Translation Elongation Factor EF-1 Alpha Research Articles

Fusarium massalimae sp. nov. (F. lateritium species complex) occurs endophytically in leaves of Handroanthus chrysotrichus

Fusarium isolates obtained as endophytes from healthy leaves of Handroanthus chrysotrichus (Bignoniaceae) in the Atlantic Forest of north-eastern Brazil were characterised morphologically and phylogenetically on the basis of partial rDNA sequences encompassing the second largest subunit of RNA polymerase II, translation elongation factor EF-1 alpha, and β-tubulin genes. A novel species within the Fusarium lateritium species complex (FLSC) is described as Fusarium massalimae. Isolates of F. pseudocircinatum of the Fusarium fujikuroi species complex (FFSC) were also isolated as endophytes from leaves of H. chrysotrichus. Mating type gene analyses suggested that obtained isolates of F. massalimae and F. pseudocircinatum are heterothallic.

Pseudoplagiostoma myracrodruonis (Pseudoplagiostomataceae, Diaporthales): a new endophytic species from Brazil

Two initially unidentifiable isolates were found when endophytic fungi from healthy leaves of Myracrodruon urundeuva (Anacardiaceae) were being investigated in Brazil. Based on phylogenetic analyses using internal transcribed spacer (ITS) and partial large subunit (LSU) of ribosomal DNA, RNA polymerase II (RPB2), translation elongation factor EF-1 alpha (TEF1), and tubulin (TUB2), the isolates were identified as members of the family Pseudoplagiostomataceae (Diaporthales). In this paper, we introduce a new species, Pseudoplagiostoma myracrodruonis, thus accepting eight species in Pseudoplagiostoma (Pseudoplagiostomataceae). The new species is proposed to accommodate endophytes characterized by a coelomycetous asexual morph, pycnidial conidiomata with lageniform to ampulliform and hyaline to pale brown phialidic conidiogenous cells that produce unicellular, ellipsoid to oblong-cylindrical, hyaline, and guttulate conidia. In the current phylogenetic analysis, P. myracrodruonis is placed in a well-supported clade in Pseudoplagiostoma (Pseudoplagiostomataceae). A discussion about the relationship of Pseudoplagiostoma species is included in the paper.

Phylogeny of the genus Fuscoporia and taxonomic assessment of the F. contigua group

ABSTRACTThe genus Fuscoporia of the Hymenochaetaceae is characterized by resupinate to pileate basidiocarps, a dimitic hyphal system with fine crystal aggregates and encrusted generative hyphae in dissepiment edge and tube trama, the presence of hymenial setae, and hyaline, thin-walled, smooth basidiospores. Members of the F. contigua group are easy to distinguish from other species of Fuscoporia because of the moderately large pores, presence of mycelial setae, and large hymenial setae. Here, we explore phylogenetic relationships among 20 species of Fuscoporia based on examination of some 90 collections sampled worldwide. Seven new species are recognized in the F. contigua group—F. americana, F. centroamericana, F. costaricana, F. latispora, F. monticola, F. septiseta, and F. sinica—described from China, Costa Rica, Mexico, and the United States. Phylogenetic analyses inferred from DNA sequences of the nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS), D1–D2 domains of nuc 28S rDNA (28S), and translation elongation factor EF-1 alpha (tef1) support the F. contigua group as one of two major clades within Fuscoporia comprising nine species worldwide.

Efficient establishment of pure cultures of yellow chanterelle Cantharellus anzutake from ectomycorrhizal root tips, and morphological characteristics of ectomycorrhizae and cultured mycelium

Abstract Species of fleshy yellow Cantharellus are known as chanterelles, which are among the most popular wild edible mycorrhizal mushrooms in the world. However, pure culture isolates of Cantharellus are rare. We report an efficient isolation technique of the Japanese golden chanterelle, Cantharellus anzutake, from its ectomycorrhizal root tips. Field-sampled fresh ectomycorrhizal root tips of C. anzutake on various hosts such as pines, spruce, and oaks were vortexed with 0.005% Tween 80 solution, surface sterilized with 1% calcium hypochlorite solution, rinsed with sterilized distilled water, and placed on modified Norkrans’ C (MNC) agar plate medium. Most ectomycorrhizal root tips of C. anzutake produced yellowish mycelial colonies within a few months. In contrast, tissue isolation from basidiomata provided limited cultures of C. anzutake but much contamination of bacteria and molds, even on media that contained antibiotics. The established C. anzutake cultures had clamp connections on the hyphae and contained intracellular oily droplets. These cultured isolates were identified as C. anzutake by sequence analysis of the rRNA internal transcribed spacer (ITS) region and translation elongation factor EF1-alpha (tef-1) genes.

Extract of Syzygium aromaticum suppress eEF1A protein expression and fungal growth.

Clove extract has therapeutic potential as an antifungal drug, yet the mechanism of action remains ambiguous. Current study aimed to address the molecular process of the antifungal activity exerted by clove extract. The antifungal assay results showed that clove extract had some effects on all of the tested yeast. Propidium iodide staining assay showed cell membrane damage in Saccharomyces cerevisiae after treatment of clove extract for 30h. Interestingly, SDS-polyacrylamide gel electrophoresis assays revealed that the protein expression of eukaryotic elongation factor 1 alpha (eEF1A) was suppressed significantly after treatment with clove extract (not pure eugenol). Transcriptional analyses revealed that the TEF1 and TEF2 genes (translation elongation factor EF-1 alpha) encoding eEF1A were not disturbed with the addition of clove extract; however, the expression of related genes EFB1 (translation elongation factor 1 subunit beta), ENO2 (phosphopyruvate hydratase ENO2), GSP1 (Ran GTPase GSP1), RPP0 (ribosomal protein P0), YEF3 (translation elongation factor EF-3), TEF4 (translation elongation factor EF1B gamma), and RPS2 (ribosomal 40S subunit protein S2) increased significantly. These results suggest that clove extract plays a role in disrupting growth and affecting yeast metabolism. eEF1A was affected by clove extract at the protein level but not at the transcriptional level. This study is based on a detailed study of antifungal action exerted by clove extract, and proposed that down-regulation of eEF1A protein expression may contribute to its antifungal activity. These results may have clinical significance for future application of clove extract as a natural antifungal agent.

Scp160p is required for translational efficiency of codon-optimized mRNAs in yeast

The budding yeast multi-K homology domain RNA-binding protein Scp160p binds to >1000 messenger RNAs (mRNAs) and polyribosomes, and its mammalian homolog vigilin binds transfer RNAs (tRNAs) and translation elongation factor EF1alpha. Despite its implication in translation, studies on Scp160p's molecular function are lacking to date. We applied translational profiling approaches and demonstrate that the association of a specific subset of mRNAs with ribosomes or heavy polysomes depends on Scp160p. Interaction of Scp160p with these mRNAs requires the conserved K homology domains 13 and 14. Transfer RNA pairing index analysis of Scp160p target mRNAs indicates a high degree of consecutive use of iso-decoding codons. As shown for one target mRNA encoding the glycoprotein Pry3p, Scp160p depletion results in translational downregulation but increased association with polysomes, suggesting that it is required for efficient translation elongation. Depletion of Scp160p also decreased the relative abundance of ribosome-associated tRNAs whose codons show low potential for autocorrelation on mRNAs. Conversely, tRNAs with highly autocorrelated codons in mRNAs are less impaired. Our data indicate that Scp160p might increase the efficiency of tRNA recharge, or prevent diffusion of discharged tRNAs, both of which were also proposed to be the likely basis for the translational fitness effect of tRNA pairing.

A review of GermanScedosporium prolificanscases from 1993 to 2007

Scedosporium prolificans is one of the most life-threatening fungal opportunistic pathogens due to its high resistance to common systemic antifungal agents. While a close relative of Pseudallescheria boydii, S. prolificans has a more limited geographic range being primarily found in Australia, USA and Spain. Infections have also been reported from several other European countries and from Chile. Twenty patients with Scedosporium prolificans infection or colonization from August 1993 to May 2007 were retrospectively reviewed in Germany. They had all been identified at or reported to the Reference Laboratory for Pseudallescheria/Scedosporium spp. in Berlin. Twelve of 13 patients with haematological disorders and/or on immunosuppressive therapy developed a fatal invasive scedosporiosis. Colonization of the respiratory tract was reported for one patient after heart-lung-transplantation, all six patients with cystic fibrosis and one with chronic sinusitis. Molecular studies of the S. prolificans isolates confirmed that parts of the 18S, the Internal Transcribed Spacer (ITS) regions and the D1/D2 domain of the 28S region of rDNA are monomorphic. However, sequencing of parts of the translation elongation factor EF1-alpha (EF-1alpha) and the chitin synthase (CHS-1) genes revealed the presence of three and two distinct genotypes, respectively. Two informative mutations were found in EF-1alpha and a single nucleotide exchange in the CHS-1 gene.

In vitro antagonism of Thielaviopsis paradoxa by Trichoderma longibrachiatum

Seventy-nine Trichoderma strains were isolated from soil taken from 28 commercial plantations of Agave tequilana cv. 'Azul' in the State of Jalisco, Mexico. Nine of these isolates produced nonvolatile metabolites that completely inhibited the growth of Thielaviopsis paradoxa on potato dextrose agar plates. These isolates were identified as Trichoderma longibrachiatum on the basis of their morphology and DNA sequence analysis of two genes (ITS rDNA and translation elongation factor EF-1alpha). Mycoparasitism of Th. paradoxa by T. longibrachiatum strains in dual cultures was examined by scanning electron microscopy. The Trichoderma hyphae grew alongside the Th. paradoxa hyphae, but penetration of Thielaviopsis hyphae by Trichoderma was no apparent. Aleurioconidia of Th. paradoxa were parasitized by Trichoderma. Both hyphae and aleurioconidia of Th. paradoxa lost turgor pressure, wrinkled, collapsed and finally disintegrated. In liquid cultures, all nine Trichoderma isolates produced proteases, beta-1,3-glucanases and chitinases that would be responsible for the degradation of Thielaviopsis hyphae. These results demonstrate that the modes of action of T. longibrachiatum involved against Th. paradoxa in vitro experiments are mycoparasitism and the production of nonvolatile toxic metabolites.

Functional and physical interactions of Krr1p, a Saccharomyces cerevisiae nucleolar protein.

The Krr1 protein of Saccharomyces cerevisiae is involved in processing of pre-rRNA and assembly of pre-ribosomal 40S subunits. To further investigate the function of Krr1p we constructed a conditional cold sensitive mutant krr1-21, and isolated seven genes from Schizosaccharomyces pombe whose products suppressed the cold sensitive phenotype of krr1-21 cells. Among the multicopy suppressors we found genes coding for translation elongation factor EF-1alpha, a putative ribose methyltransferase and five genes encoding ribosomal proteins. Using the tandem affinity purification (TAP) method we identified thirteen S. cerevisiae ribosomal proteins interacting with Krr1p. Taken together, these results indicate that Krr1p interacts functionally as well as physically with ribosomal proteins. Northern blot analysis revealed that changes in the level of krr1-21 mRNA were accompanied by similar changes in the level of mRNAs of genes encoding ribosomal proteins. Thus, Krr1p and the genes encoding ribosomal proteins it interacts with seem to be coordinately regulated at the level of transcription.

Functional and physical interactions of Krr1p, a Saccharomyces cerevisiae nucleolar protein.

The Krr1 protein of Saccharomyces cerevisiae is involved in processing of pre-rRNA and assembly of pre-ribosomal 40S subunits. To further investigate the function of Krr1p we constructed a conditional cold sensitive mutant krr1-21, and isolated seven genes from Schizosaccharomyces pombe whose products suppressed the cold sensitive phenotype of krr1-21 cells. Among the multicopy suppressors we found genes coding for translation elongation factor EF-1alpha, a putative ribose methyltransferase and five genes encoding ribosomal proteins. Using the tandem affinity purification (TAP) method we identified thirteen S. cerevisiae ribosomal proteins interacting with Krr1p. Taken together, these results indicate that Krr1p interacts functionally as well as physically with ribosomal proteins. Northern blot analysis revealed that changes in the level of krr1-21 mRNA were accompanied by similar changes in the level of mRNAs of genes encoding ribosomal proteins. Thus, Krr1p and the genes encoding ribosomal proteins it interacts with seem to be coordinately regulated at the level of transcription.

An expression system for the functional analysis of pheromone genes in the tetrapolar basidiomycete Schizophyllum commune.

The investigation of putative pheromone genes of basidiomycetes has been difficult since the small open reading frames are essentially annotated on the basis of a C-terminal farnesylation signal. In order to identify the functional reading frame, expression of small DNA fragments in the fungus is necessary. The expression system developed in the presented paper allows fusion to the promoter of the tef1 gene encoding the constitutively and highly expressed translation elongation factor EF1alpha. This system has been shown to be functional using an easily selectable gene, ura1. The application to identification of functional pheromone genes has been shown with the newly detected bap2(4) gene. The Bap2(4) pheromone is the first Balpha pheromone gene activating only a single receptor specificity.

The root of the tree of life in the light of the covarion model.

A few duplicated genes have been found useful to root the universal tree of life. Despite controversial results, the consensus led to locate the root in the eubacterial branch. However, we demonstrated (Philippe and Forterre 1999) that all these markers were in fact unsuitable for any firm conclusion, mainly because of their high level of mutational saturation, which masks a major part of the phylogenetic signal. But then, the very persistence of signal for events as early as the separation of the three domains becomes puzzling. This paradox was studied here for translation elongation factor proteins, EF-1alpha and EF-2, which appeared to be one of the least confusing markers. We showed that these proteins do not conform to a classical rate-across-sites pattern, as those modeled by a gamma law, but rather to a covarion-based model, because the evolutionary rate of a given position often changes between taxonomic groups. Conservation of the very ancient signal can thus be better explained by the covarion model: a substitution can occur in deep branches, and the position remains constant afterward, as "fossilized" by a change of covation. As no reconstruction method has up to now taken into account this complex model, we devised a simple method for extracting the phylogenetic signal, by considering the variability of sequence positions within predefined phylogenetic groups. We showed that noise quantitatively prevailed upon signal. Parsimony will produce erroneous topologies, because it has to minimize primarily the number of steps of the noise. In contrast, our method effectively concentrated the signal and was more suitable for inferring ancient events. We consequently found the eubacterial rooting to be presumably due to a long branch attraction artifact, because of the higher evolutionary rate of Eubacteria for these proteins. Among the two other rooting possibilities, the eukaryotic rooting appeared to be more supported, although not enough to be conclusive.

Integrative transformation of the dimorphic yeast arxula adeninivorans LS3 based on hygromycin B resistance.

A transformation system has been developed for the dimorphic yeast Arxula adeninivorans based on a stable integration of the donor DNA into ribosomal DNA. For this purpose a cassette was constructed which contains the E. coli hph gene, conferring hygromycin B resistance, fused to the 5' expression signals of the A. adeninivorans TEF1 gene, encoding the translation elongation factor EF-1alpha, and the transcription termination region of the Saccharomyces cerevisiae PHO5 gene. This cassette was fused into the 25S rDNA of A. adeninivorans. Linearization of this vector was required for high transformation frequencies. The vector was integrated in multiple copies into the 25S rDNA by homologous recombination. Copy number was not altered even after the growth of transformants for 15 generations under non-selective growth conditions. Microscopical analyses revealed that integration of the transformed plasmid did not influence the dimorphism, which is triggered at 42 degrees C for both transformed and non-transformed cells.

Deregulated expression of the PU.1 transcription factor blocks murine erythroleukemia cell terminal differentiation.

Murine erythroleukemia (MEL) cells are transformed erythroid precursors that are blocked from completing the late stages of erythroid differentiation. A frequent event in the generation of these malignant cells is deregulation of the hematopoietic-specific transcription factor PU.1 (Spi-1) by retroviral insertion of the spleen-focus-forming virus component of Friend virus. During chemically induced reinitiation of MEL cell terminal differentiation, expression of PU.1 is rapidly down-regulated, suggesting that PU.1 might interfere with processes required for terminal differentiation of erythroid precursors. To investigate the role of PU.1 in erythroid differentiation we transfected MEL cells with a PU.1 cDNA controlled by the eucaryotic translation elongation factor EF1 alpha promoter. Deregulated expression of PU.1 blocked chemically induced differentiation and terminal cell division. Deregulated expression of two other protooncogenes, c-myc and c-myb, also has been shown to block MEL differentiation. We present evidence that PU.1 inhibits terminal differentiation at an earlier step than c-Myc and c-Myb. Thus reinitiation of MEL cell terminal differentiation appears to be controlled by an ordered program of turning off several protooncogenes. Down-regulation of PU.1 may be a very early step in this program.

Didemnin binds to the protein palmitoyl thioesterase responsible for infantile neuronal ceroid lipofuscinosis.

The marine natural product didemnin B, currently in clinical trials as an antitumor agent, has several potent biological activities apparently mediated by distinct mechanisms. Our initial investigation of didemnin B resulted in the discovery of its GTP-dependent binding of the translation elongation factor EF1 alpha. This finding is consistent with the protein synthesis inhibitory activity of didemnin B observed at intermediate concentrations. To begin to dissect the mechanisms involved in the cytostatic and immunosuppressive activities of didemnin B, observed at low concentrations, additional didemnin-binding proteins were sought. Here we report the purification of a 36-kDa glycosylated didemnin-binding protein from bovine brain lysate. Cloning of the human cDNA encoding this protein revealed a strong sequence similarity with palmitoyl protein thioesterase (PPT), an enzyme that removes palmitate from H-Ras and the G alpha s subunits of heterotrimeric GTP-binding proteins in vitro. Mutations in PPT have recently been shown to be responsible for infantile neuronal ceroid lipofuscinosis, which is a severe brain disorder characterized by progressive loss of brain function and early death.

Cloning and characterization of a TEF gene for elongation factor 1? from the yeast Arxula adeninivorans

The translation elongation factor EF-1 alpha appears to play a major role in the control of cell proliferation and ageing in higher eukaryotes. Here we report the cloning of the TEF1 gene encoding the elongation factor 1 alpha of the dimorphic yeast Arxula adeninivorans Ls3. The gene is localized on chromosome 2 from Arxula adeninivorans, comprises 1380 bp and encodes a protein containing 459 amino acids. In contrast to other fungi, a second TEF gene encoding an identical, or nearly identical, polypeptide could not be identified. The transcriptional activity of the TEF1 gene did not change during mycelial growth, whereas a slight decrease could be detected during the yeast growth.

Translational arrest in hypoxic potato tubers is correlated with the aberrant association of elongation factor EF-1 alpha with polysomes.

Translation elongation factor EF-1 alpha became stably associated with potato tuber polysomes at the onset of hypoxia, coincident with a sharp rise in lactate and decrease in tissue pH. This aberrant association of EF-1 alpha with polysomes also occurred when aerobic tuber extracts were acidified in vitro. Upon resumption of protein synthesis, an increase in the steady-state levels of EF-1 alpha, and expression of an EF-1 alpha/GUS transgene was observed. These results indicate that translational arrest results from to the failure of EF-1 alpha to dissociate from ribosomes during the elongation cycle, and that restoration of protein synthesis is coordinated with expression of EF-1 alpha.

Protein translation elongation factor-1 alpha from Trypanosoma brucei binds calmodulin.

The following study examines the calmodulin (CaM) branch of the calcium signal pathway in the protozoan parasite Trypanosoma brucei. To accomplish this goal, a subset of cytosolic CaM-binding proteins (CaMBPs) was partially purified by a combination of DE52 and CaM-Sepharose affinity chromatography. Monoclonal antibodies (CBP-KK1) were used to clone the cDNA for a 53-kDa CaMBP from a lambda ZAP expression library of the metacyclic stage of T. brucei. The deduced amino acid sequence of clone CaMBP-12B had 81% overall amino acid identity to the translation elongation factor-1 alpha (EF-1 alpha) from Euglena gracilis and 76% identity to the rabbit EF-1 alpha. Rabbit EF-1 alpha was recognized by CBP-KK1 and was shown to bind to CaM-Sepharose in a calcium-dependent manner. By contrast, the complex of EF-1 alpha beta gamma did not bind CaM-Sepharose. A heterobifunctional sulfhydryl derivative of CaM (N-succinimidyl 3-(2-pyridyldithio)propionate-CaM) formed reducible cross-links with EF-1 alpha in solution but not with the complex of EF-1 alpha beta gamma. Biotinylated CaM bound weakly to trypanosome and rabbit EF-1 alpha in a gel overlay assay. This report demonstrates the direct interaction between CaM and the translation elongation factor EF-1 alpha.

A survey of transcripts expressed specifically in root nodules of broadbean (Vicia faba L.).

More than 600 potentially nodule-specific clones have been detected by differential hybridization of a broadbean cDNA library constructed from root nodule poly(A)+ RNA. These isolated cDNAs belong to at least 28 different clone groups containing cross-hybridizing sequences. The number of clones within a clone group varies from about 200 to only one single clone. Northern hybridization experiments revealed nodule-specific transcripts for 14 clone groups and markedly nodule-enhanced transcripts for another 7 clone groups. Sequence homologies indicate that three transcript sequences code for different leghemoglobins. Two other transcripts encode a nodule-specific sucrose synthase and a nodule-enhanced asparagine synthetase, respectively. Four deduced gene products are proline-rich, two of them being the homologues of PsENOD2 and PsENOD12. The third proline-rich protein (PRP) is composed of similar amino acid repeats as the nodule-specific PsENOD12 but is expressed in nodules and roots in comparable amounts. The fourth PRP is a nodule-enhanced extensin-type protein built up by Ser-Pro4 repeats. Two further nodule-specific transcripts encode gene products showing some similarity to structural glycine-rich proteins. Additionally, transcripts could be identified for broadbean homologues of the nodulins MsNOD25, PsENOD3 and PsENOD5 and transcripts specifying a nodule-enhanced lipoxygenase and a translation elongation factor EF-1 alpha, which is expressed in all broadbean tissues tested.

The activation process of Arabidopsis thaliana A1 gene encoding the translation elongation factor EF-1 alpha is conserved among angiosperms.

In Arabidopsis thaliana, the activation process of the A1 EF-1 alpha gene depends on several elements. Using the GUS reporter gene, transient expression experiments have shown that mutations of upstream cis-acting elements of the A1 promoter, or the deletion of an intron located within the 5' non-coding region, similarly affect expression in dicot or monocot protoplasts. The results reported here strongly suggest that this 5' intron is properly spliced in Zea mays. We show that two trans-acting factors, specifically interacting with an upstream activating sequence (the TEF 1 box), are present in nuclear extracts prepared from A. thaliana, Brassica rapa, Nicotiana tabacum and Z. mays. In addition, a DNA sequence homologous to the TEF 1 box, found at approximately the same location within a Lycopersicon esculentum EF-1 alpha promoter, interacts with the same trans-acting factors. Homologies found between the A. thaliana and L. esculentum TEF 1 box sequences have allowed us to define mutations of this upstream element which affect the interaction with the corresponding trans-acting factors. These results support the notion that the activation processes of A. thaliana EF-1 alpha genes have been conserved among angiosperms and provide interesting data on the functional structure of the TEF 1 box.