Reliability of gene expression ratios for cDNA microarrays in multiconditional experiments with a reference design

In a typical gene expression profiling experiment with multiple conditions, a common reference sample is used for co-hybridization with the samples to yield expression ratios. Differential expression for any other sample pair can then be calculated by assembling the ratios from their hybridizations with the reference. In this study we test the validity of this approach. Differential expression of a sample pair (i, j) was obtained in two ways: directly, by hybridizations of sample i versus j, and indirectly, by multiplying the expression ratios for hybridizations of sample i versus pool and pool versus sample j. We performed gene expression profiling using amphibian embryos (Xenopus laevis). Every sample combination of four different stages and a pool was profiled. Direct and indirect values were compared and used as the quality criterion for the data. Based on this criterion, 82% of all ratios were found to be sufficiently accurate. To increase the reliability of the signals, several widely used filtering techniques were tested. Filtering by differences of repeated hybridizations was found to be the optimal filter. Finally, we compared microarray-based gene expression profiles with the corresponding expression patterns obtained by whole-mount in situ hybridizations, resulting in a 90% correspondence.     

Analysis of fast dynamic processes in living cells: high-resolution and high-speed dual-color imaging combined with automated image analysis

The generation of spectral mutants of the green fluorescent protein (GFP) set the stage for multiple-color imaging in living cells. However, the use of this technique has been limited by a spectral overlap of the available GFP mutants and/or by insufficient resolution in both time and space. Using a new setup for dual-color imaging, we demonstrate here the visualization of small, fast moving vesicular structures with a high time resolution. Two GFP-fusion proteins were generated: human chromogranin B, a secretory granule matrix protein, and phogrin, a secretory granule membrane protein. They were tagged with enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), respectively. Both fusion proteins were cotransfected in Vero cells, a cell line from green monkey kidney. EYFP and ECFP were excited sequentially at high time rates using a monochromator. Charged coupled device (CCD)-based image acquisition resulted in 5-8 dual-color images per second, with a resolution sufficient to detect transport vesicles in mammalian cells. Under these conditions, a fully automated time-resolved analysis of the movement of color-coded objects was achieved. The development of specialized software permitted the analysis of the extent of colocalization between the two differentially labeled sets of cellular structures over time. This technical advance will provide an important tool to study the dynamic interactions of subcellular structures in living cells.     

Dynamics of HIV-1 Assembly and Release

Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1–2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of ∼5×10⁻³ s⁻¹, corresponding to 8–9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at ∼1,500±700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics.     

Genome sequencing of pediatric medulloblastoma links catastrophic DNA rearrangements with TP53 mutations

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Highlights? Complex chromosomal alterations (chromothripsis) observed in medulloblastomas ? Cancers with such alterations harbor TP53 mutations ? Context-specific link between the status of p53 and likelihood of chromothripsis ? p53 status and chromothripsis also correlate with aggressive acute myeloid leukemia