BackgroundWhen a stop codon is located in the ribosomal A-site, the termination complex promotes release of the polypeptide and dissociation of the 80S ribosome. In eukaryotes two proteins eRF1 and eRF3 play a crucial function in the termination process. The essential GTPase Sup35p, the eRF3 release factor of Saccharomyces cerevisiae is highly conserved. In particular, we observed that all eRF3 homologs share a potential phosphorylation site at threonine 341, suggesting a functional role for this residue. The goal of this study was to determine whether this residue is actually phosphorylated in yeast and if it is involved in the termination activity of the protein.ResultsWe detected no phosphorylation of the Sup35 protein in vivo. However, we show that it is phosphorylated by the cAMP-dependent protein kinase A on T341 in vitro. T341 was mutated to either alanine or to aspartic acid to assess the role of this residue in the activity of the protein. Both mutant proteins showed a large decrease of GTPase activity and a reduced interaction with eRF1/Sup45p. This was correlated with an increase of translational readthrough in cells carrying the mutant alleles. We also show that this residue is involved in functional interaction between the N- and C-domains of the protein.ConclusionOur results point to a new critical residue involved in the translation termination activity of Sup35 and in functional interaction between the N- and C-domains of the protein. They also raise interesting questions about the relation between GTPase activity of Sup35 and its essential function in yeast.

BackgroundFlatfish metamorphosis involves major physiological and morphological changes. Due to its importance in aquaculture and as a model for developmental studies, some gene expression studies have focused on the understanding of this process using quantitative real-time PCR (qRT-PCR) technique. Therefore, adequate reference genes for accurate normalization are required.ResultsThe stability of 12 potential reference genes was examined during larval development in Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus) to determine the most suitable genes for qRT-PCR analysis. Transcription levels of genes encoding β-Actin (ACTB), glyceraldehyde-3P-dehydrogenase (GAPDH), annexin A2 (ANXA2), glutathione S-transferase (GST), ornithine decarboxylase (ODC), hypoxanthine phosphoribosyltransferase (HPRT1), ubiquitin (UBQ), elongation factor 1 alpha (eEF1A1), 18S ribosomal RNA, and the ribosomal proteins S4 (RPS4) and L13a (RPL13a) were quantitated. Two paralogous genes for ACTB were analyzed in each of both flatfish species. In addition, two paralogous genes for GAPDH were studied in Senegalese sole. RPL13a represented non-orthologous genes between both flatfish species. GeNorm and NormFinder analyses for expression stability revealed RPS4, UBQ and eEF1A1 as the most stable genes in Senegalese sole, Atlantic halibut and in a combined analysis. In all cases, paralogous genes exhibited differences in expression stability.ConclusionThis work suggests RPS4, UBQ, and eEF1A1 genes as useful reference genes for accurate normalization in qRT-PCR studies in Senegalese sole and Atlantic halibut larvae. The congruent results between both species in spite of the drastic differences in larval development suggest that selected housekeeping genes (HKGs) could be useful in other flatfish species. However, the finding of paralogous gene copies differentially expressed during development in some HKGs underscores the necessity to identify orthologous genes.

BackgroundPioglitazone was reported to improve hepatic steatosis and necroinflammation in human studies. To investigate whether the hepato-protective effect of pioglitazone was associated with an improvement of antioxidant defense mechanism, oxidative DNA damage and repair activity were determined in a high fat diet model. Male C57BL/6 mice were respectively fed with a 30% fat diet, the same diet with pioglitazone 100 mg/kg/day, or a chow diet as control for 8 weeks. Tissue oxidative stress was indicated by malondialdehyde concentration. Oxidative DNA damage was detected by immunohistochemical 8-oxoG staining. Enzymatic antioxidant defense was detected by the real-time PCR of superoxide dismutase (Sod1, Sod2) and DNA glycosylase (Ogg1, MutY). Oxidative DNA repair was detected by immunohistochemical staining and western blotting of OGG1 expression.ResultsOur results show that hepatic steatosis was induced by a high-fat diet and improved by adding pioglitazone. Malondialdehyde concentration and 8-oxoG staining were strongly increased in the high-fat diet group, but attenuated by pioglitazone. Gene expressions of antioxidant defense mechanism: Sod1, Sod2, Ogg1 and MutY significantly decreased in the high-fat diet group but reversed by pioglitazone co-administration.ConclusionThe attenuation of hepatic oxidative DNA damage by pioglitazone in a high-fat diet may be mediated by up-regulation of the antioxidant defense mechanism and oxidative DNA repair activity. The diminution of oxidative damage may explain the clinical benefit of pioglitazone treatment in patients with non-alcoholic fatty liver disease.

BackgroundAs an epigenetic regulator, the transcriptional intermediary factor 1β (TIF1β)/KAP1/TRIM28) has been linked to gene expression and chromatin remodeling at specific loci by association with members of the heterochromatin protein 1 (HP1) family and various other chromatin factors. The interaction between TIF1β and HP1 is crucial for heterochromatin formation and maintenance. The HP1-box, PXVXL, of TIF1β is responsible for its interaction with HP1. However, the underlying mechanism of how the interaction is regulated remains poorly understood.ResultsThis work demonstrates that TIF1β is phosphorylated on Ser473, the alteration of which is dynamically associated with cell cycle progression and functionally linked to transcriptional regulation. Phosphorylation of TIF1β/Ser473 coincides with the induction of cell cycle gene cyclin A2 at the S-phase. Interestingly, chromatin immunoprecipitation demonstrated that the promoter of cyclin A2 gene is occupied by TIF1β and that such occupancy is inversely correlated with Ser473 phosphorylation. Additionally, when HP1β was co-expressed with TIF1β/S473A, but not TIF1β/S473E, the colocalization of TIF1β/S473A and HP1β to the promoters of Cdc2 and Cdc25A was enhanced. Non-phosphorylated TIF1β/Ser473 allowed greater TIF1β association with the regulatory regions and the consequent repression of these genes. Consistent with possible inhibition of TIF1β's corepressor function, the phosphorylation of the Ser473 residue, which is located near the HP1-interacting PXVXL motif, compromised the formation of TIF1β-HP1 complex. Finally, we found that the phosphorylation of TIF1β/Ser473 is mediated by the PKCδ pathway and is closely linked to cell proliferation.ConclusionThe modulation of HP1β-TIF1β interaction through the phosphorylation/de-phosphorylation of TIF1β/Ser473 may constitute a molecular switch that regulates the expression of particular genes. Higher levels of phosphorylated TIF1β/Ser473 may be associated with the expression of key regulatory genes for cell cycle progression and the proliferation of cells.

Backgroundthis study set out to examine the effects of the treatment with 1,25-dihydroxyvitamin D3 (1,25D3) [150 IU/Kg (3.75 μg/Kg) one a day, for 15 days] to non-diabetic rats and in rats rendered diabetic by a single injection of streptozotocin [65 mg/kg].Resultstreatment with 1,25D3 to non-diabetic rats did not affect the biochemical parameters measured in the plasma and urine of these animals. Likewise, insulin receptor expression in the kidney, liver, or adipose tissue and insulin-stimulated glucose transport in adipocytes from these animals were not affected either. Treatment with 1,25D3 to streptozotocin-induced diabetic rats did not correct the hyperglycemia, hypoinsulinemia, glycosuria or ketonemia induced by the diabetes, although it partially reversed the over-expression of the insulin receptor gene in the liver and adipose tissue, without altering the normal expression of this gene in the kidney. These effects were accompanied by a normalization of the number of insulin receptors without altering receptor affinity but improving the insulin response to glucose transport in adipocytes from these diabetic animals. Moreover, a computer search in the rat insulin receptor promoter revealed the existence of two candidate vitamin D response element (VDRE) sequences located at -256/-219 bp and -653/-620 bp, the first overlapped by three and the second by four AP-2-like sites.Conclusionthese genomic actions of 1,25D3 could represent beneficial effects associated with the amelioration of diabetes via mechanisms that possibly involve direct transcriptional activation of the rat insulin receptor gene. The candidate VDREs identified may respond to 1,25D3 via activation of the vitamin D receptor, although this remains to be investigated.

BackgroundRegulation of the expression of particular genes can rely on mechanisms that are different from classical transcriptional and translational control. The LY6G5B and LY6G6D genes encode LY-6 domain proteins, whose expression seems to be regulated in an original fashion, consisting of an intron retention event which generates, through an early premature stop codon, a non-coding transcript, preventing expression in most cell lines and tissues.ResultsThe MHC LY-6 non-coding transcripts have shown to be stable and very abundant in the cell, and not subject to Nonsense Mediated Decay (NMD). This retention event appears not to be solely dependent on intron features, because in the case of LY6G5B, when the intron is inserted in the artificial context of a luciferase expression plasmid, it is fully spliced but strongly stabilises the resulting luciferase transcript. In addition, by quantitative PCR we found that the retained and spliced forms are differentially expressed in tissues indicating an active regulation of the non-coding transcript. EST database analysis revealed that these genes have an alternative expression pathway with the formation of Transcription Induced Chimeras (TIC). This data was confirmed by RT-PCR, revealing the presence of different transcripts that would encode the chimeric proteins CSNKβ-LY6G5B and G6F-LY6G6D, in which the LY-6 domain would join to a kinase domain and an Ig-like domain, respectively.ConclusionIn conclusion, the LY6G5B and LY6G6D intron-retained transcripts are not subjected to NMD and are more abundant than the properly spliced forms. In addition, these genes form chimeric transcripts with their neighbouring same orientation 5' genes. Of interest is the fact that the 5' genes (CSNKβ or G6F) undergo differential splicing only in the context of the chimera (CSNKβ-LY6G5B or G6F-LY6G6C) and not on their own.

BackgroundCCAAT/Enhancer Binding Proteinδ (C/EBPδ) is a member of the highly conserved C/EBP family of leucine zipper (bZIP) proteins. C/EBPδ is highly expressed in G0 growth arrested mammary epithelial cells (MECs) and "loss of function" alterations in C/EBPδ have been associated with impaired contact inhibition, increased genomic instability and increased cell migration. Reduced C/EBPδ expression has also been reported in breast cancer and acute myeloid leukemia (AML). C/EBPδ functions as a transcriptional activator, however, only a limited number of C/EBPδ target genes have been reported. As a result, the role of C/EBPδ in growth control and the potential mechanisms by which "loss of function" alterations in C/EBPδ contribute to tumorigenesis are poorly understood. The goals of the present study were to identify C/EBPδ target genes using Chromatin Immunoprecipitation coupled with a CpG Island (HCG12K) Array gene chip ("ChIP-chip") assay and to assess the expression and potential functional roles of C/EBPδ target genes in growth control.ResultsChIP-chip assays identified ~100 C/EBPδ target gene loci which were classified by gene ontology (GO) into cell adhesion, cell cycle regulation, apoptosis, signal transduction, intermediary metabolism, gene transcription, DNA repair and solute transport categories. Conventional ChIP assays validated the ChIP-chip results and demonstrated that 14/14 C/EBPδ target loci were bound by C/EBPδ in G0 growth arrested MCF-12A MECs. Gene-specific RT-PCR analysis also demonstrated C/EBPδ-inducible expression of 14/14 C/EBPδ target genes in G0 growth arrested MCF-12A MECs. Finally, expression of endogenous C/EBPδ and selected C/EBPδ target genes was also demonstrated in contact-inhibited G0 growth arrested nontransformed human MCF-10A MECs and in mouse HC11 MECs. The results demonstrate consistent activation and downstream function of C/EBPδ in growth arrested human and murine MECs.ConclusionC/EBPδ target genes were identified by a global gene array approach and classified into functional categories that are consistent with biological contexts in which C/EBPδ is induced, such as contact-mediated G0 growth arrest, apoptosis, metabolism and inflammation. The identification and validation of C/EBPδ target genes provides new insights into the mechanistic role of C/EBPδ in mammary epithelial cell biology and sheds new light on the potential impact of "loss of function" alterations in C/EBPδ in tumorigenesis.

BackgroundBrown algae are plant multi-cellular organisms occupying most of the world coasts and are essential actors in the constitution of ecological niches at the shoreline. Ectocarpus siliculosus is an emerging model for brown algal research. Its genome has been sequenced, and several tools are being developed to perform analyses at different levels of cell organization, including transcriptomic expression analyses. Several topics, including physiological responses to osmotic stress and to exposure to contaminants and solvents are being studied in order to better understand the adaptive capacity of brown algae to pollution and environmental changes. A series of genes that can be used to normalise expression analyses is required for these studies.ResultsWe monitored the expression of 13 genes under 21 different culture conditions. These included genes encoding proteins and factors involved in protein translation (ribosomal protein 26S, EF1alpha, IF2A, IF4E) and protein degradation (ubiquitin, ubiquitin conjugating enzyme) or folding (cyclophilin), and proteins involved in both the structure of the cytoskeleton (tubulin alpha, actin, actin-related proteins) and its trafficking function (dynein), as well as a protein implicated in carbon metabolism (glucose 6-phosphate dehydrogenase). The stability of their expression level was assessed using the Ct range, and by applying both the geNorm and the Normfinder principles of calculation.ConclusionComparisons of the data obtained with the three methods of calculation indicated that EF1alpha (EF1a) was the best reference gene for normalisation. The normalisation factor should be calculated with at least two genes, alpha tubulin, ubiquitin-conjugating enzyme or actin-related proteins being good partners of EF1a. Our results exclude actin as a good normalisation gene, and, in this, are in agreement with previous studies in other organisms.

BackgroundInositol is a key cellular metabolite for many organisms. Cryptococcus neoformans is an opportunistic pathogen which primarily infects the central nervous system, a region of high inositol concentration, of immunocompromised individuals. Through the use of myo-inositol oxygenase C. neoformans can catabolize inositol as a sole carbon source to support growth and viability.ResultsThree myo-inositol oxygenase gene sequences were identified in the C. neoformans genome. Differential regulation was suggested by computational analyses of the three gene sequences. This included examination of the upstream regulatory regions, identifying ORE/TonE and UASINO sequences, conserved introns/exons, and in frame termination sequences. Homology modeling of the proteins encoded by these genes revealed key differences in the myo-inositol active site.ConclusionThe results suggest there are two functional copies of the myo-inositol oxygenase gene in the C. neoformans genome. The functional genes are differentially expressed in response to environmental inositol concentrations. Both the upstream regulatory regions of the genes and the structure of the specific proteins suggest that MIOX1 would function when inositol concentrations are low, whereas MIOX2 would function when inositol concentrations are high.

BackgroundAlthough much is known about molecular mechanisms that prevent re-initiation of DNA replication on newly replicated DNA during a single cell cycle, knowledge is sparse regarding the regions that are most susceptible to re-replication when those mechanisms are bypassed and regarding the extents to which checkpoint pathways modulate re-replication. We used microarrays to learn more about these issues in wild-type and checkpoint-mutant cells of the fission yeast, Schizosaccharomyces pombe.ResultsWe found that over-expressing a non-phosphorylatable form of the replication-initiation protein, Cdc18 (known as Cdc6 in other eukaryotes), drove re-replication of DNA sequences genome-wide, rather than forcing high level amplification of just a few sequences. Moderate variations in extents of re-replication generated regions spanning hundreds of kilobases that were amplified (or not) ~2-fold more (or less) than average. However, these regions showed little correlation with replication origins used during S phase. The extents and locations of amplified regions in cells deleted for the checkpoint genes encoding Rad3 (ortholog of human ATR and budding yeast Mec1) and Cds1 (ortholog of human Chk2 and budding yeast Rad53) were similar to those in wild-type cells. Relatively minor but distinct effects, including increased re-replication of heterochromatic regions, were found specifically in cells lacking Rad3. These might be due to Cds1-independent roles for Rad3 in regulating re-replication and/or due to the fact that cells lacking Rad3 continued to divide during re-replication, unlike wild-type cells or cells lacking Cds1. In both wild-type and checkpoint-mutant cells, regions near telomeres were particularly susceptible to re-replication. Highly re-replicated telomere-proximal regions (50–100 kb) were, in each case, followed by some of the least re-replicated DNA in the genome.ConclusionThe origins used, and the extent of replication fork progression, during re-replication are largely independent of the replication and DNA-damage checkpoint pathways mediated by Cds1 and Rad3. The fission yeast pattern of telomere-proximal amplification adjacent to a region of under-replication has also been seen in the distantly-related budding yeast, which suggests that subtelomeric sequences may be a promising place to look for DNA re-replication in other organisms.