The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

IL9 maps to mouse chromosome 13 and human chromosome 5

Mouse and human cDNA clones encoding the T-cell and mast cell growth factor P40, now designated IL-9, were used to identify DNA restriction fragment length polymorphisms (RFLPs) in sets of somatic cell hybrids and between inbred strains of mice and interspecific backcross progeny. Segregation of mouse and human chromosomes among somatic cell hybrids indicated a location on mouse chromosome 13 and human chromosome 5. RFLPs were identified among inbred strains of mice. Analysis of chromosome 13 alleles for Tcrg, Dhfr, and Il-9 in an interspecific cross between Mus musculus and NFS/N or C58/J mice indicates that IL-9 is distal to Tcrg and Proximal to Dhfr.     

Inferring landscape resistance to gene flow when genetic drift is spatially heterogeneous

In connectivity models, land cover types are assigned cost values characterizing their resistance to species movements. Landscape genetic methods infer these values from the relationship between genetic differentiation and cost distances. The spatial heterogeneity of population sizes, and consequently genetic drift, is rarely included in this inference although it influences genetic differentiation. Similarly, migration rates and population spatial distributions potentially influence this inference. Here, we assessed the reliability of cost value inference under several migration rates, population spatial patterns and degrees of population size heterogeneity. Additionally, we assessed whether considering intra-population variables, here using gravity models, improved the inference when drift is spatially heterogeneous. We simulated several gene flow intensities between populations with varying local sizes and spatial distributions. We then fit gravity models of genetic distances as a function of (i) the ‘true’ cost distances driving simulations or alternative cost distances, and (ii) intra-population variables (population sizes, patch areas). We determined the conditions making the identification of the ‘true’ costs possible and assessed the contribution of intra-population variables to this objective. Overall, the inference ranked cost scenarios reliably in terms of similarity with the ‘true’ scenario (cost distance Mantel correlations), but this ‘true’ scenario rarely provided the best model goodness of fit. Ranking inaccuracies and failures to identify the ‘true’ scenario were more pronounced when migration was very restricted (<4 dispersal events/generation), population sizes were most heterogeneous and some populations were spatially aggregated. In these situations, considering intra-population variables helps identify cost scenarios reliably, thereby improving cost value inference from genetic data.     

Response of citrus trees to modified radiation regime in semi-arid conditions

Citrus trees are characterized by a large canopy and low hydraulicconductivity. In Israel's semi-arid summer climate this couldcause transpiration to exceed water uptake and cause temporaryexcessive water deficits. It was hypothesized that reductionof radiative load would reduce transpiration and thus reducedeficits. Net radiation of lemon trees in the hottest season was reducedby shading hedgerows with reflective nets for approximatelyone month in both 1994 and 1995. Stem sap flow and climate variableswere measured continuously. Daily courses of leaf conductanceand leaf water potentials were measured on selected days. Midday net radiation below the dense and sparse shade net treatmentswas 47% and 73% of that above the control trees. Midday ‘sunlit’leaf temperatures below the nets were reduced by 2.7 and 1.6C,respectively. The reduction in net radiation caused large changes in leafconductance. Average midday sunlit leaf conductance measuredin 1995 under the dense and sparse treatments and control were4.1, 2.9 and 1.8mm s^–1, respectively (significantly differentat P <0.01). Similar differences in sunlit leaf conductancewere found in 1994. Shade leaf conductance was not affectedby the treatments. Daily total and midday sap flow under the dense net were reducedby 6–7% and 10–11%, respectively. Sap flow underthe sparse net did not change significantly in 1994, but in1995 daily and midday sap flows were reduced by 6% and 7%, respectively.Midday leaf water potentials increased by 0.2 and 0.1 MPa underdense shade in 1994 and 1995, respectively. Under sparse shademidday leaf water potentials increased by 0.1 MPa in 1994, butdid not change significantly in 1995. A modified Penman-Monteith model evaluated transpiration ifleaf conductance were constant in the different radiation environments.At leaf conductance levels found in the unshaded trees, denseshade was estimated to cause a 25% reduction in transpiration,while leaf conductance values found in trees under the denseshade would lead to an increase in transpiration of more than35% in unshaded trees. The ability of the tree to maintain almost constant transpirationin different radiation environments and thus avoid water deficitby adjusting the conductance of sunlit leaves is discussed interms of environmental influences and significance to the plant'swater balance. Key words: Tree transpiration, stomatal closure, climate modification, citrus     

Gain of function of mutant p53 by coaggregation with multiple tumor suppressors

Many p53 missense mutations possess dominant-negative activity and oncogenic gain of function. We report that for structurally destabilized p53 mutants, these effects result from mutant-induced coaggregation of wild-type p53 and its paralogs p63 and p73, thereby also inducing a heat-shock response. Aggregation of mutant p53 resulted from self-assembly of a conserved aggregation-nucleating sequence within the hydrophobic core of the DNA-binding domain, which becomes exposed after mutation. Suppressing the aggregation propensity of this sequence by mutagenesis abrogated gain of function and restored activity of wild-type p53 and its paralogs. In the p53 germline mutation database, tumors carrying aggregation-prone p53 mutations have a significantly lower frequency of wild-type allele loss as compared to tumors harboring nonaggregating mutations, suggesting a difference in clonal selection of aggregating mutants. Overall, our study reveals a novel disease mechanism for mutant p53 gain of function and suggests that, at least in some respects, cancer could be considered an aggregation-associated disease.     

PIAS1-mediated sumoylation of focal adhesion kinase activates its autophosphorylation

Focal adhesion kinase (FAK) is a protein tyrosine kinase enriched in focal adhesions, which plays a critical role in integrin-dependent cell motility and survival. The crucial step in its activation is autophosphorylation on Tyr-397, which promotes the recruitment of several enzymes including Src family kinases and the activation of multiple signaling pathways. We found in a yeast two-hybrid screen that the N-terminal domain of FAK interacted with protein inhibitor of activated STAT1 (PIAS1). This interaction was confirmed and shown to be direct using in vitro assays. PIAS1 was co-immunoprecipitated with FAK from transfected cells and brain extracts. PIAS1 has recently been recognized as a small ubiquitin-like modifier (SUMO) ligase. In the presence of PIAS1 and SUMO-1, FAK was sumoylated in intact cells, whereas PYK2, a closely related enzyme, was not. Sumoylation occurred on Lys-152, a residue conserved in FAK during evolution. Sumoylated FAK, like PIAS1, was recovered predominantly from the nuclear fraction. Sumoylation did not require the catalytic activity or autophosphorylation of FAK. In contrast, sumoylation increased dramatically the ability of FAK to autophosphorylate in intact cells and in immune precipitate kinase assays. Endogenous FAK was sumoylated in the presence of PIAS1 and SUMO-1 independently of cell adhesion, and autophosphorylation of sumoylated FAK was persistently increased in suspended cells. These observations show that sumoylation controls the activity of a protein kinase and suggest that FAK may play a novel role in signaling between the plasma membrane and the nucleus.     

CRISPR/Cas9-mediated efficient targeted mutagenesis has the potential to accelerate the domestication of <Emphasis Type="Italic">Coffea canephora</Emphasis>

Genome editing, which is an unprecedented technological breakthrough, has provided a valuable means of creating targeted mutations in plant genomes. In this study, we developed a genomic web tool to identify all gRNA target sequences in the coffee genome, along with potential off-targets. In all, 8,145,748 CRISPR guides were identified in the draft genome of Coffea canephora corresponding to 5,338,568 different sequences and, of these, 4,655,458 were single, and 514,591 were covering exons. The proof of concept was established by targeting the phytoene desaturase gene (CcPDS) using the Agrobacterium tumefaciens transformation technique and somatic embryogenesis as the plant regeneration method. An analysis of the RNA-guided genome-editing events showed that 22.8% of the regenerated plants were heterozygous mutants and 7.6% were homozygous mutants. Mutation efficiency at the target site was estimated to be 30.4%. We demonstrated that genome editing by the CRISPR/Cas9 method is an efficient and reliable way of knocking out genes of agronomic interest in the coffee tree, opening up the way for coffee molecular breeding. Our results also showed that the use of somatic embryogenesis, as the method for regenerating genome-edited plants, could restrict the choice of targeted genes to those that are not essential to the embryo development and germination steps.