RESUMEN
Parafrancisella adeliensis, a Francisella-like endosymbiont, was found to reside in the cytoplasm of an Antarctic strain of the bipolar ciliate species, Euplotes petzi. To inquire whether Euplotes cells collected from distant Arctic and peri-Antarctic sites host Parafrancisella bacteria, wild-type strains of the congeneric bipolar species, E. nobilii, were screened for Parafrancisella by in situ hybridization and 16S gene amplification and sequencing. Results indicate that all Euplotes strains analyzed contained endosymbiotic bacteria with 16S nucleotide sequences closely similar to the P. adeliensis 16S gene sequence. This finding suggests that Parafrancisella/Euplotes associations are not endemic to Antarctica, but are common in both the Antarctic and Arctic regions.
Asunto(s)
Euplotes , Francisella , Filogenia , Euplotes/genética , Euplotes/microbiología , Citoplasma , Regiones AntárticasRESUMEN
Ribosome synthesis is a complex process that involves a large set of protein trans-acting factors, among them DEx(D/H)-box helicases. These are enzymes that carry out remodelling activities onto RNAs by hydrolysing ATP. The nucleolar DEGD-box protein Dbp7 is required for the biogenesis of large 60S ribosomal subunits. Recently, we have shown that Dbp7 is an RNA helicase that regulates the dynamic base-pairing between the snR190 small nucleolar RNA and the precursors of the ribosomal RNA within early pre-60S ribosomal particles. As the rest of DEx(D/H)-box proteins, Dbp7 has a modular organization formed by a helicase core region, which contains conserved motifs, and variable, non-conserved N- and C-terminal extensions. The role of these extensions remains unknown. Herein, we show that the N-terminal domain of Dbp7 is necessary for efficient nuclear import of the protein. Indeed, a basic bipartite nuclear localization signal (NLS) could be identified in its N-terminal domain. Removal of this putative NLS impairs, but does not abolish, Dbp7 nuclear import. Both N- and C-terminal domains are required for normal growth and 60S ribosomal subunit synthesis. Furthermore, we have studied the role of these domains in the association of Dbp7 with pre-ribosomal particles. Altogether, our results show that the N- and C-terminal domains of Dbp7 are important for the optimal function of this protein during ribosome biogenesis.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ARN Helicasas DEAD-box/metabolismo , Ribosomas/metabolismo , ARN Ribosómico/metabolismo , Proteínas Nucleares/genética , Proteínas Ribosómicas/metabolismo , Precursores del ARN/genéticaRESUMEN
Calcium (Ca2+) is a universal second messenger that plays a key role in cellular signaling. However, Ca2+ signals are transduced with the help of Ca2+-binding proteins, which serve as sensors, transducers, and elicitors. Among the collection of these Ca2+-binding proteins, calmodulin (CaM) emerged as the prototypical model in eukaryotic cells. This is a small protein that binds four Ca2+ ions and whose functions are multiple, controlling many essential aspects of cell physiology. CaM is universally distributed in eukaryotes, from multicellular organisms, such as human and land plants, to unicellular microorganisms, such as yeasts and ciliates. Here, we review most of the information gathered on CaM in Paramecium, a group of ciliates. We condense the information here by mentioning that mature Paramecium CaM is a 148 amino acid-long protein codified by a single gene, as in other eukaryotic microorganisms. In these ciliates, the protein is notoriously localized and regulates cilia function and can stimulate the activity of some enzymes. When Paramecium CaM is mutated, cells show flawed locomotion and/or exocytosis. We further widen this and additional information in the text, focusing on genomic data.
RESUMEN
The Ca2+-sensor protein calmodulin (CaM) is a major regulator of multiple cell functions. A unique and puzzling feature of human, and all so far investigated mammals, is the presence of three distinct CaM genes on different chromosomes, which code for identical proteins. How this case of apparent genetic redundancy evolved and why it could be to the advantage of the mammalian organisms is not well established. With a main focus on humans, this article aims to review existing literature addressing how the genes nonetheless differ in function. Clearly, the three CaM genes are differentially expressed in different tissues, during development, in response to different stimuli, and other factors including environmental conditions. As shown in hippocampal neurons, different mRNAs from the CAM genes may even localize differently within the same cell. Regulation of CaM gene expression is achieved by a variety of regulatory elements present in the three genes, including different promotor/insulator elements and 3'- and 5'-noncoding regions differing in length and sequence, as well as regulation by epigenetic factors and miRNAs. Here, we hypothesize that predicted differences in mRNA stability and translational efficiency due to divergent codon usage could play an additional regulatory role as the three genes differ markedly in their use of synonymous codons. CALM3, predicted to produce a relatively stable mRNA may be important where the transcription level is low or transiently absent, e.g. during spermatogenesis. In contrast, CALM2 with a predicted much shorter mRNA half-life, may provide better temporal control of CaM levels. Deciphering the underlying mechanisms responsible for all this complexity may help to understand why this unique multigenic arrangement may be an advantage for the optimal spatio-temporal expression of CaM in higher eukaryotes. Finally, we discuss the expression of the CaM genes in selected human pathologies, and how mutations in these genes are responsible for the appearance of serious congenital syndromes, mainly affecting the heart, and although less known, possibly also affecting the functionality of the central nervous system and other organs.
Asunto(s)
Calmodulina , Estabilidad del ARN , Animales , Humanos , Calmodulina/genética , Calmodulina/metabolismo , Codón , Uso de Codones , Mamíferos/metabolismo , MicroARNs , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Chaperonas Moleculares/metabolismo , ARN Nucleolar Pequeño/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Emparejamiento Base , ARN Helicasas DEAD-box/genética , Chaperonas Moleculares/genética , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Biogénesis de Organelos , Pliegue del ARN , Precursores del ARN/química , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Ribosómico/química , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Nucleolar Pequeño/genética , Subunidades Ribosómicas Grandes de Eucariotas/química , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The epidermal growth factor receptor (EGFR) harbors a calmodulin (CaM)-binding domain (CaM-BD) and a CaM-like domain (CaM-LD) upstream and downstream, respectively, of the tyrosine kinase (TK) domain. We demonstrate in this paper that deletion of the positively charged CaM-BD (EGFR/CaM-BD∆) inactivated the TK activity of the receptor. Moreover, deletion of the negatively charged CaM-LD (EGFR/CaM-LD∆), leaving a single negative residue (glutamate), reduced the activity of the receptor. In contrast, substituting the CaM-LD with a histidine/valine-rich peptide (EGFR/InvCaM-LD) caused full inactivation. We also demonstrated using confocal microscopy and flow cytometry that the chimera EGFR-green fluorescent protein (GFP)/CaM-BD∆, the EGFR/CaM-LD∆, and EGFR/InvCaM-LD mutants all bind tetramethylrhodamine-labelled EGF. These EGFR mutants were localized at the plasma membrane as the wild-type receptor does. However, only the EGFR/CaM-LD∆ and EGFR/InvCaM-LD mutants appear to undergo ligand-dependent internalization, while the EGFR-GFP/CaM-BD∆ mutant seems to be deficient in this regard. The obtained results and in silico modelling studies of the asymmetric structure of the EGFR kinase dimer support a role of a CaM-BD/CaM-LD electrostatic interaction in the allosteric activation of the EGFR TK.
Asunto(s)
Calmodulina/metabolismo , Membrana Celular/metabolismo , Animales , Células CHO , Señalización del Calcio/fisiología , Línea Celular , Cricetulus , Activación Enzimática/fisiología , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Unión Proteica/fisiología , Dominios Proteicos/fisiología , Proteínas Tirosina Quinasas/metabolismoRESUMEN
In eukaryotes, the beak structure of 40S subunits is formed by the protrusion of the 18S rRNA helix 33 and three ribosomal proteins: eS10, eS12 and eS31. The exact role of these proteins in ribosome biogenesis is not well understood. While eS10 is an essential protein encoded by two paralogous genes in Saccharomyces cerevisiae, eS12 and eS31 are not essential proteins encoded by the single-copy genes RPS12 and UBI3, respectively. Here, we have analysed the contribution of yeast eS12 to ribosome biogenesis and compared it with that of eS31. Polysome analysis reveals that deletion of either RPS12 or UBI3 results in equivalent 40S deficits. Analysis of pre-rRNA processing indicates that eS12, akin to eS31, is required for efficient processing of 20S pre-rRNA to mature 18S rRNA. Moreover, we show that the 20S pre-rRNA accumulates within cytoplasmic pre-40S particles, as deduced from FISH experiments and the lack of nuclear retention of 40S subunit reporter proteins, in rps12∆ and ubi3∆ cells. However, these particles containing 20S pre-rRNA are not efficiently incorporated into polyribosomes. We also provide evidence for a genetic interaction between eS12 or eS31 and the late-acting 40S assembly factors Enp1 and Ltv1, which appears not to be linked to the dynamics of their association with or release from pre-40S particles in the absence of either eS12 or eS31. Finally, we show that eS12- and eS31-deficient ribosomes exhibit increased levels of translational misreading. Altogether, our data highlight distinct important roles of the beak region during ribosome assembly and function.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Eliminación de Gen , Fenotipo , Procesamiento Proteico-Postraduccional , Procesamiento Postranscripcional del ARN , Transporte de ARN , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismoRESUMEN
In Saccharomyces cerevisiae, more than 250 trans-acting factors are involved in the maturation of 40S and 60S ribosomal subunits. The expression of most of these factors is transcriptionally coregulated to ensure correct ribosome production under a wide variety of environmental and intracellular conditions. Here, we identified the essential nucleolar Pol5 protein as a novel trans-acting factor required for the synthesis of 60S ribosomal subunits. Pol5 weakly and/or transiently associates with early to medium pre-60S ribosomal particles. Depletion of and temperature-sensitive mutations in Pol5 result in a deficiency of 60S ribosomal subunits and accumulation of half-mer polysomes. Both processing of 27SB pre-rRNA to mature 25S rRNA and release of pre-60S ribosomal particles from the nucle(ol)us to the cytoplasm are impaired in the Pol5-depleted strain. Moreover, we identified the genes encoding ribosomal proteins uL23 and eL27A as multicopy suppressors of the slow growth of a temperature-sensitive pol5 mutant. These results suggest that Pol5 could function in ensuring the correct folding of 25S rRNA domain III; thus, favoring the correct assembly of these two ribosomal proteins at their respective binding sites into medium pre-60S ribosomal particles. Pol5 is homologous to the human tumor suppressor Myb-binding protein 1A (MYBBP1A). However, expression of MYBBP1A failed to complement the lethal phenotype of a pol5 null mutant strain though interfered with 60S ribosomal subunit biogenesis.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Procesamiento Postranscripcional del ARNRESUMEN
The study of the draft genome of an Antarctic marine ciliate, Euplotes petzi, revealed foreign sequences of bacterial origin belonging to the γ-proteobacterium Francisella that includes pathogenic and environmental species. TEM and FISH analyses confirmed the presence of a Francisella endocytobiont in E. petzi. This endocytobiont was isolated and found to be a new species, named F. adeliensis sp. nov.. F. adeliensis grows well at wide ranges of temperature, salinity, and carbon dioxide concentrations implying that it may colonize new organisms living in deeply diversified habitats. The F. adeliensis genome includes the igl and pdp gene sets (pdpC and pdpE excepted) of the Francisella pathogenicity island needed for intracellular growth. Consistently with an F. adeliensis ancient symbiotic lifestyle, it also contains a single insertion-sequence element. Instead, it lacks genes for the biosynthesis of essential amino acids such as cysteine, lysine, methionine, and tyrosine. In a genome-based phylogenetic tree, F. adeliensis forms a new early branching clade, basal to the evolution of pathogenic species. The correlations of this clade with the other clades raise doubts about a genuine free-living nature of the environmental Francisella species isolated from natural and man-made environments, and suggest to look at F. adeliensis as a pioneer in the Francisella colonization of eukaryotic organisms.
Asunto(s)
Euplotes/microbiología , Francisella/aislamiento & purificación , Regiones Antárticas , Elementos Transponibles de ADN , Euplotes/fisiología , Francisella/clasificación , Francisella/genética , Francisella/fisiología , Genoma Bacteriano , Filogenia , Agua de Mar/microbiología , SimbiosisRESUMEN
The exon junction complex (EJC) is a key element of the splicing machinery. The EJC core is composed of eIF4A3, MAGO, Y14 and MLN51. Few accessory proteins, such as CWC22 or UPF3, bind transiently to the EJC. The EJC has been implicated in the control of the splicing of long introns. To ascertain whether the EJC controls the splicing of short introns, we used Paramecium tetraurelia as a model organism, since it has thousands of very tiny introns. To elucidate whether EJC affects intron splicing in P. tetraurelia, we searched for EJC protein-coding genes, and silenced those genes coding for eIF4A3, MAGO and CWC22. We found that P. tetraurelia likely assembles an active EJC with only three of the core proteins, since MLN51 is lacking. Silencing of eIF4A3 or CWC22 genes, but not that of MAGO, caused lethality. Silencing of the MAGO gene caused either an increase, decrease, or no change in intron retention levels of some intron-containing mRNAs used as reporters. We suggest that a fine-tuning expression of EJC genes is required for steady intron removal in P. tetraurelia. Taking into consideration our results and those published by others, we conclude that the EJC controls splicing independently of the intron size.
Asunto(s)
Proteínas Nucleares/metabolismo , Paramecium tetraurelia/genética , Empalme del ARN , Silenciador del Gen , Intrones , Proteínas Nucleares/genética , Paramecium tetraurelia/crecimiento & desarrollo , Paramecium tetraurelia/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , ARN Mensajero/genética , ARN Protozoario/genéticaRESUMEN
Pelomyxa palustris is a giant anaerobic/microaerobic amoeba, characterized by a number of exceptional cytological and physiological features, among them the presumed absence of energy producing organelles and the presence of endosymbiotic bacteria. These endosymbionts have been previously distinguished as: a large rectangular-shaped Gram-variable rod with a central cleft; a slender Gram-negative rod; and a slender Gram-positive rod. Using DNA extracted from P. palustris cysts, we have obtained three SSU rRNA gene sequences. We have determined that these sequences are affiliated to three different prokaryotic genera: Methanosaeta (a methanogenic archaea), Syntrophorhabdus (a syntrophic Gram-negative bacteria) and Rhodococcus (an aerobic chemoorganotrophic Gram-positive bacteria). To our knowledge, it is the first time that Syntrophorhabdus has been described as an endosymbiont in association with a methanogen. Strikingly, no traces of Methanobacterium formicicum could be detected, despite this methanogen had allegedly been isolated from trophozoites of P. palustris. It seems that the host and the endosymbionts have established a multipartite syntrophic consortium resembling to some extent those found in sewage treatment plants.
Asunto(s)
Archamoebae/microbiología , Deltaproteobacteria/fisiología , Methanosarcinales/fisiología , Rhodococcus/fisiología , Simbiosis , Archamoebae/fisiología , Deltaproteobacteria/clasificación , Deltaproteobacteria/genética , Deltaproteobacteria/aislamiento & purificación , Methanosarcinales/clasificación , Methanosarcinales/genética , Methanosarcinales/aislamiento & purificación , Filogenia , ARN de Archaea/genética , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Rhodococcus/clasificación , Rhodococcus/genética , Rhodococcus/aislamiento & purificación , Análisis de Secuencia de ARNRESUMEN
We identified the Grb7 family members, Grb10 and Grb14, as Ca2+ -dependent CaM-binding proteins using Ca2+ -dependent CaM-affinity chromatography as we previously did with Grb7. The potential CaM-binding sites were identified and experimentally tested using fluorescent-labeled peptides corresponding to these sites. The apparent affinity constant of these peptides for CaM, and the minimum number of calcium ions bound to CaM that are required for effective binding to these peptides were also determined. We prepared deletion mutants of the three adaptor proteins lacking the identified sites and determined that they lost or strongly diminished their CaM-binding capacity following the sequence Grb7 > > Grb14 > Grb10. More than one CaM-binding site and/or accessory CaM-binding sites appear to exist in Grb10 and Grb14, as compared to a single one present in Grb7.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Señalización del Calcio , Calmodulina/metabolismo , Proteína Adaptadora GRB10/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Sitios de Unión , Calmodulina/química , Cromatografía de Afinidad , Secuencia Conservada , Proteína Adaptadora GRB10/química , Proteína Adaptadora GRB10/genética , Proteína Adaptadora GRB7/química , Proteína Adaptadora GRB7/genética , Proteína Adaptadora GRB7/metabolismo , Eliminación de Gen , Células HEK293 , Humanos , Cinética , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Homología Estructural de ProteínaRESUMEN
Nonsense-mediated decay recognises mRNAs containing premature termination codons. One of its components, UPF3, is a molecular link bridging through its binding to the exon junction complex nonsense-mediated decay and splicing. In protists UPF3 has not been identified yet. We report that Paramecium tetraurelia bears an UPF3 gene and that it has a role in nonsense-mediated decay. Interestingly, the identified UPF3 has not conserved the essential amino acids required to bind the exon junction complex. Though, our data indicates that this ciliate bears genes coding for core proteins of the exon junction complex.
Asunto(s)
Codón sin Sentido , Paramecium/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Paramecium/química , Proteínas Protozoarias/química , Alineación de SecuenciaRESUMEN
Ferrous ion biooxidation is a process with many promising industrial applications: mainly regeneration of ferric ion as an oxidizing reagent in bioleaching processes and depuration of acid mine drainage. The flooded packed-bed bioreactor (FPB) is the design that leads to the highest biooxidation rate. In this bioreactor, biomass is immobilized in a biofilm that consists of an inorganic matrix, formed by precipitated ferric compounds, in the pores of which cells are attached. This biofilm covers the surface of particles (siliceous stone) that form the bed. The chemical stability of this inorganic matrix defines the widest possible pH range in FPBs. At pH below 1, ferric matrix is dissolved and cells are washed out. At pH higher than 2, ferric ion precipitates massively, greatly hindering mass transfer to cells. Thus, among other parameters, pH is recognised as a key factor for operational control in FPBs. This paper aims to explain the effect of pH on FPB operation, with an emphasis on microbial population behaviour. FPBs seeded with mixed inocula were assayed in the pH range from 2.3 to 0.8 and the microbial population was characterised. The microbial consortium in the bioreactor is modified by pH; at pH above 1.3 Acidithiobacillus ferrooxidans is the dominant microorganism, whereas at pH below 1.3 Leptospirillum ferrooxidans dominates. Inoculum can be adapted to acidity during continuous operation by progressively decreasing the pH of the inlet solution. Thus, in the pH range from 2.3 to 1, the biotic factor does not compromise the bioreactor performance.
Asunto(s)
Reactores Biológicos , Compuestos Ferrosos/metabolismo , Acidithiobacillus/metabolismo , Bacterias/metabolismo , Teorema de Bayes , Biopelículas , Biomasa , Células Inmovilizadas , Compuestos Férricos/metabolismo , Concentración de Iones de Hidrógeno , Oxidación-Reducción , FilogeniaRESUMEN
Peritrich ciliates have been traditionally subdivided into two orders, Sessilida and Mobilida within the subclass Peritrichia. However, all the existing small subunit (SSU) rRNA phylogenetic trees showed that the sessilids and mobilids did not branch together. To shed some light on this disagreement, we tested whether or not the classic Peritrichia is a monophyletic group by assessing the reliability of the SSU rRNA phylogeny in terms of congruency with alpha-tubulin phylogeny. For this purpose, we obtained 10 partial alpha-tubulin sequences from peritrichs and built phylogenetic trees based on alpha-tubulin nucleotide and amino acid data. A phylogenetic tree from the alpha-tubulin and SSU rRNA genes in combination was also constructed and compared with that from the SSU rRNA gene using a similar species sampling. Our results show that the mobilids and sessilids are consistently separated in all trees, which reinforces the idea that the peritrichs do not constitute a monophyletic group. However, in all alpha-tubulin gene trees, the urceolariids and trichodiniids do not group together, suggested mobilids may not be a monophyletic group.
Asunto(s)
Oligohimenóforos/clasificación , Filogenia , Proteínas Protozoarias/genética , Subunidades Ribosómicas Pequeñas/genética , Tubulina (Proteína)/genética , ADN Protozoario/genética , Datos de Secuencia Molecular , Oligohimenóforos/genética , ARN Ribosómico/genéticaRESUMEN
The parasitic protozoan Entamoeba histolytica relies on a very dynamic cytoskeleton in order to invade and survive in host tissues. Identification of cytoskeletal elements is key to understanding these processes. Here we present the characterization of EhLimA, the first LIM protein of E. histolytica. EhLimA consists of a single LIM domain at its N terminus and exhibits the highest degree of homology with DdLimE from Dictyostelium discoideum. Immunofluorescence localization of EhLimA using anti-EhLimA antibodies revealed that EhLimA is highly concentrated at the plasma membrane of cells. Silencing or overexpression of the EhLimA gene did not have a significant effect on the growth or morphology of the parasite. EhLimA associates with the cytoskeleton as demonstrated by the enrichment of the protein in cytoskeleton fractions as well as in pull-down assays that revealed that cytoskeleton association involves interaction with actin. EhLimA binding to actin was shown to be dependent on the N-terminal LIM domain of EhLimA, as removal of even half of the LIM domain resulted in almost complete inhibition of the binding to actin. We also found that a portion of EhLimA floats to the lower-density regions of a sucrose gradient together with portions of the Gal-lectin light subunit and actin. Treatment of cells with the cholesterol-sequestering agent digitonin resulted in increased solubility of EhLimA. These results indicate that in addition to cytoskeletal association, EhLimA may also associate with lipid rafts in the parasite plasma membrane and suggest that EhLimA may be part of the molecular system connecting the actin cytoskeleton to membrane rafts.
Asunto(s)
Membrana Celular/metabolismo , Entamoeba histolytica/metabolismo , Microdominios de Membrana/metabolismo , Proteínas Protozoarias/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/química , Clonación Molecular , Citoesqueleto/química , Citoesqueleto/metabolismo , Silenciador del Gen , Microdominios de Membrana/química , Datos de Secuencia Molecular , Proteínas Protozoarias/análisis , Proteínas Protozoarias/genéticaRESUMEN
Based on morphological characters, peritrich ciliates (Class Olygohymenophorea, Subclass Peritrichia) have been subdivided into the Orders Sessilida and Mobilida. Molecular phylogenetic studies on peritrichs have been restricted to members of the Order Sessilida. In order to shed more light into the evolutionary relationships within peritrichs, the complete small subunit rRNA (SSU rRNA) sequences of four mobilid species, Trichodina nobilis, Trichodina heterodentata, Trichodina reticulata, and Trichodinella myakkae were used to construct phylogenetic trees using maximum parsimony, neighbor joining, and Bayesian analyses. Whatever phylogenetic method used, the peritrichs did not constitute a monophyletic group: mobilid and sessilid species did not cluster together. Similarity in morphology but difference in molecular data led us to suggest that the oral structures of peritrichs are the result of evolutionary convergence. In addition, Trichodina reticulata, a Trichodina species with granules in the center of the adhesive disc, branched separately from its congeners, Trichodina nobilis and Trichodina heterodentata, trichodinids without such granules. This indicates that granules in the adhesive disc might be a phylogenetic character of high importance within the Family Trichodinidae.
Asunto(s)
Genes de ARNr/genética , Oligohimenóforos/genética , Filogenia , Animales , Oligohimenóforos/clasificaciónRESUMEN
Macronuclear deoxyribonucleic acid (DNA) in hypotrichous ciliates consists of a set of linear molecules ranging in size from 0.5 to several tens of kilobases and typically carrying a single gene. Each minichromosome is present at a ploidy of >or=1,000 per macronucleus. These molecules are known as gene-sized molecules. Multigene molecules are also present, but are still poorly described. In analyzing the encystment-excystment cycle of Sterkiella histriomuscorum, we have characterized a set of 21 macronuclear molecules both at the DNA and complementary DNA (cDNA) levels. On a total of 23 validated coding sequences, we mapped the 5' and 3' untranslated regions for a subset of 10 and 18 transcripts, respectively. A combination of DNA and cDNA data allows us to precisely determine several structural features of macronuclear chromosomes, such as the organization of multigene molecules, an intron content higher than expected, and a conserved sequence surrounding the initiation transcription site. It also reveals one coding sequence containing a transcribed 10-bp element that displays the characteristic features of internal eliminated sequences (IES). Its presence in a fraction of the minichromosomes carrying this gene raises the possibility of an incomplete IES excision process during the development of the S. histriomuscorum macronucleus.
Asunto(s)
Cromosomas/genética , Cilióforos/genética , Orden Génico , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Southern Blotting , Cilióforos/crecimiento & desarrollo , ADN/genética , ADN Complementario/genética , Expresión Génica , Intrones , Datos de Secuencia Molecular , Sitio de Iniciación de la TranscripciónRESUMEN
Entamoeba histolytica is a phagocytic cell with numerous vesicles of different sizes and shapes but without a well-defined Golgi apparatus. Despite this, genes implied in membrane trafficking have been identified in the genome of this parasite. One of these genes is homologous to the N-ethylmaleimide sensitive fusion factor (NSF), whose protein has been shown to play an important role in vesicle fusion in other eukaryotic cells. In this report, we investigated the NSF homologue gene from a pathogenic E. histolytica, characterized its protein product and two of its activities, ATPase and in vitro intra-Golgi transport. The finding of an active NSF protein in E. histolytica indicates that a simple or primordial Golgi apparatus probably exists in this microorganism, as has been proposed by others.