNAs showed that (13) C-labelled RNAs at mixing ratios of 0.05-0.001% had definitely accumulated in the heaviest fraction. Consequently, high-throughput sequencing provided up to 500-fold higher sensitivity for screening of (13) C-labelled RNA than T-RFLP. Ultra-high-sensitivity rRNA-SIP represents a clear advance towards a more complete understanding of microbial ecosystem function, including the ecophysiology of rare microorganisms in various natural environments.

Inga I. Hitzeroth - One of the best experts on this subject based on the ideXlab platform.

  • Purification of Virus-Like Particles (VLPs) from Plants.
    Methods in molecular biology (Clifton N.J.), 2016
    Co-Authors: Albertha R. Van Zyl, Inga I. Hitzeroth
    Abstract:

    Viral coat proteins expressed in plants often form virus-like particles (VLPs) which are good vaccine candidates as they are safe and highly immunogenic and can be easily purified. The VLPs can be purified by rate-zonal density Centrifugation which is based on the size of the VLP or they can be purified by Isopycnic Centrifugation which is a fast and simple method and results in isolation of VLPs with the same density. Details on how to apply both rate-zonal and Isopycnic Centrifugation for VLP purification from plants are provided in this chapter.

Holger Jeske - One of the best experts on this subject based on the ideXlab platform.

  • Properties of African Cassava Mosaic Virus Capsid Protein Expressed in Fission Yeast
    Viruses, 2016
    Co-Authors: Katharina Hipp, Benjamin Schäfer, Gabi Kepp, Holger Jeske
    Abstract:

    The capsid proteins (CPs) of geminiviruses combine multiple functions for packaging the single-stranded viral genome, insect transmission and shuttling between the nucleus and the cytoplasm. African cassava mosaic virus (ACMV) CP was expressed in fission yeast, and purified by SDS gel electrophoresis. After tryptic digestion of this protein, mass spectrometry covered 85% of the amino acid sequence and detected three N-terminal phosphorylation sites (threonine 12, serines 25 and 62). Differential Centrifugation of cell extracts separated the CP into two fractions, the supernatant and pellet. Upon Isopycnic Centrifugation of the supernatant, most of the CP accumulated at densities typical for free proteins, whereas the CP in the pellet fraction showed a partial binding to nucleic acids. Size-exclusion chromatography of the supernatant CP indicated high order complexes. In DNA binding assays, supernatant CP accelerated the migration of ssDNA in agarose gels, which is a first hint for particle formation. Correspondingly, CP shifted ssDNA to the expected densities of virus particles upon Isopycnic Centrifugation. Nevertheless, electron microscopy did not reveal any twin particles, which are characteristic for geminiviruses.