Eukaryotic Elongation Research Articles

Tilletia puccinelliae, a new species of reticulate-spored bunt fungus infecting Puccinellia distans

A shipment of Fults alkaligrass seed (Puccinellia distans) grown in Washington state containing bunted florets was intercepted by quarantine officials at China’s Tianjin Entry-Exit Quarantine and Inspection Bureau. The bunted florets were filled with irregularly shaped, reticulately ornamented teliospores that germinated in a manner characteristic of systemically infecting Tilletia spp. on grass hosts in subfamily Pooideae. Based on morphological characters and a multigene phylogenetic analysis of the ITS region rDNA, eukaryotic translation elongation factor 1 alpha and a region of the second largest subunit of RNA polymerase II including a putative intein, the Puccinellia bunt is genetically distinct from known species of Tilletia and is proposed as a new species, T. puccinelliae.

Generation of a Zinc Finger Protein ZPR1 Mutant That Constitutively Interacted with Translation Elongation Factor 1α

Zinc finger protein ZPR1 (ZPR1) binds to eukaryotic translation elongation factor 1alpha (eEF1alpha) in response to growth stimuli, and is also involved in transcription and cell cycle regulation. In this study, we characterized the interaction of ZPR1 and eEF1alpha and generated a ZPR1 mutant that constitutively interacted with eEF1alpha. ZPR1-DeltaA (Delta193-246) bound to eEF1alpha independently of Zn(2+) in vivo. This study indicates that ZPR1-DeltaA (Delta193-246) is a useful tool to provide structural insights into ZPR1 and to investigate the biological significance of the interaction between ZPR1 and eEF1alpha.

Influence of cation–π interactions to the structural stability of prokaryotic and eukaryotic translation elongation factors

We have investigated the role of cation-pi interactions on translation elongation factors. In our investigation, an average of four significant cation-pi interactions were found, that is, an average of one cation-pi interaction per 44 residues in the ten elongation factors were observed. The analysis on the influence of short (< + or - 4), medium (> + or - 4 to < + or - 20) and long (>20) range contacts showed that cation-pi interactions are mainly formed by medium and long-range contacts. Arg-Tyr pair was found largest in number but energetic contribution of Arg-Trp pair was found most. Preferred secondary structural conformation analysis of the residues involved in cation-pi interaction indicates that the cationic Arg prefers to be in helix and Lys having equal probability for helix and strand, whereas the aromatic Phe and Trp were found mostly in helix while Tyr in strand regions. The cation-pi interaction residues involved in these proteins were found highly conserved with 48.86% residues having conservation score of > or = 6. Analysis of secondary structure preference of the energetically significant cation-pi residues in different solvent accessible range indicates that most of the pi residues are found buried or partially buried whereas cationic residues were found mostly at the protein surface. The results presented in this study will be useful for structural stability studies in translation elongation factors.

PROCOV: maximum likelihood estimation of protein phylogeny under covarion models and site-specific covarion pattern analysis

BackgroundThe covarion hypothesis of molecular evolution holds that selective pressures on a given amino acid or nucleotide site are dependent on the identity of other sites in the molecule that change throughout time, resulting in changes of evolutionary rates of sites along the branches of a phylogenetic tree. At the sequence level, covarion-like evolution at a site manifests as conservation of nucleotide or amino acid states among some homologs where the states are not conserved in other homologs (or groups of homologs). Covarion-like evolution has been shown to relate to changes in functions at sites in different clades, and, if ignored, can adversely affect the accuracy of phylogenetic inference.ResultsPROCOV (protein covarion analysis) is a software tool that implements a number of previously proposed covarion models of protein evolution for phylogenetic inference in a maximum likelihood framework. Several algorithmic and implementation improvements in this tool over previous versions make computationally expensive tree searches with covarion models more efficient and analyses of large phylogenomic data sets tractable. PROCOV can be used to identify covarion sites by comparing the site likelihoods under the covarion process to the corresponding site likelihoods under a rates-across-sites (RAS) process. Those sites with the greatest log-likelihood difference between a 'covarion' and an RAS process were found to be of functional or structural significance in a dataset of bacterial and eukaryotic elongation factors.ConclusionCovarion models implemented in PROCOV may be especially useful for phylogenetic estimation when ancient divergences between sequences have occurred and rates of evolution at sites are likely to have changed over the tree. It can also be used to study lineage-specific functional shifts in protein families that result in changes in the patterns of site variability among subtrees.

Identifying stable reference genes for evaluation of antioxidative enzyme gene expression in auditory cortex and cochlea of young and old Fischer 344 rats

Conclusion. This study suggests that the validation of reference genes is important for the accurate evaluation of differential gene expression in age-related hearing loss (AHL). Objectives. To identify the most stable reference gene in the auditory cortex and cochlea of young and old rats. Materials and methods. mRNA levels of some common reference genes such as β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C, hypoxanthine phosphoribosyl-transferase and eukaryotic translation elongation factor (EF) were quantified in the auditory cortex and cochlea of young and old rats by quantitative RT-PCR, and then their stability was evaluated using GeNorm and Normfinder software. Results. There were some variations in the expression of reference genes found in the auditory cortex and cochlea of rats, wherein EF and GAPDH were identified as the most stable reference genes in auditory cortex and cochlea, respectively. mRNA levels of Cu/Zn-superoxide dismutase (SOD), Mn-SOD, catalase and glutathione peroxidase in the auditory cortex and cochlea do not vary significantly in young and old rats, when they are normalized with EF or GAPDH, the valid reference genes, respectively. However, Cu/Zn-SOD and catalase levels differ significantly in the auditory cortex when they are normalized with β-actin, an invalid reference gene, resulting in the misinterpretation of the target gene expression levels.

Immunoscreening of urinary bladder cancer cDNA library and identification of potential tumor antigen

To identify potential tumor antigen by immunoscreening the urinary bladder cancer cDNA library with monoclonal antibodies. Monoclonal antibodies were prepared. A cDNA expression library was constructed from bladder cancer cell line BLZ211. Immunogenic proteins were identified by immunoscreening the cDNA library with ten monoclonal antibodies. The cDNA library of BLZ211 cells was established using lambdaZAP as vector. The titer of unamplified cDNA library was 1.39x10(6) pfu/ml with a recombinant rate of 97.72%, and titer of amplified one was 8.4x10(9) pfu/ml. After immunoscreening, ten positive clones representing ten different antigens were identified, which include two proteins with unknown function; coactosin-like 1, eukaryotic translation elongation factor, HNRPA1, histidine triad nucleotide binding protein, KRT7, LCN2, TSTA3, zinc finger protein, C11orf48 and HSPC148. The cDNA library was of high quality and can be used in further study. By immunoscreening the bladder cancer cDNA library with ten monoclonal antibodies, we identified ten immunogenic proteins that otherwise would not have been identified as potential diagnostic marker and vaccinogens of bladder cancer using the gene discovery effort.

Дослідження експресії різних субодиниць еукаріотного фактора елонгації трансляції eEF1 у гліальних пухлинах головного мозку людини

Дослідження експресії різних субодиниць еукаріотного фактора елонгації трансляції eEF1 у гліальних пухлинах головного мозку людини

Effect of acute respiratory acidosis on translation signaling in skeletal muscle

We previously demonstrated that acute respiratory acidosis depresses protein synthesis by approximately 22% in skeletal muscle. However, the molecular mechanisms of the depressive effect of acidosis on muscle protein synthesis are not well known. In this study, we examined the effect of acidosis on the regulation of mRNA translation initiation and elongation by assessing the phosphorylation states of the translation repressor 4E‐BP1, eukaryotic initiation factor 2 (eIF2) and eukaryotic elongation factor‐2 (eEF‐2). Rats (n = 10) were made acidotic by increasing the CO2 content of inspired air to 12% for 1 h. Similar animals breathing normal air served as controls (n=10). Gastrocnemious muscles were harvested and the phosphorylation states of 4E‐BP1, eIF2 and eEF‐2 were assessed by immunoblot analysis. The results showed that 4E‐BP1 phosphorylation (γ‐form/total) was 28% lower in acidotic animals (P &lt; 0.05), while no effect of acidosis could be detected on the phosphorylation status of eIF2 (p‐Ser51/total) and eEF‐2 (p‐Thr56/total) (P = NS). The results suggest that the depression of muscle protein synthesis by acute respiratory acidosis may be mediated in part by downregulation of the mRNA binding step in translation initiation.Supported by NIH grants DK54991 and 5‐MO1‐RR‐10710

Proteomic analysis of 6-nitrotryptophan-containing proteins in peroxynitrite-treated PC12 cells

Abstract. We identified 6-nitrotryptophan-containing proteins in peroxynitrite-treated PC12 cell, a neuron-like cell, by proteomic analysis using a specific polyclonal antibody which we have developed by using 6-nitrotryptophan-containing synthetic peptides as an antigen. Western blot analysis showed nine immunoreactive spots of newly appeared or significantly increased signals in the peroxynitrite-treated cells compared with that of the untreated cells in two-dimensional gel electrophoresis. We analyzed the proteins corresponding to these spots by trypsin digestion and LC-MS/MS. These spots contained seven proteins such as M2 pyruvate kinase, mitochondrial aconitase 2, eukaryotic translation elongation factor 2, pyruvate carboxylase, sarcosine dehydrogenase, vinculin and heat-shock protein 90α. Several proteins contained functional tryptophan residues that interact with other molecules. We speculate that the nitration of tryptophan residues in the neuron-like cell during neuroinflammation may affect cellular energy metabolism, protein synthesis, and stress response and may modulate the specific interaction of proteins with other molecules.

Transgene Persistence and Cell Turnover in the Diarthrodial Joint: Implications for Gene Therapy of Chronic Joint Diseases

Local gene therapy for chronic joint diseases requires prolonged transgenic expression, but this has not been reliably achieved in animal models. Using normal and immunocompromised animals, we examined the capacity of various cell types in joint tissues to maintain and express exogenous transgenes after direct intra-articular gene delivery. We found that transgenic expression could persist for the lifetime of the animal but required precise immunological compatibility between the vector, transgene product, and host. It was not dependent on vector integration or promoter origin. We identified two phenotypically distinct sub-populations of genetically modified cells within the joint: (i) transient cells, with a half-life of a few weeks, and (ii) stable cells that reside in the joint tissues indefinitely. Contrary to the prevailing assumption, the transient sub-population was composed almost exclusively of synovial fibroblasts, indicating that the synovium is not an appropriate tissue upon which to base a long-term therapy. Instead, fibroblasts in the ligaments, tendons, and capsule emerged as the primary cell types capable of sustained therapeutic transgene expression. This study sheds new light on the cellular dynamics of articular tissues and suggests that cell turnover and immune reactivity are the key determinants in achieving sustained transgenic expression intra-articularly.

Novel Radiation Response Genes Identified in Gene-Trapped MCF10A Mammary Epithelial Cells

We have used a gene-trapping strategy to screen human mammary epithelial cells for radiation response genes. Relative mRNA expression levels of five candidate genes in MCF10A cells were analyzed, both with and without exposure to radiation. In all five cases, the trapped genes were significantly down-regulated after radiation treatment. Sequence analysis of the fusion transcripts identified the trapped genes: (1) the human androgen receptor, (2) the uncharacterized DREV1 gene, which has known homology to DNA methyltransferases, (3) the human creatine kinase gene, (4) the human eukaryotic translation elongation factor 1 beta 2, and (5) the human ribosomal protein L27. All five genes were down-regulated significantly after treatment with varying doses of ionizing radiation (0.10 to 4.0 Gy) and at varying times (2-30 h after treatment). The genes were also analyzed in human fibroblast and lymphoblastoid cell lines to determine whether the radiation response being observed was cell-type specific. The results verified that the observed radiation response was not a cell-type-specific phenomenon, suggesting that the genes play essential roles in the radiation damage control pathways. This study demonstrates the potential of the gene-trap approach for the identification and functional analysis of novel radiation response genes.

260 COMPARISON OF HOUSEKEEPING GENE EXPRESSION IN INDIVIDUAL 8-CELL STAGE MOUSE EMBRYOS PRODUCED UNDER DIFFERENT CONDITIONS

Real-time PCR has the potential to accurately quantify the mRNA level of selected genes in single cells and individual pre-implantation-stage embryos. The goal of our study was to examine the variations in gene expression within individual embryos of the same stage and between embryos of the same stage but from different sources. In our study, we determined expression level of the 7 most commonly used housekeeping genes in 8-cell-stage mouse embryos produced under different culture conditions. Messenger RNA of 6 embryos each that was derived in vivo, or cultured in vitro from the zygote stage, or derived from oocytes activated parthenogenetically and developed in vitro were extracted individually followed by reverse transcription into cDNA. Optimized real-time PCR was performed for cytoplasmic beta-actin (Actb), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), H2A histone family, member Z (H2afz), hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1), ubiquitin C (Ubc), peptidylprolyl isomerase A (cyclophilin A) (Ppia), and eukaryotic translation elongation factor 1 epsilon 1 (Eef1e1) genes. The results were analyzed, and the percentage standard error of the mean relative expression value was compared for all genes. All 7 genes were presented above the detection limit in all samples. One or two individual embryos showed 2- to 4-fold higher mRNA levels than the average for all genes in the group. The embryos cultured in vitro showed much higher expression levels of H2afz, Ppia, and Eef1e1 genes than those in the in vivo group. The parthenogenetic group was similar to the in vivo group in expression of Actb, H2afz, Hprt, and Eef1e1 genes, but showed significant differences (P &amp;lt; 0.05; Student's t-test) compared to the in vitro group (Table 1). The percent standard error of the mean decreased gradually as the number of samples was increased. The 6 individual embryos in similar groups showed relatively low variability compared to embryos at similar stage but produced in different conditions. Interestingly, the parthenogenetic embryos showed a level of gene expression comparable to that of the in vivo ones, notwithstanding their culture in vitro. In conclusion, morphological observations and similarity in developmental stage alone cannot guarantee the uniformity of embryo samples, and a minimum of 4–6 replicates per treatment is needed. Moreover, we showed that culture condition itself has an effect on housekeeping gene expression, which, if neglected, might result in misinterpretation of data. Table 1.Relative expression values of the different culture groups (mean ±SE; n =6 embryos) This work was supported by EU FP6 (MEXT-CT-2003-509582 and 518240), Wellcome Trust (Grant No. 070246), and Hungarian National Science Fund (OTKA) (Grant No. T046171).

Сравнительный анализ функциональной активности нативной и модифицированной трипсином форм эукариотического фактора элонгации трансляции 1А

Сравнительный анализ функциональной активности нативной и модифицированной трипсином форм эукариотического фактора элонгации трансляции 1А

Получение и характеристика моноклональных антител против эукариотического трансляционного фактора элонгации 1А

Получение и характеристика моноклональных антител против эукариотического трансляционного фактора элонгации 1А

Изучение различных физико-химических характеристик неканонического комплекса эукариотического фактора элонгации eEF-lA с деацилированной тРНК

Изучение различных физико-химических характеристик неканонического комплекса эукариотического фактора элонгации eEF-lA с деацилированной тРНК

Purification of eukaryotic cytoplasmic elongation factor 2 and organellar elongation factor G by an affinity binding procedure

A rapid affinity binding procedure for obtaining highly purified organellar elongation factor G (EF-G) and cytoplasmic elongation factor 2 (EF-2) in excellent yields from whole cell extracts is described. The procedure involves the addition of ribosomes, GTP, and fusidic acid to a crude cell extract. The fusidic acid stabilizes the formation of a translocase—GDP—ribosome complex which can be recovered from the extract by high-speed centrifugation. The translocase is then released from the ribosomes by a high salt wash. To purify organellar EF-G, 70 S ribosomes from Escherichia coli are used. If 80 S ribosomes such as those from wheat germ are used instead of 70 S ribosomes, EF-2 can be selectively purified from the cell extract. This procedure has been applied to the purification of both cytoplasmic and chloroplast translocases of Euglena gracilis . Chloroplast EF-G and cytoplasmic EF-2 can be separated from each other and from the vast majority of the proteins in the postribosomal supernatant with yields in both cases of 60% or greater.