p53 and Translation Attenuation Regulate Distinct Cell Cycle Checkpoints during Endoplasmic Reticulum (ER) Stress

Cell cycle checkpoints ensure that proliferation occurs only under permissive conditions, but their role in linking nutrient availability to cell division is incompletely understood. Protein folding within the endoplasmic reticulum (ER) is exquisitely sensitive to energy supply and amino acid sources because deficiencies impair luminal protein folding and consequently trigger ER stress signaling. Following ER stress, many cell types arrest within the G₁ phase, although recent studies have identified a novel ER stress G₂ checkpoint. Here, we report that ER stress affects cell cycle progression via two classes of signal: an early inhibition of protein synthesis leading to G₂ delay involving CHK1 and a later induction of G₁ arrest associated both with the induction of p53 target genes and loss of cyclin D1. We show that substitution of p53/47 for p53 impairs the ER stress G₁ checkpoint, attenuates the recovery of protein translation, and impairs induction of NOXA, a mediator of cell death. We propose that cell cycle regulation in response to ER stress comprises redundant pathways invoked sequentially first to impair G₂ progression prior to ultimate G₁ arrest.     

Alterations in striatal synaptic transmission are consistent across genetic mouse models of Huntington's disease

Since the identification of the gene responsible for HD (Huntington''s disease), many genetic mouse models have been generated. Each employs a unique approach for delivery of the mutated gene and has a different CAG repeat length and background strain. The resultant diversity in the genetic context and phenotypes of these models has led to extensive debate regarding the relevance of each model to the human disorder. Here, we compare and contrast the striatal synaptic phenotypes of two models of HD, namely the YAC128 mouse, which carries the full-length huntingtin gene on a yeast artificial chromosome, and the CAG140 KI (knock-in) mouse, which carries a human/mouse chimaeric gene that is expressed in the context of the mouse genome, with our previously published data obtained from the R6/2 mouse, which is transgenic for exon 1 mutant huntingtin. We show that striatal MSNs (medium-sized spiny neurons) in YAC128 and CAG140 KI mice have similar electrophysiological phenotypes to that of the R6/2 mouse. These include a progressive increase in membrane input resistance, a reduction in membrane capacitance, a lower frequency of spontaneous excitatory postsynaptic currents and a greater frequency of spontaneous inhibitory postsynaptic currents in a subpopulation of striatal neurons. Thus, despite differences in the context of the inserted gene between these three models of HD, the primary electrophysiological changes observed in striatal MSNs are consistent. The outcomes suggest that the changes are due to the expression of mutant huntingtin and such alterations can be extended to the human condition.     

Novel gene and gene model detection using a whole genome open reading frame analysis in proteomics

Background  

Defining the location of genes and the precise nature of gene products remains a fundamental challenge in genome annotation. Interrogating tandem mass spectrometry data using genomic sequence provides an unbiased method to identify novel translation products. A six-frame translation of the entire human genome was used as the query database to search for novel blood proteins in the data from the Human Proteome Organization Plasma Proteome Project. Because this target database is orders of magnitude larger than the databases traditionally employed in tandem mass spectra analysis, careful attention to significance testing is required. Confidence of identification is assessed using our previously described Poisson statistic, which estimates the significance of multi-peptide identifications incorporating the length of the matching sequence, number of spectra searched and size of the target sequence database.     

A novel mutator of Escherichia coli carrying a defect in the dgt gene, encoding a dGTP triphosphohydrolase

A novel mutator locus in Escherichia coli was identified from a collection of random transposon insertion mutants. Several mutators in this collection were found to have an insertion in the dgt gene, encoding a previously characterized dGTP triphosphohydrolase. The mutator activity of the dgt mutants displays an unusual specificity. Among the six possible base pair substitutions in a lacZ reversion system, the G·C→C·G transversion and A·T→G·C transition are strongly enhanced (10- to 50-fold), while a modest effect (two- to threefold) is also observed for the G·C→A·T transition. Interestingly, a two- to threefold reduction in mutant frequency (antimutator effect) is observed for the G·C→T·A transversion. In the absence of DNA mismatch repair (mutL) some of these effects are reduced or abolished, while other effects remain unchanged. Analysis of these effects, combined with the DNA sequence contexts in which the reversions take place, suggests that alterations of the dGTP pools as well as alterations in the level of some modified dNTP derivatives could affect the fidelity of in vivo DNA replication and, hence, account for the overall mutator effects.     

EPR study of non-covalent spin labeled serum albumin and hemoglobin

Electron Paramagnetic Resonance (EPR) was used to investigate the Tempyo spin label (3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy) as a report group for the interactions and the conformational changes of lyophilized bovine serum albumin (BSA) and bovine hemoglobin (BH), as function of pH values in the range 2.5-11. The EPR spectra are similar with those of other non-covalently spin label porphyrins in frozen solution at very low temperatures. This behavior indicated a possible spin-spin interaction between the hemic iron and the nitroxide group. The changes in the EPR spectra as function of the pH are discussed in terms of conformational changes of the proteins. Spectral simulations and magnetic EPR parameters reveal the following: (i) one single paramagnetic species, with Gaussian line shape, was used for the best fits of experimental spectra in the case of serum albumin samples; and (ii) a weighted sum of Lorentzian and Gaussian line shape in the case of hemoglobin samples. The representation of correlation time vs. pH, reveals a dependence of degree of immobilization of spin label on the conformational changes of proteins in acidic and basic environment.     

Co-occurring grass species differ in their associated microbial community composition in a temperate native grassland

Background and aims

Specific associations exist between plant species and the soil microbial community and these associations vary between habitat types and different plant groups. However, there is evidence that the associations are highly specific. Hence, we aimed to determine the specificity of plant-microbe relationships amongst co-occurring grass species in a temperate grassland.

Methods and results

We examined the broad microbial groups of bacteria and fungi as well as a specific fungal group, the arbuscular mycorrhizal community amongst two dominant C₃ and C₄ species and one sub-dominant C₃ species using terminal restriction fragment length polymorphism (T-RFLP) analysis. We found that the two dominant species were more similar to each other in their bacterial and arbuscular mycorrhizal community composition than either was to the sub-dominant species, but not in their fungal community composition. We also found no clear evidence that those differences were directly linked to soil chemical properties.

Conclusions

Our results demonstrate that co-occurring grass species have a distinct soil microbial community and T-RFLP analysis is able to detect plant species effect on the microbial community composition on an extremely local scale, providing an insight into the differences in the response of bacterial, fungal and arbuscular mycorrhizal communities to different, but similar and co-occurring, plant species.     

Sib mating designs for mapping quantitative trait loci

The power to separate the variance of a quantitative trait locus (QTL) from the polygenic variance is determined by the variability of genes identical by descent (IBD) at the QTL. This variability may increase with inbreeding. Selfing, the most extreme form of inbreeding, increases the variability of the IBD value shared by siblings, and thus has a higher efficiency for QTL mapping than random mating. In self-incompatible organisms, sib mating is the closest form of inbreeding. Similar to selfing, sib mating may also increase the power of QTL detection relative to random mating. In this study, we develop an IBD-based method under sib mating designs for QTL mapping. The efficiency of sib mating is then compared with random mating. Monte Carlo simulations show that sib mating designs notably increase the power for QTL detection. When power is intermediate, the power to detect a QTL using full-sib mating is, on average, 7% higher than under random mating. In addition, the IBD-based method proposed in this paper can be used to combine data from multiple families. As a result, the estimated QTL parameters can be applied to a wide statistical inference space relating to the entire reference population. This revised version was published online in July 2006 with corrections to the Cover Date.     

The fission yeast MO25 protein functions in polar growth and cell separation

Proteins of the MO25 family are widely conserved but their function has not been characterized in detail. Human MO25 is a cofactor of LKB1, a conserved protein kinase with roles in cell polarity in nematodes, flies and mammalian cells. Furthermore, the budding yeast MO25 homologue, Hym1, is important for cell separation and morphogenesis. We have characterized Pmo25p, the MO25 homologue in the fission yeast Schizosaccharomyces pombe. Pmo25p is an essential protein required for polar growth; in its absence the actin cytoskeleton becomes depolarized and cells adopt a round morphology. In addition, pmo25 mutants are defective in cell separation. Both functions of Pmo25p appear to be mediated by the Orb6p–Mob2p kinase complex. Pmo25p shows no distinct localization during interphase, but it is recruited to one of the two spindle pole bodies during anaphase and to the division site during cytokinesis. The septation initiation network (SIN) regulates the localization of Pmo25p, suggesting that it regulates Pmo25p function during cell division.     

Regulation of the gibberellin pathway by auxin and DELLA proteins

The synthesis and deactivation of bioactive gibberellins (GA) are regulated by auxin and by GA signalling. The effect of GA on its own pathway is mediated by DELLA proteins. Like auxin, the DELLAs promote GA synthesis and inhibit its deactivation. Here, we investigate the relationships between auxin and DELLA regulation of the GA pathway in stems, using a pea double mutant that is deficient in DELLA proteins. In general terms our results demonstrate that auxin and DELLAs independently regulate the GA pathway, contrary to some previous suggestions. The extent to which DELLA regulation was able to counteract the effects of auxin regulation varied from gene to gene. For Mendel’s LE gene (PsGA3ox1) no counteraction was observed. However, for another synthesis gene, a GA 20-oxidase, the effect of auxin was weak and in WT plants appeared to be completely over-ridden by DELLA regulation. For a key GA deactivation (2-oxidase) gene, PsGA2ox1, the up-regulation induced by auxin deficiency was reduced to some extent by DELLA regulation. A second pea 2-oxidase gene, PsGA2ox2, was up-regulated by auxin, in a DELLA-independent manner. In Arabidopsis also, one 2-oxidase gene was down-regulated by auxin while another was up-regulated. Monitoring the metabolism pattern of GA₂₀ showed that in Arabidopsis, as in pea, auxin can promote the accumulation of bioactive GA.     

The BRCA1 Tumor Suppressor Binds to Inositol 1,4,5-Trisphosphate Receptors to Stimulate Apoptotic Calcium Release

The inositol 1,4,5-trisphosphate receptor (IP₃R) is a ubiquitously expressed endoplasmic reticulum (ER)-resident calcium channel. Calcium release mediated by IP₃Rs influences many signaling pathways, including those regulating apoptosis. IP₃R activity is regulated by protein-protein interactions, including binding to proto-oncogenes and tumor suppressors to regulate cell death. Here we show that the IP₃R binds to the tumor suppressor BRCA1. BRCA1 binding directly sensitizes the IP₃R to its ligand, IP₃. BRCA1 is recruited to the ER during apoptosis in an IP₃R-dependent manner, and, in addition, a pool of BRCA1 protein is constitutively associated with the ER under non-apoptotic conditions. This is likely mediated by a novel lipid binding activity of the first BRCA1 C terminus domain of BRCA1. These findings provide a mechanistic explanation by which BRCA1 can act as a proapoptotic protein.