ABSTRACT
Entamoeba histolytica, the protozoan responsible for human amoebiasis, exhibits a great genome plasticity that is probably related to homologous recombination events. It contains the RAD52 epistasis group genes, including Ehrad51 and Ehrad54, and the Ehblm gene, which are key homologous recombination factors in other organisms. Ehrad51 and Ehrad54 genes are differentially transcribed in trophozoites when DNA double-strand breaks are induced by ultraviolet-C irradiation. Moreover, the EhRAD51 recombinase is overexpressed at 30 min in the nucleus. Here, we extend our analysis of the homologous recombination mechanism in E. histolytica by studying EhRAD51, EhRAD54, and EhBLM expression in response to DNA damage. Bioinformatic analyses show that EhRAD54 has the molecular features of homologous proteins, indicating that it may have similar functions. Western blot assays evidence the differential expression of EhRAD51, EhRAD54, and EhBLM at different times after DNA damage, suggesting their potential roles in the different steps of homologous recombination in this protozoan.
Subject(s)
DNA Repair , Entamoeba histolytica/metabolism , Homologous Recombination , Protozoan Proteins/physiology , Amino Acid Sequence , Animals , Cell Nucleus/chemistry , Consensus Sequence , Cytoplasm/chemistry , DNA Breaks, Double-Stranded , DNA Helicases/chemistry , DNA Helicases/genetics , DNA Helicases/physiology , DNA Repair/genetics , DNA, Protozoan/genetics , DNA, Protozoan/radiation effects , Entamoeba histolytica/genetics , Entamoeba histolytica/radiation effects , Genes, Protozoan , Homologous Recombination/genetics , Humans , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Rad51 Recombinase/genetics , Rad51 Recombinase/physiology , RecQ Helicases/genetics , RecQ Helicases/physiology , Sequence Alignment , Sequence Homology, Amino Acid , Time Factors , Ultraviolet RaysABSTRACT
In eukaryotes, polyadenylation of pre-mRNA 3' end is essential for mRNA export, stability and translation. Taking advantage of the knowledge of genomic sequences of Entamoeba histolytica, the protozoan responsible for human amoebiasis, we previously reported the putative polyadenylation machinery of this parasite. Here, we focused on the predicted protein that has the molecular features of the 25 kDa subunit of the Cleavage Factor Im (CFIm25) from other organisms, including the Nudix (nucleoside diphosphate linked to another moiety X) domain, as well as the RNA binding domain and the PAP/PAB interacting region. The recombinant EhCFIm25 protein (rEhCFIm25) was expressed in bacteria and used to generate specific antibodies in rabbit. Subcellular localization assays showed the presence of the endogenous protein in nuclear and cytoplasmic fractions. In RNA electrophoretic mobility shift assays, rEhCFIm25 was able to form specific RNA-protein complexes with the EhPgp5 mRNA 3´ UTR used as probe. In addition, Pull-Down and LC/ESI-MS/MS tandem mass spectrometry assays evidenced that the putative EhCFIm25 was able to interact with the poly(A) polymerase (EhPAP) that is responsible for the synthesis of the poly(A) tail in other eukaryotic cells. By Far-Western experiments, we confirmed the interaction between the putative EhCFIm25 and EhPAP in E. histolytica. Taken altogether, our results showed that the putative EhCFIm25 is a conserved RNA binding protein that interacts with the poly(A) polymerase, another member of the pre-mRNA 3' end processing machinery in this protozoan parasite.
Subject(s)
Entamoeba histolytica/genetics , Exoribonucleases/genetics , Poly A/genetics , Protein Subunits/genetics , RNA-Binding Proteins/genetics , mRNA Cleavage and Polyadenylation Factors/genetics , Amino Acid Sequence , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cytoplasm/genetics , Cytoplasm/metabolism , Entamoeba histolytica/metabolism , Exoribonucleases/metabolism , Molecular Sequence Data , Poly A/metabolism , Protein Subunits/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , mRNA Cleavage and Polyadenylation Factors/metabolismABSTRACT
Como sabemos, la placa dentobacteriana (PDB) contiene diversos microorganismos que son factores predisponentes de diversas patologías bucales como la enfermedad periodontal y la caries, sta última relacionada con el aumento principalmente de uno de los tres estreptococos considerados como cariogénicos; Streptococcus mutans [..] El objetivo de este proyecto es evaluar el efecto de las tinturas madres Calendula officinallis y Echinacea angustifolia, para que inhiban el desarrollo del Streptococcus mutans. La investigación es prospectiva, experimental, comparativa y longitudinal. Se estudia "in vitro" la cepa de Streptococcus mutans G536, obtenida en medios de cultivo como son: Gelosa Sangre (GS), KF con TTC al 1% con tinturas y diluciones homeopáticas a 1:1; 1:3, 1:6; A los medios de cultivo se les agregó la tintura madre substituyendo el agua en forma proporcional. El medio de cultivo con Calendula officinalis mostró un mayor efecto inhibitorio que el medio de cultivo de Echinacea angustifolia, sin embargo, ambas presentaron mayor inhibición en las diluciones 1/1 y 1/3 como se ha reportado en investigaciones anteriores con Streptococcus de la Placa Dentobacteriana y tinturas homeopáticas. (AU)
Subject(s)
Anti-Bacterial Agents , Echinacea/adverse effects , /adverse effects , Streptococcus mutans , Dentistry , Mother Tincture , Dental Caries , Anti-Infective Agents, Local , HomeopathyABSTRACT
Como sabemos, la placa dentobacteriana (PDB) contiene diversos microorganismos que son factores predisponentes de diversas patologías bucales como la enfermedad periodontal y la caries, sta última relacionada con el aumento principalmente de uno de los tres estreptococos considerados como cariogénicos; Streptococcus mutans [..] El objetivo de este proyecto es evaluar el efecto de las tinturas madres Calendula officinallis y Echinacea angustifolia, para que inhiban el desarrollo del Streptococcus mutans. La investigación es prospectiva, experimental, comparativa y longitudinal. Se estudia "in vitro" la cepa de Streptococcus mutans G536, obtenida en medios de cultivo como son: Gelosa Sangre (GS), KF con TTC al 1% con tinturas y diluciones homeopáticas a 1:1; 1:3, 1:6; A los medios de cultivo se les agregó la tintura madre substituyendo el agua en forma proporcional. El medio de cultivo con Calendula officinalis mostró un mayor efecto inhibitorio que el medio de cultivo de Echinacea angustifolia, sin embargo, ambas presentaron mayor inhibición en las diluciones 1/1 y 1/3 como se ha reportado en investigaciones anteriores con Streptococcus de la Placa Dentobacteriana y tinturas homeopáticas.
