The rate of the molecular clock and the cost of gratuitous protein synthesis

The nature of the protein molecular clock, the protein-specific rate of amino acid substitutions, is among the central questions of molecular evolution. Protein expression level is the dominant determinant of the clock rate in a number of organisms. It has been suggested that highly expressed proteins evolve slowly in all species mainly to maintain robustness to translation errors that generate toxic misfolded proteins. Here we investigate this hypothesis experimentally by comparing the growth rate of Escherichia coli expressing wild type and misfolding-prone variants of the LacZ protein. We show that the cost of toxic protein misfolding is small compared to other costs associated with protein synthesis. Complementary computational analyses demonstrate that there is also a relatively weaker, but statistically significant, selection for increasing solubility and polarity in highly expressed E. coli proteins. Although we cannot rule out the possibility that selection against misfolding toxicity significantly affects the protein clock in species other than E. coli, our results suggest that it is unlikely to be the dominant and universal factor determining the clock rate in all organisms. We find that in this bacterium other costs associated with protein synthesis are likely to play an important role. Interestingly, our experiments also suggest significant costs associated with volume effects, such as jamming of the cellular environment with unnecessary proteins

Transthyretin regulates thyroid hormone levels in the choroid plexus, but not in the brain parenchyma: study in a transthyretin-null mouse model

6 pages, 3 figures, 2 tables.-- Presented in part in abstract form at the 26th Annual Meeting of the European Thyroid Association, Milan, Italy, August 28 to September 1, 1999.-- This is part of the Ph.D. thesis of J.A.P., University of Porto, Porto, Portugal.Transthyretin (TTR) is the major T4-binding protein in rodents. Using a TTR-null mouse model we asked the following questions. 1) Do other T4 binding moieties replace TTR in the cerebrospinal fluid (CSF)? 2) Are the low whole brain total T4 levels found in this mouse model associated with hypothyroidism, e.g. increased 5'-deiodinase type 2 (D2) activity and RC3-neurogranin messenger RNA levels? 3) Which brain regions account for the decreased total whole brain T4 levels? 4) Are there changes in T3 levels in the brain? Our results show the following. 1) No other T4-binding protein replaces TTR in the CSF of the TTR-null mice. 2) D2 activity is normal in the cortex, cerebellum, and hippocampus, and total brain RC3-neurogranin messenger RNA levels are not altered. 3) T4 levels measured in the cortex, cerebellum, and hippocampus are normal. However T4 and T3 levels in the choroid plexus are only 14% and 48% of the normal values, respectively. 4) T3 levels are normal in the brain parenchyma. The data presented here suggest that TTR influences thyroid hormone levels in the choroid plexus, but not in the brain. Interference with the blood-choroid-plexus-CSF-TTR-mediated route of T4 entry into the brain caused by the absence of TTR does not produce measurable features of hypothyroidism. It thus appears that TTR is not required for T4 entry or for maintenance of the euthyroid state in the mouse brain.This work was supported by Grants PRAXIS (Portugal) SAU/2/96 and BIA/459/94.Peer reviewe

Mre11 Protein Complex Prevents Double-Strand Break Accumulation during Chromosomal DNA Replication

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RNA stabilizes transcription-Dependent Chromatin Loops Induced By Nuclear Hormones

We show that transcription induced by nuclear receptors for estrogen (e2) or retinoic acid (RA) is associated with formation of chromatin loops that juxtapose the 5’ end (containing the promoter) with the enhancer and the 3′ polyA addition site of the target gene. We nd three loop con gurations which change as a function of time after induction: 1. RA or E2-induced loops which connect the 5′ end, the enhancer and the 3′ end of the gene, and are stabilized by RNA early after induction; 2. E2-independent loops whose stability does not require RNA; 3. Loops detected only by treatment of chromatin with RNAse H1 prior to hormonal induction. RNAse H1 digests RNA that occludes the relevant restriction sites, thus preventing detection of these loops. R-loops at the 5′ and 3′ ends of the RA or e2-target genes were demonstrated by immunoprecipitation with anti-DNA-RNA hybrid antibodies as well as by sensitivity to RNAse H1. The cohesin RAD21 subunit is preferentially recruited to the target sites upon RA or e2 induction of transcription. R21 binding to chromatin is eliminated by RNAse H1. We identi ed e2-induced and RNase H1-sensitive antisense RNAs located at the 5′ and 3′ ends of the e2-induced transcription unit which stabilize the loops and RAD21 binding to chromatin. This is the rst report of chromatin loops that form after gene induction that are maintained by RNA:DNA hybrids

DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism

We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5' and 3' ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells

Muscle expression of human retinol-binding protein (RBP). Suppression of the visual defect of RBP knockout mice.

Mice lacking retinol-binding protein (RBP) have low circulating retinol levels. They have severe visual defects due to a low content of retinol or retinyl esters in the eye. A transgenic mouse strain that expresses human RBP under the control of the muscle creatine kinase promoter in the null background was generated. The exogenous protein bound retinol and transthyretin in the circulation and effectively delivered retinol to the eye. Thus, RBP expressed from an ectopic source suppresses the visual phenotype, and retinoids accumulate in the eye. No human RBP was found in the retinal pigment epithelium of the transgenic mice, indicating that retinol uptake by the eye does not entail endocytosis of the carrier RBP

ARP2/3- and resection-coupled genome reorganization facilitates translocations [preprint]

DNA end-resection and nuclear actin-based movements orchestrate clustering of double-strand breaks (DSBs) into homology-directed repair (HDR) domains. Here, we analyze how actin nucleation by ARP2/3 affects damage-dependent and -independent 3D genome reorganization and facilitates pathologic repair. We observe that DNA damage, followed by ARP2/3-dependent establishment of repair domains enhances local chromatin insulation at a set of damage-proximal boundaries and affects compartment organization genome-wide. Nuclear actin polymerization also promotes interactions between DSBs, which in turn facilitates aberrant intra- and inter-chromosomal rearrangements. Notably, BRCA1 deficiency, which decreases end-resection, DSB mobility, and subsequent HDR, nearly abrogates recurrent translocations between AsiSI DSBs. In contrast, loss of functional BRCA1 yields unique translocations genome-wide, reflecting a critical role in preventing spontaneous genome instability and subsequent rearrangements. Our work establishes that the assembly of DSB repair domains is coordinated with multiscale alterations in genome architecture that enable HDR despite increased risk of translocations with pathologic potential

High-coverage methylation data of a gene model before and after DNA damage and homologous repair

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles

The v-Ki-Ras Oncogene Alters cAMP Nuclear Signaling by Regulating the Location and the Expression of cAMP-dependent Protein Kinase IIβ

The v-Ki-Ras oncoprotein dedifferentiates thyroid cells and inhibits nuclear accumulation of the catalytic subunit of cAMP-dependent protein kinase. After activation of v-Ras or protein kinase C, the regulatory subunit of type II protein kinase A, RIIbeta, translocates from the membranes to the cytosol. RIIbeta mRNA and protein were eventually depleted. These effects were mimicked by expressing AKAP45, a truncated version of the RII anchor protein, AKAP75. Because AKAP45 lacks membrane targeting domains, it induces the translocation of PKAII to the cytoplasm. Expression of AKAP45 markedly decreased thyroglobulin mRNA levels and inhibited accumulation of C-PKA in the nucleus. Our results suggest that: 1) The localization of PKAII influences cAMP signaling to the nucleus; 2) Ras alters the localization and the expression of PKAII; 3) Translocation of PKAII to the cytoplasm reduces nuclear C-PKA accumulation, resulting in decreased expression of cAMP-dependent genes, including RIIbeta, TSH receptor, and thyroglobulin. The loss of RIIbeta permanently down-regulates thyroid-specific gene expression