Yeast ribosomal protein L7 and its homologue Rlp7 are simultaneously present at distinct sites on pre-60S ribosomal particles

Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C₁ and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7^Lb within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.     

Large-scale exploration of growth inhibition caused by overexpression of genomic fragments in Saccharomyces cerevisiae

We have screened the genome of Saccharomyces cerevisiae for fragments that confer a growth-retardation phenotype when overexpressed in a multicopy plasmid with a tetracycline-regulatable (Tet-off) promoter. We selected 714 such fragments with a mean size of 700 base-pairs out of around 84,000 clones tested. These include 493 in-frame open reading frame fragments corresponding to 454 distinct genes (of which 91 are of unknown function), and 162 out-of-frame, antisense and intergenic genomic fragments, representing the largest collection of toxic inserts published so far in yeast.     

Metagenomes of the picoalga Bathycoccus from the Chile coastal upwelling

Among small photosynthetic eukaryotes that play a key role in oceanic food webs, picoplanktonic Mamiellophyceae such as Bathycoccus, Micromonas, and Ostreococcus are particularly important in coastal regions. By using a combination of cell sorting by flow cytometry, whole genome amplification (WGA), and 454 pyrosequencing, we obtained metagenomic data for two natural picophytoplankton populations from the coastal upwelling waters off central Chile. About 60% of the reads of each sample could be mapped to the genome of Bathycoccus strain from the Mediterranean Sea (RCC1105), representing a total of 9 Mbp (sample T142) and 13 Mbp (sample T149) of non-redundant Bathycoccus genome sequences. WGA did not amplify all regions uniformly, resulting in unequal coverage along a given chromosome and between chromosomes. The identity at the DNA level between the metagenomes and the cultured genome was very high (96.3% identical bases for the three larger chromosomes over a 360 kbp alignment). At least two to three different genotypes seemed to be present in each natural sample based on read mapping to Bathycoccus RCC1105 genome.     

Gene functionalities and genome structure in Bathycoccus prasinos reflect cellular specializations at the base of the green lineage

ABSTRACT: BACKGROUND: Bathycoccus prasinos is an extremely small cosmopolitan marine green alga whose cells are covered with intricate spider's web patterned scales that develop within the Golgi cisternae before their transport to the cell surface. The objective of this work is to sequence and analyze its genome, and to present a comparative analysis with other known genomes of the green lineage. RESULTS: Its small genome of 15Mb consists of 19 chromosomes and lacks transposons. Although 70% of all B. prasinos genes share similarities with other Viridiplantae genes, up to 428 genes were probably acquired by horizontal gene transfer, mainly from other eukaryotes. Two chromosomes, one big and one small, are atypical, an unusual synapomorphic feature within the Mamiellales. Genes on these atypical outlier chromosomes show lower GC content and a significant fraction of putative horizontal gene transfer genes. Whereas the small outlier chromosome lacks colinearity with other Mamiellales and contains many unknown genes without homologs in other species, the big outlier shows a higher intron content, increased expression levels and a unique clustering pattern of housekeeping functionalities. Four gene families are highly expanded in B. prasinos, including sialyltransferases, sialidases, ankyrin repeats and zinc ion-binding genes, and we hypothesize that these genes are associated with the process of scale biogenesis. CONCLUSION: The minimal genomes of the Mamiellophyceae provide a baseline for evolutionary and functional analyses of metabolic processes in green plants.     

Evidence that a higher ATP cost of muscular contraction contributes to the lower mechanical efficiency associated with COPD: preliminary findings

Impaired metabolism in peripheral skeletal muscles potentially contributes to exercise intolerance in chronic obstructive pulmonary disease (COPD). We used (31)P-magnetic resonance spectroscopy ((31)P-MRS) to examine the energy cost and skeletal muscle energetics in six patients with COPD during dynamic plantar flexion exercise compared with six well-matched healthy control subjects. Patients with COPD displayed a higher energy cost of muscle contraction compared with the controls (control: 6.1 ± 3.1% of rest·min(-1)·W(-1), COPD: 13.6 ± 8.3% of rest·min(-1)·W(-1), P = 0.01). Although, the initial phosphocreatine resynthesis rate was also significantly attenuated in patients with COPD compared with controls (control: 74 ± 17% of rest/min, COPD: 52 ± 13% of rest/min, P = 0.04), when scaled to power output, oxidative ATP synthesis was similar between groups (6.5 ± 2.3% of rest·min(-1)·W(-1) in control and 7.8 ± 3.9% of rest·min(-1)·W(-1) in COPD, P = 0.52). Therefore, our results reveal, for the first time that in a small subset of patients with COPD a higher ATP cost of muscle contraction may substantially contribute to the lower mechanical efficiency previously reported in this population. In addition, it appears that some patients with COPD have preserved mitochondrial function and normal energy supply in lower limb skeletal muscle.     

Exogenous myristic acid acylates proteins in cultured rat hepatocytes

Fatty acid acylation is a functionally important modification of proteins. In the liver, however, acylated proteins remain largely unknown. This work was aimed at investigating fatty acid acylation of proteins in cultured rat hepatocytes. Incubation of these cells with [9,10-3H] myristic acid followed by two-dimensional electrophoresis separation of the delipidated cellular proteins and autoradiography evidenced the reproducible and selective incorporation of radioactivity from the precursor into 18 well-resolved proteins in the 10--120 kDa range and the 4--7 pH range. Radiolabeling of these proteins resulted from covalent linkage to the precursor [9,10-3H] myristic acid or to its elongation product, palmitic acid. The majority of the covalent linkages between the proteins and the fatty acids were broken by base hydrolysis, which indicated that the linkage was of thioester or ester-type. Only one of the studied proteins was attached to myristic acid via an amide linkage which resisted the basic treatment but was broken by acid hydrolysis. After incubation with [9,10-3H] palmitic acid, only two proteins previously detected with myristic acid were radiolabeled. Finally, the identified acylated proteins may be grouped into two classes: proteins involved in signal transduction (the alpha subunit of a heterotrimeric G protein and several small G proteins) and cytoskeletal proteins (cytokeratins, actin).     

Acute Respiratory Failure in Critically Ill Patients with Interstitial Lung Disease

Background

Patients with chronic known or unknown interstitial lung disease (ILD) may present with severe respiratory flares that require intensive management. Outcome data in these patients are scarce.

Patients and Methods

Clinical and radiological features were collected in 83 patients with ILD-associated acute respiratory failure (ARF). Determinants of hospital mortality and response to corticosteroid therapy were identified by logistic regression.

Results

Hospital and 1-year mortality rates were 41% and 54% respectively. Pulmonary hypertension, computed tomography (CT) fibrosis and acute kidney injury were independently associated with mortality (odds ratio (OR) 4.55; 95% confidence interval (95%CI) (1.20–17.33); OR, 7.68; (1.78–33.22) and OR 10.60; (2.25–49.97) respectively). Response to steroids was higher in patients with shorter time from hospital admission to corticosteroid therapy. Patients with fibrosis on CT had lower response to steroids (OR, 0.03; (0.005–0.21)). In mechanically ventilated patients, overdistension induced by high PEEP settings was associated with CT fibrosis and hospital mortality.

Conclusion

Mortality is high in ILD-associated ARF. CT and echocardiography are valuable prognostic tools. Prompt corticosteroid therapy may improve survival.