170 research outputs found

    Relative importance of βcyto- and γcyto-actin in primary mouse embryonic fibroblasts

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    The highly homologous β (βcyto) and γ (γcyto) cytoplasmic actins are hypothesized to carry out both redundant and unique essential functions, but studies using targeted gene knockout and siRNA-mediated transcript knockdown to examine βcyto- and γcyto-isoform--specific functions in various cell types have yielded conflicting data. Here we quantitatively characterized actin transcript and protein levels, as well as cellular phenotypes, in both gene- and transcript-targeted primary mouse embryonic fibroblasts. We found that the smooth muscle αsm-actin isoform was the dominantly expressed actin isoform in WT primary fibroblasts and was also the most dramatically up-regulated in primary βcyto- or β/γcyto-actin double-knockout fibroblasts. Gene targeting of βcyto-actin, but not γcyto-actin, led to greatly decreased cell proliferation, decreased levels of cellular ATP, and increased serum response factor signaling in primary fibroblasts, whereas immortalization induced by SV40 large T antigen supported fibroblast proliferation in the absence of βcyto-actin. Consistent with in vivo gene-targeting studies in mice, both gene- and transcript-targeting approaches demonstrate that the loss of βcyto-actin protein is more disruptive to primary fibroblast function than is the loss of γcyto-actin

    Essential nucleotide- and protein-dependent functions of Actb/β-actin

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    The highly similar cytoplasmic β- and γ-actins differ by only four functionally similar amino acids, yet previous in vitro and in vivo data suggest that they support unique functions due to striking phenotypic differences between Actb and Actg1 null mouse and cell models. To determine whether the four amino acid variances were responsible for the functional differences between cytoplasmic actins, we gene edited the endogenous mouse Actb locus to translate γ-actin protein. The resulting mice and primary embryonic fibroblasts completely lacked β-actin protein, but were viable and did not present with the most overt and severe cell and organismal phenotypes observed with gene knockout. Nonetheless, the edited mice exhibited progressive high-frequency hearing loss and degeneration of actin-based stereocilia as previously reported for hair cell-specific Actb knockout mice. Thus, β-actin protein is not required for general cellular functions, but is necessary to maintain auditory stereocilia

    Potentiation of curing by a broad-host-range self-transmissible vector for displacing resistance plasmids to tackle AMR

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    Plasmids are potent vehicles for spread of antibiotic resistance genes in bacterial populations and often persist in the absence of selection due to efficient maintenance mechanisms. We previously constructed non-conjugative high copy number plasmid vectors that efficiently displace stable plasmids from enteric bacteria in a laboratory context by blocking their replication and neutralising their addiction systems. Here we assess a low copy number broad-host-range self-transmissible IncP-1 plasmid as a vector for such curing cassettes to displace IncF and IncK plasmids. The wild type plasmid carrying the curing cassette displaces target plasmids poorly but derivatives with deletions near the IncP-1 replication origin that elevate copy number about two-fold are efficient. Verification of this in mini IncP-1 plasmids showed that elevated copy number was not sufficient and that the parB gene, korB, that is central to its partitioning and gene control system, also needs to be included. The resulting vector can displace target plasmids from a laboratory population without selection and demonstrated activity in a mouse model although spread is less efficient and requires additional selection pressure

    ACCESS-OM2 v1.0: a global ocean-sea ice model at three resolutions

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    We introduce ACCESS-OM2, a new version of the ocean–sea ice model of the Australian Community Climate and Earth System Simulator. ACCESS-OM2 is driven by a prescribed atmosphere (JRA55-do) but has been designed to form the ocean–sea ice component of the fully coupled (atmosphere–land–ocean–sea ice) ACCESS-CM2 model. Importantly, the model is available at three different horizontal resolutions: a coarse resolution (nominally 1∘ horizontal grid spacing), an eddy-permitting resolution (nominally 0.25∘), and an eddy-rich resolution (0.1∘ with 75 vertical levels); the eddy-rich model is designed to be incorporated into the Bluelink operational ocean prediction and reanalysis system. The different resolutions have been developed simultaneously, both to allow for testing at lower resolutions and to permit comparison across resolutions. In this paper, the model is introduced and the individual components are documented. The model performance is evaluated across the three different resolutions, highlighting the relative advantages and disadvantages of running ocean–sea ice models at higher resolution. We find that higher resolution is an advantage in resolving flow through small straits, the structure of western boundary currents, and the abyssal overturning cell but that there is scope for improvements in sub-grid-scale parameterizations at the highest resolution
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