Enzyme-catalyzed, post-translational modifications of core histones have been implicated in the complex changes in gene expression that drive early mammalian development. However, until recently the small number of cells available from the preimplantation embryo itself has prevented quantitative analysis of histone modifications at key regulator genes. The possible involvement of histone modifications in the embryo's response to extracellular signals, or as determinants of cell fate or lineage progression, remains unclear. Here we describe the use of a recently-developed chromatin immunoprecipitation technique (CChIP) to assay histone modification levels at key regulator genes (Pou5f1, Nanog, Cdx2, Hoxb1, Hoxb9) as mouse embryos progress from 8-cell to blastocyst in culture. Only by the blastocyst stage, when the embryonic (Inner Cell Mass) and extra-embryonic (Trophoblast) lineages are compared, do we see the expected association between histone modifications previously linked to active and silent chromatin, and transcriptional state. To explore responses to an environmental signal, we exposed embryos to the histone deacetylase inhibitor, anti-epileptic and known teratogen valproic acid (VPA), during progression from 8-cell to morula stage. Such treatment increased H4 acetylation and H3 lysine 4 methylation at the promoters of Hoxb1 and Hoxb9, but not the promoters of Pou5f1, Nanog,Cdx2 or the housekeeping gene Gapdh. Despite the absence of detectable Hoxb transcription, these VPA-induced changes were heritable, following removal of the inhibitor, at least until the blastocyst stage. The selective hyperacetylation of Hoxb promoters in response to a histone deacetylase inhibitor, suggests that Hox genes have a higher turnover of histone acetates than other genes in the preimplantation embryo. To explain the heritability, through mitosis, of VPA-induced changes in histone modification at Hoxb promoters, we describe how an epigenetic feed-forward loop, based on cross-talk between H3 acetylation and H3K4 methylation, might generate a persistently increased steady-state level of histone acetylation in response to a transient signal.
19564914
Lymphoid specific helicase (Lsh) belongs to the family of SNF2/helicases. Disruption of Lsh leads to developmental growth retardation and premature aging in mice. However, the specific effect of Lsh on human cellular senescence remains unknown. Herein, we report that Lsh overexpression delays cell senescence by silencing p16(INK4a) in human fibroblasts. The patterns of p16(INK4a) and Lsh expression during cell senescence present the inverse correlation. We also find that Lsh requires histone deacetylase (HDAC) activity to repress p16(INK4a) and treatment with trichostatin A (TSA) is sufficient to block the repressor effect of Lsh. Moreover, overexpression of Lsh is correlated with deacetylation of histone H3 at the p16 promoter, and TSA treatment in Lsh-expressing cells reverses the acetylation status of histones. Additionally, we demonstrate an interaction between Lsh, histone deacetylase 1 (HDAC1) and HDAC2 in vivo. Furthermore, we demonstrate that Lsh interacts in vivo with the p16 promoter and recruits HDAC1. Our data suggest that Lsh represses endogenous p16(INK4a) expression by recruiting HDAC to establish a repressive chromatin structure at the p16(INK4a) promoter, which in turn delays cell senescence.
19561196
Regulation of chromatin structure through post-translational modifications of histones (e.g., acetylation) has emerged as an important mechanism to translate a variety of environmental stimuli, including drugs of abuse, into specific changes in gene expression. Since alterations in gene expression are thought to contribute to the development and maintenance of the addicted state, recent efforts are aimed at identifying how drugs of abuse alter chromatin structure and the enzymes which regulate it. This review discusses how drugs of abuse alter histone acetylation in brain reward regions, through which enzymes this occurs, and ultimately what role histone acetylation plays in addiction-related behaviors.
19560043
The structural assembly of synapses can be accomplished in a rapid time frame, although most nascent synapses formed during early development are not fully functional and respond poorly to presynaptic action potentials. The mechanisms that are responsible for this delay in synapse maturation are unknown. Histone deacetylases (HDACs) regulate the activity state of chromatin and repress gene expression through the removal of acetyl groups from histones. Class I HDACs, which include HDAC1 and HDAC2, are expressed in the CNS, although their specific role in neuronal function has not been studied. To delineate the contribution of HDAC1 and HDAC2 in the brain, we have used pharmacological inhibitors of HDACs and mice with conditional alleles to HDAC1 and HDAC2. We found that a decrease in the activities of both HDAC1 and HDAC2 during early synaptic development causes a robust facilitation of excitatory synapse maturation and a modest increase in synapse numbers. In contrast, in mature neurons a decrease in HDAC2 levels alone was sufficient to attenuate basal excitatory neurotransmission without a significant change in the numbers of detectable nerve terminals. Therefore, we propose that HDAC1 and HDAC2 form a developmental switch that controls synapse maturation and function acting in a manner dependent on the maturational states of neuronal networks.
19553468
Linker histones H1 are key modulators of chromatin structure. Tightness of their binding to DNA is regulated by posttranslational modifications. In this study we have analyzed posttranslational modifications of five major variants of H1 in human tissue - H1.0, H1.2, H1.3, H1.4, and H1.5. To improve sequence coverage, tryptic peptides of H1 were separated by HPLC and the individual fractions were analyzed using a peptide on-chip implementation of nanoelectrospray (TriVersa), coupled to a linear ion trap-Orbitrap hybrid instrument. For quantitative analysis of lysine methylation, ionization efficiencies of methylated and non-methylated peptides were determined using synthetic peptides. Our analysis revealed that monomethylation of lysine residues alongside with phosphorylation of serine and threonine residues is the major modification of H1 in tissue. We found that most prominent methylation sites are in the N-terminal tail and the globular domain of H1. In the C- terminal domains we identified only few less abundant methylation sites. Quantitative analysis revealed that up to 25% of H1.4 is methylated at K-26 in human tissues. Another prominent methylation site was mapped to K-27 in H1.5, which resembles K-26 site in H1.4. In H1.0 five less abundant (<1% of H1.0) sites were identified. Analysis of patient matched pairs of cancer and adjacent normal breast demonstrated high variation between individuals.
19552482
Histone modification is an important mechanism in oncogenesis and development of hematologic malignancies. Acetylation of lysine residues on histones and opening chromatin are correlated with activation of genes, whereas lysine residues methylation can result in either activation or repression on expressions of chromatin. The main point of all is deacytylation of histone mediated by histone deacetylases (HDACs). HDAC inhibitors are divided into 4 categories: short-chain fatty acids, hydroxamic acids, cyclic tetrapeptides and benzamides, owning different mechanisms in HDAC inhibition. Many kinds of I/II phase clinical tests showed that all these HDAC inhibitors have obviously therapeutic efficacies in treatment of hematologic malignancies with low poisons. Combination of HDAC inhibitors with DNA demethylation drugs can decrease DNA methylation, increase histone acetylation and recover antioncogene expression. As important parts of epigenetics, histone acetylation and HDAC inhibitors possess positive prospects in treatment of hematologic malignancies. In this review the advances of study on mechanisms of histone modification, HDAC inhibitors and their use in treatment of hematologic malignancies are summaried.
19549415
Genomic imprinting arises from allele-specific epigenetic modifications that are established during gametogenesis and that are maintained throughout somatic development. These parental-specific modifications include DNA methylation and post-translational modifications to histones, which create allele-specific active and repressive domains at imprinted regions. Through the use of a high-density genomic tiling array, we generated DNA and histone methylation profiles at 11 imprinted gene clusters in the mouse from DNA and from chromatin immunoprecipitated from sperm, heart, and cerebellum. Our analysis revealed that despite high levels of differential DNA methylation at non-CpG islands within these regions, imprinting control regions (ICRs) and secondary differentially methylated regions (DMRs) were identified by an overlapping pattern of H3K4 trimethylation (active chromatin) and H3K9 trimethylation (repressive chromatin) modifications in somatic tissue, and a sperm differentially methylated region (sDMR; sperm not equal somatic tissue). Using these features as a common signature of DMRs, we identified 11 unique regions that mapped to known imprinted genes, to uncharacterized genes, and to intergenic regions flanking known imprinted genes. A common feature among these regions was the presence of a CpG island and an array of tandem repeats. Collectively, this study provides a comprehensive analysis of DNA methylation and histone H3K4me3 and H3K9me3 modifications at imprinted gene clusters, and identifies common epigenetic and genetic features of regions regulating genomic imprinting.
19542493
Every cell has to duplicate its entire genome during S-phase of the cell cycle. After replication, the newly synthesized DNA is rapidly assembled into chromatin. The newly assembled chromatin 'matures' and adopts a variety of different conformations. This differential packaging of DNA plays an important role for the maintenance of gene expression patterns and has to be reliably copied in each cell division. Posttranslational histone modifications are prime candidates for the regulation of the chromatin structure. In order to understand the maintenance of chromatin structures, it is crucial to understand the replication of histone modification patterns. To study the kinetics of histone modifications in vivo, we have pulse-labeled synchronized cells with an isotopically labeled arginine ((15)N(4)) that is 4 Da heavier than the naturally occurring (14)N(4) isoform. As most of the histone synthesis is coupled with replication, the cells were arrested at the G1/S boundary, released into S-phase and simultaneously incubated in the medium containing heavy arginine, thus labeling all newly synthesized proteins. This method allows a comparison of modification patterns on parental versus newly deposited histones. Experiments using various pulse/chase times show that particular modifications have considerably different kinetics until they have acquired a modification pattern indistinguishable from the parental histones.
19541851
In order to explore the conservation/divergence of transcriptional regulation in the platyhelminth parasite Schistosoma mansoni, we are studying the structures and functions of transcriptional mediators and in particular histone-modifying enzymes. Reversible histone acetylation changes chromatin structure and modulates gene transcription. The removal of acetyl residues from histones and other proteins is catalyzed by histone deacetylases (HDACs) that are under increasing study as therapeutic targets, both in cancer and parasitic diseases. In order to determine the extent and importance of histone acetylation in S. mansoni, we tested the effects of three histone deacetylase inhibitors (HDACi) on both larval and adult worms in culture. Trichostatin A (TSA), valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) inhibited global HDAC activity at all life-cycle stages. TSA and VPA, but not SAHA, caused mortality of schistosomula and adults, with TSA showing the most rapid effect. Moreover, TSA caused an increase in apoptosis in schistosomula shown by the TUNEL assay and an increase in caspase 3/7 activity. Both TSA and VPA were shown to cause an increase in general levels of protein acetylation in schistosomes; more particularly of histone 4 whereas histone 3 acetylation was less affected. In the case of TSA treatment this histone hyperacetylation was correlated with the increased expression of caspases 3 and 7 transcripts. Finally, quantitative chromatin immunoprecipitation showed that the proximal promoter region of the S. mansoni caspase 7 gene was hyperacetylated on histone H4 after TSA treatment.
19538992
The ability of HSV to persist in cells depends on the extent of viral gene expression, which may be controlled by epigenetic mechanisms. We used quiescent infection with the viral mutants, d109 and d106, to explore the effects of cell type and the presence of the viral protein ICP0 on the expression and chromatin structure of the HCMV, tk and gC promoters on the viral genome. Expression from the HCMV promoter on the d109 genome decreased with time and was considerably less in HEL than in Vero cells. Expression from the HCMV promoter in d106 was considerably more abundant than from d109 and this increased with time in both cell types. The same pattern of expression was seen on the tk and gC genes on the viral genomes, although the levels of tk and gC RNA were approximately 10(2) and 10(5) fold lower than wild type virus in d106 and d109, respectively. In micrococcal nuclease digestion experiments, nucleosomes were evident on the d109 genome and the amount of total H3 as determined by chromatin immunoprecipitation (ChIP) was considerably greater on d109 than d106 genomes. The acetylation of histone H3 on the d106 genomes was evident at early and late times post infection in Veros cells, but only at late times in HEL cells. The same pattern was observed for H3 acetylated on lysine 9 (H3AcK9). Trimethylation of H3K9 (H3me3K9) on d109 genomes was only evident at late times post infection in Vero cells, while it was observed both early and late in HEL cells. Heterochromatin Protein 1gamma (HP1gamma) was only generally present on d109 genomes at late times post infection of HEL cells. The observations of chromatin structure correlate with the expression patterns of the three analyzed genes on the quiescent genomes. Therefore, several mechanisms generally affect the expression and contribute to the silencing of persisting genomes. These are the abundance of nucleosomes, the acetylation state of the histones, and heterochromatin. The extent by which these different mechanisms contribute to repression varies in different cell types and is counteracted by the presence of ICP0.
19535445
Most malignant features of cancer cells are triggered by activated oncogenes and the loss of tumor suppressors due to mutation or epigenetic inactivation. It is still unclear, to what extend the escape of emerging cancer cells from recognition and elimination by the immune system is determined by similar mechanisms. We compared the transcriptomes of HCT116 colorectal cancer cells deficient in DNA methyltransferases (DNMTs) and of cells, in which the RAS pathway as the major growth-promoting signaling system is blocked by inhibition of MAPK. We identified the MHC Class I genes HLA-A1/A2 and the ULBP2 gene encoding 1 of the 8 known ligands of the activating NK receptor NKG2D among a cluster of immune genes up-regulated under the conditions of both DNMT-deficiency and MEK-inhibition. Bisulphite sequencing analyses of HCT116 with DNMT deficiency or after MEK-inhibition showed that de-methylation of the ULPB2 promoter correlated with its enhanced surface expression. The HLA-A promoters were not methylated indicating that components of the HLA assembly machinery were also suppressed in DNMT-deficient and MEK-inhibited cells. Increased HLA-A2 surface expression was correlated with enhanced recognition and lysis by A2-specific CTL. On the contrary, elevated ULBP2 expression was not reflected by enhanced recognition and lysis by NK cells. Cosuppression of HLA Class I and NKG2D ligands and genes encoding peptide transporters or proteasomal genes mediates a strong functional link between RAS activation, DNMT activity and disruption of the antigen presenting system controlling immune recognition in colorectal cancer cells. (c) 2009 UICC.
19569244
To clarify genome-wide DNA methylation profiles during hepatocarcinogenesis, bacterial artificial chromosome (BAC) array-based methylated CpG island amplification was performed on 126 tissue samples. The average numbers of BAC clones showing DNA hypo- or hypermethylation increased from non-cancerous liver tissue obtained from patients with hepatocellular carcinomas (HCCs) (N) to HCCs. N appeared to be at the precancerous stage, showing DNA methylation alterations that were correlated with the future development of HCC. Using Wilcoxon test, 25 BAC clones, whose DNA methylation status was inherited by HCCs from N and were able to discriminate 15 N samples from 10 samples of normal liver tissue obtained from patients without HCCs (C) with 100% sensitivity and specificity, were identified. The criteria using the 25 BAC clones were able to discriminate 24 additional N samples from 26 C samples in the validation set with 95.8% sensitivity and 96.2% specificity. Using Wilcoxon test, 41 BAC clones, whose DNA methylation status was able to discriminate patients who survived more than 4 years after hepatectomy from patients who suffered recurrence within 6 months and died within a year after hepatectomy, were identified. The DNA methylation status of the 41 BAC clones was correlated with the cancer-free and overall survival rates of HCC patients. Multivariate analysis revealed that satisfying the criteria using the 41 BAC clones was an independent predictor of overall outcome. Genome-wide alterations of DNA methylation may participate in hepatocarcinogenesis from the precancerous stage, and DNA methylation profiling may provide optimal indicators for carcinogenetic risk estimation and prognostication. (c) 2009 UICC.
19569176
Dietary restriction (DR) increases lifespan in a range of evolutionarily distinct species. The polyphenol resveratrol may be a dietary mimetic of some effects of DR. The pivotal role of the mammalian histone deacetylase (HDAC) Sirt1, and its homologue in other organisms, in mediating the effects of both DR and resveratrol on lifespan/ageing suggests it may be the common conduit through which these dietary interventions influence ageing. We propose the novel hypothesis that effects of DR relevant to lifespan extension include maintenance of DNA methylation patterns through Sirt1-mediated epigenetic effects, and proffer the view that dietary components, including resveratrol, may mimic these actions.
19568959
The developmental program of T helper and regulatory T cell lineage commitment is governed by both genetic and epigenetic mechanisms. The principal events, signaling pathways and the lineage determining factors involved have been extensively studied in the past ten years. Recent studies have elucidated the important role of chromatin remodeling and epigenetic changes for proper regulation of gene expression of lineage-specific cytokines. These include DNA methylation and histone modifications in epigenomic reprogramming during T helper cell development and effector T cell functions. This review discusses the basic epigenetic mechanisms and the role of transcription factors for the differential cytokine gene regulation in the T helper lymphocyte subsets.
19568591
Colorectal cancers (CRC)--and probably all cancers--are caused by alterations in genes. This includes activation of oncogenes and inactivation of tumor suppressor genes (TSGs). There are many ways to achieve these alterations. Oncogenes are frequently activated by point mutation, gene amplification, or changes in the promoter (typically caused by chromosomal rearrangements). TSGs are typically inactivated by mutation, deletion, or promoter methylation, which silences gene expression. About 15% of CRC is associated with loss of the DNA mismatch repair system, and the resulting CRCs have a unique phenotype that is called microsatellite instability, or MSI. This paper reviews the types of genetic alterations that can be found in CRCs and hepatocellular carcinoma (HCC), and focuses upon the epigenetic alterations that result in promoter methylation and the CpG island methylator phenotype (CIMP). The challenge facing CRC research and clinical care at this time is to deal with the heterogeneity and complexity of these genetic and epigenetic alterations, and to use this information to direct rational prevention and treatment strategies.
19568590
BACKGROUND: Insulators and domain boundaries both shield genes from adjacent enhancers and inhibit intrusion of heterochromatin into transgenes. Previous studies examined the functional mechanism of the MYC insulator element MINE and its CTCF binding sites in the context of transgenes that were randomly inserted into the genome by transfection. However, the contribution of CTCF binding sites to both gene regulation and maintenance of chromatin has not been tested at the endogenous MYC gene. METHODOLOGY/PRINCIPAL FINDINGS: To determine the impact of CTCF binding on MYC expression, a series of mutant human chromosomal alleles was prepared in homologous recombination-efficient DT40 cells and individually transferred by microcell fusion into murine cells. Functional tests reported here reveal that deletion of CTCF binding elements within the MINE does not impact the capacity of this locus to correctly organize an 'accessible' open chromatin domain, suggesting that these sites are not essential for the formation of a competent, transcriptionally active locus. Moreover, deletion of the CTCF site at the MYC P2 promoter reduces transcription but does not affect promoter acetylation or serum-inducible transcription. Importantly, removal of either CTCF site leads to DNA methylation of flanking sequences, thereby contributing to progressive loss of transcriptional activity. CONCLUSIONS: These findings collectively demonstrate that CTCF-binding at the human MYC locus does not repress transcriptional activity but is required for protection from DNA methylation.
19568426
Temozolomide (TMZ) exerts its cytotoxic effects by methylating guanine in DNA, resulting in a mismatch with thymine. We studied possible enhancement of the cytotoxic activity of several other targeted drugs in four lung cancer cell lines by TMZ. the data are in relation to O(6)-alkylguanine-DNA-alkyltransferase (AGT) expression, gene methylation, cell cycle distribution and adduct formation. Synergism/additivity was found with O(6)-BG), gemcitabine, lonafarnib and paclitaxel, but not with platinum analogs and topoisomerase-inhibitors. O(6)-BG enhanced TMZ-induced accumulation in the G2/m-phase by increasing formation and retention of the O(6)-methyldeoxyguanosine adducts. TMZ combinations with drugs showing a different individual effect on the cell cycle (e.g. gemcitabine-induced S-phase) were most effective. The results show that O(6)-BG enhanced the TMZ effect in all cell lines. TMZ enhanced the cytotoxicity of gemcitabine, paclitaxel and lonafarnib in most cell lines, possibly by affecting the cell cycle, supporting possible application of TMZ in the treatment of lung cancer.
19567356
The aberrant expression of DNA methyltransferase 1 (DNMT1) in cloned embryos has been implicated as a possible factor in the improper donor genome reprogramming during nuclear transfer. DNMT1 is responsible for maintaining DNA methylation and the subsequent differentiation status of somatic cells. The presence of DNMT1 transcript in the donor cell may contribute to perpetuation of the highly methylated status of the somatic nuclei in cloned embryos. The objective of the present study was to determine the methylation pattern of cloned embryos reconstructed with cells treated with DNMT1-specific small interfering RNA (siRNA). Bovine fibroblasts were transfected with a DNMT1-specific siRNA under optimised conditions. The expression patterns of DNMT1 were characterised by Q-PCR using the DeltaDeltaC(T) method. The level of DNMT1 was successfully decreased in bovine fibroblast cells using a DNMT1-specific siRNA. Additionally, reduction in the expression of DNMT1 mRNA and DNMT1 protein led to a moderate hypomethylation pattern in the siRNA-treated cells. The use of siRNA-treated cells as donor nuclei during nuclear transplantation induced a reduction in methylation levels compared with controls but did not reduce methylation levels to that of IVF embryos. Further studies are required to determine if this level of reduced methylation is sufficient to improve subsequent development.
19567221
The present study investigated the global pattern of two histone modifications and methylation of DNA during in vitro maturation of bovine oocytes retrieved from follicles of two different sizes (<2 mm and 2-8 mm). The methylation status of histone H3 at position lysine K9 (H3K9 me2), the acetylation status of histone H4 at position lysine K12 (H4K12ac) and the methylation of DNA were assessed by immunocytochemistry. In parallel, the relative abundance of mRNAs coding for proteins specifically involved in reprogramming, including HLA-B associated transcript 8 (G9A), suppressor of variegation 3-9 homolog 1 (SUV39H1), the somatic isoform of DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3b (DNMT3b) and zygote arrest 1 (ZAR1) was determined by RT-PCR. The alpha-H3K9 me2 signal was present in the GV stage and remained detectable until the end of the maturation period. alpha-H4K12ac antibody gave a stronger signal in GV and GVBD oocytes and markedly decreased after GVBD. The signal showing the methylation of DNA was present during the entire maturation period. The five transcripts showed a gene-specific expression profile. Results revealed the global patterns of H3K9 me2, H4K12ac, DNA methylation and the mRNA pool profiles of genes critically involved in epigenetic modifications during bovine oocyte maturation and their possible relationship with the acquisition of oocyte developmental competence and follicular development.
19567217
1. Ghrelin is a multi-functional peptide hormone which affects various processes including growth hormone and insulin release, appetite regulation, gut motility, metabolism and cancer cell proliferation. Ghrelin is produced in the stomach and in other normal and pathological cell types. It may act as an endocrine or autocrine/paracrine factor. 2. This article reviews recent findings in the study of ghrelin and its receptor, which suggest that the ghrelin gene locus may give rise to a number of functional molecules, (peptides and RNA transcripts) in addition to ghrelin. 3. The ghrelin gene encodes a precursor protein, preproghrelin, from which ghrelin and other potentially active peptides are derived by alternative mRNA splicing and/or proteolytic processing. The metabolic role of the peptide obestatin, derived from the preproghrelin C-terminal region, is controversial. However, obestatin has direct effects on cell proliferation. 4. The regulation of ghrelin expression and the mechanisms through which the peptide products arise are unclear. We have recently re-examined the organisation of the ghrelin gene and identified several novel exons and transcripts. One transcript, which lacks the ghrelin-coding region of preproghrelin, contains the coding sequence of obestatin. 5. Furthermore, we have identified an overlapping gene on the antisense strand of ghrelin, GHRLOS, which generates transcripts that may function as non-coding regulatory RNAs or code for novel, short bioactive peptides. 6. The identification of these novel ghrelin-gene related transcripts and peptides raises critical questions regarding their physiological function and their potential role in obesity, diabetes and cancer.
19566830
FASTR3D is a web-based search tool that allows the user to fast and accurately search the PDB database for structurally similar RNAs. Currently, it allows the user to input three types of queries: (i) a PDB code of an RNA tertiary structure (default), optionally with specified residue range, (ii) an RNA secondary structure, optionally with primary sequence, in the dot-bracket notation and (iii) an RNA primary sequence in the FASTA format. In addition, the user can run FASTR3D with specifying additional filtering options: (i) the released date of RNA structures in the PDB database, and (ii) the experimental methods used to determine RNA structures and their least resolutions. In the output page, FASTR3D will show the user-queried RNA molecule, as well as user-specified options, followed by a detailed list of identified structurally similar RNAs. Particularly, when queried with RNA tertiary structures, FASTR3D provides a graphical display to show the structural superposition of the query structure and each of identified structures. FASTR3D is now available online at http://bioalgorithm.life.nctu.edu.tw/FASTR3D/.
19435878
Recently, artificial microRNA (amiRNA) has become a promising RNA interference (RNAi) technology. Here, we describe a flexible and reliable method for constructing both single- and multi-amiRNA expression vectors. Two universal primers, together with two specific primers carrying the encoding sequence of amiRNA were designed and utilized to synthesize the functional amiRNA cassette through a one-step PCR. With appropriate restriction sites, the synthesized amiRNA cassettes can be cloned into any site of different destination vectors. Using the method, we constructed both single- and multi-amiRNA expression vectors to target three reporter genes, which code firefly luciferase (Fluc), enhanced green fluorescent protein (EGFP) and beta-galactosidase (LacZ), respectively. The expressions of three genes were all specifically inhibited by either the corresponding single- or the multi-amiRNA expression vector in 293T cells. And the RNAi efficiency of each amiRNA produced by both single- and multi-amiRNA expression vectors was comparable.
19564946
The room-temperature X-ray structures of ubiquitin (PDB code 1ubq) and of the RNA-binding domain of nonstructural protein 1 of influenza A virus (PDB code 1ail) solved at 1.8 and 1.9 A resolution, respectively, were used to investigate whether a set of conformations rather than a single X-ray structure provides better agreement with both the X-ray data and the observed (13)C(alpha) chemical shifts in solution. For this purpose, a set of new conformations for each of these proteins was generated by fitting them to the experimental X-ray data deposited in the PDB. For each of the generated structures, which show R and R(free) factors similar to those of the deposited X-ray structure, the (13)C(alpha) chemical shifts of all residues in the sequence were computed at the DFT level of theory. The sets of conformations were then evaluated by their ability to reproduce the observed (13)C(alpha) chemical shifts by using the conformational average root-mean-square-deviation (ca-r.m.s.d.). For ubiquitin, the computed set of conformations is a better representation of the observed (13)C(alpha) chemical shifts in terms of the ca-r.m.s.d. than a single X-ray-derived structure. However, for the RNA-binding domain of nonstructural protein 1 of influenza A virus, consideration of an ensemble of conformations does not improve the agreement with the observed (13)C(alpha) chemical shifts. Whether an ensemble of conformations rather than any single structure is a more accurate representation of a protein structure in the crystal as well as of the observed (13)C(alpha) chemical shifts is determined by the dispersion of coordinates, in terms of the all-atom r.m.s.d. among the generated models; these generated models satisfy the experimental X-ray data with accuracy as good as the PDB structure. Therefore, generation of an ensemble is a necessary step to determine whether or not a single structure is sufficient for an accurate representation of both experimental X-ray data and observed (13)C(alpha) chemical shifts in solution.
19564690
A complete tree with roots, trunk and crown remains an appropriate model to represent all steps of life's development, from the emergence of a unique genetic code up to the last universal common ancestor and its further radiation. Catalytic closure of a mixture of prebiotic polymers is a heuristic alternative to the RNA world. Conjectures about emergence of life in an infinite multiverse should not confuse probability with possibility.
19524037
In eukaryotes, selective derepression of mRNA translation through altered utilization of upstream open reading frames (uORFs) or internal ribosomal entry sites (IRES) regulatory motifs following exposure to stress is regulated at the initiation stage through the increased phosphorylation of the eukaryotic initiation factor 2 on its alpha subunit (eIF2alpha). While there is only one known eIF2alpha kinase in yeast, general control nonderepressible 2 (GCN2), mammals have evolved to express at least four: GCN2, heme-regulated inhibitor kinase (HRI), double-stranded RNA-activated protein kinase (PKR) and PKR-like ER-resident kinase (PERK). So far, the main known distinction among these four kinases is their activation in response to different acute stressors. In the present study, we used the in situ perfused mouse liver model and hybridization array analyses to assess the general translational response to stress regulated by two of these kinases, GCN2 and PERK, and to differentiate between the downstream effects of activating GCN2 versus PERK. The resulting data showed that at least 2.5% of mouse liver mRNAs are subject to derepressed translation following stress. In addition, the data demonstrated that the eIF2alpha kinases, GCN2 and PERK, differentially regulate mRNA transcription and translation, which in the latter case suggests that increased eIF2alpha phosphorylation is not sufficient for derepression of translation. These findings open an avenue for more focused future research towards groups of mRNAs that code for the early cellular stress response proteins. Key words: mRNA translation, ER stress, amino acid deprivation, eIF2 phosphorylation.
19509078
BACKGROUND: High-throughput molecular approaches for gene expression profiling, such as Serial Analysis of Gene Expression (SAGE), Massively Parallel Signature Sequencing (MPSS) or Sequencing-by-Synthesis (SBS) represent powerful techniques that provide global transcription profiles of different cell types through sequencing of short fragments of transcripts, denominated sequence tags. These techniques have improved our understanding about the relationships between these expression profiles and cellular phenotypes. Despite this, more reliable datasets are still necessary. In this work, we present a web-based tool named S3T: Score System for Sequence Tags, to index sequenced tags in accordance with their reliability. This is made through a series of evaluations based on a defined rule set. S3T allows the identification/selection of tags, considered more reliable for further gene expression analysis. RESULTS: This methodology was applied to a public SAGE dataset. In order to compare data before and after filtering, a hierarchical clustering analysis was performed in samples from the same type of tissue, in distinct biological conditions, using these two datasets. Our results provide evidences suggesting that it is possible to find more congruous clusters after using S3T scoring system. CONCLUSION: These results substantiate the proposed application to generate more reliable data. This is a significant contribution for determination of global gene expression profiles. The library analysis with S3T is freely available at http://gdm.fmrp.usp.br/s3t/. S3T source code and datasets can also be downloaded from the aforementioned website.
19500384
The Basic Local Alignment Search Tool (BLAST) is a keystone of bioinformatics due to its performance and user-friendliness. Beginner and intermediate users will learn how to design and submit blastn and Megablast searches on the Web pages at the National Center for Biotechnology Information. We map nucleic acid sequences to genomes, find identical or similar mRNA, expressed sequence tag, and noncoding RNA sequences, and run Megablast searches, which are much faster than blastn. Understanding results is assisted by taxonomy reports, genomic views, and multiple alignments. We interpret expected frequency thresholds, biological significance, and statistical significance. Weak hits provide no evidence, but hints for further analyses. We find genes that may code for homologous proteins by translated BLAST. We reduce false positives by filtering out low-complexity regions. Parsed BLAST results can be integrated into analysis pipelines. Links in the output connect to Entrez, PUBMED, structural, sequence, interaction, and expression databases. This facilitates integration with a wide spectrum of biological knowledge.
19496060
The origin of the genetic code in the context of an RNA world is a major problem in the field of biophysical chemistry. In this paper, we describe how the polymerization of amino acids along RNA templates can be affected by the properties of both molecules. Considering a system without enzymes, in which the tRNAs (the translation adaptors) are not loaded selectively with amino acids, we show that an elementary translation governed by a Michaelis-Menten type of kinetics can follow different polymerization regimes: random polymerization, homopolymerization and coded polymerization. The regime under which the system is running is set by the relative concentrations of the amino acids and the kinetic constants involved. We point out that the coding regime can naturally occur under prebiotic conditions. It generates partially coded proteins through a mechanism which is remarkably robust against non-specific interactions (mismatches) between the adaptors and the RNA template. Features of the genetic code support the existence of this early translation system.
19492048
Targeted gene silencing by RNA interference allows the study of gene function in plants and animals. In cell culture and small animal models, genetic screens can be performed-even tissue-specifically in Drosophila-with genome-wide RNAi libraries. However, a major problem with the use of RNAi approaches is the unavoidable false-positive error caused by off-target effects. Until now, this is minimized by computational RNAi design, comparing RNAi to the mutant phenotype if known, and rescue with a presumed ortholog. The ultimate proof of specificity would be to restore expression of the same gene product in vivo. Here, we present a simple and efficient method to rescue the RNAi-mediated knockdown of two independent genes in Drosophila. By exploiting the degenerate genetic code, we generated Drosophila RNAi Escape Strategy Construct (RESC) rescue proteins containing frequent silent mismatches in the complete RNAi target sequence. RESC products were no longer efficiently silenced by RNAi in cell culture and in vivo. As a proof of principle, we rescue the RNAi-induced loss of function phenotype of the eye color gene white and tracheal defects caused by the knockdown of the heparan sulfate proteoglycan syndecan. Our data suggest that RESC is widely applicable to rescue and validate ubiquitous or tissue-specific RNAi and to perform protein structure-function analysis.
19483100