RESUMO
In this paper we cloned, sequenced and expressed a novel Entamoeba histolytica alcohol dehydrogenase gene (Ehadh3). Ehadh3 has a predicted 383 amino acids open reading frame, encoding for a 42.3 kDa protein. The deduced amino acid sequence showed 24 to 26% identity to other type III alcohol dehydrogenases found in prokaryotic and lower eukaryotic organisms, but not in mammalia. There are at least two Ehadh3 gene copies in the genome, but only a 1.2 kb transcript was detected. The EhADH3 fusion protein showed a NADP+(-)dependent ADH activity. Ehadh3 may be a good target for the developing of anti-E. histolytica drugs, without producing damage to the human.
Assuntos
Oxirredutases do Álcool/genética , Entamoeba histolytica/genética , Genes de Protozoários , Proteínas de Protozoários/genética , Oxirredutases do Álcool/classificação , Sequência de Aminoácidos , Anaerobiose , Animais , Bactérias/enzimologia , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Entamoeba histolytica/enzimologia , Dados de Sequência Molecular , Fases de Leitura Aberta , Saccharomyces cerevisiae/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
The molecular karyotype of 3 clones derived from strain HM1:IMSS of Entamoeba histolytica was studied by transverse alternating field electrophoresis. 11-20 bands ranging between 0.3 and over 3 Mb were resolved. Hybridization with total DNA detected highly repetitive sequences in the slow-migrating molecules, while non-repetitive sequences were located in the intermediate and fast-migrating molecules. rDNA, tubulin, actin, cysteine proteases DNA fragments, and a variable DNA sequence (EhVR1) located the respective genes mainly in the 1.3-1.5-Mb region, although they differed in the three clones. Two-dimensional transverse alternating field electrophoresis showed that more than one high-molecular weight molecule may comigrate in a single DNA band. rDNA, and EhVR1 hybridized with slow-migrating bands in a characteristic ladder pattern. Most of the bands recognized by EhVR1 seems to be linear molecules, although exonuclease III-resistant bands also hybridized with EhVR1, suggesting the presence of circles.
Assuntos
DNA de Protozoário/genética , Entamoeba histolytica/genética , Actinas/genética , Animais , Mapeamento Cromossômico , Clonagem Molecular , Cisteína Endopeptidases/genética , DNA de Protozoário/isolamento & purificação , DNA Ribossômico/genética , Genes de Protozoários , Genótipo , Fenótipo , Polimorfismo Genético , Sequências Repetitivas de Ácido Nucleico , Tubulina (Proteína)/genéticaRESUMO
We have examined the sodium dodecyl sulfate (SDS)-induced autoproteolysis of E. invadens (PZ and IP101) and E. moshkovskii (FIC and Laredo) trophozoite lysates. Heat-treated lysates containing parahydroxy-mercuribenzoate (pHMB) of all four strains had undegraded protein patterns. Unheated pHMB-lacking lysates of PZ had two (99 and 90 kDa) and IP101 lysates had three (45, 99, 90 kDa) major proteins, whereas FIC and Laredo lysates had only one (90 kDa). Heat-treatment changed the remaining proteins to smaller ones: 37 in PZ and IP101, 33 and 37 kDa in FIC and Laredo. Unheated lysates run on gelatin-containing "substrate" gels had gelatinases whose sizes were higher in lysates containing pHMB and allowed us to detect a 200 kDa gelatinase in E. invadens strains. Our results indicate that SDS induces immediate autoproteolysis by CPs in non-histolytica trophozoites, whose proteinases appear to be processed by self-digestion, and Entamoeba proteinases vary considerably under the various conditions used to obtain and characterize them.
Assuntos
Autólise , Endopeptidases/metabolismo , Entamoeba/enzimologia , Proteínas de Protozoários/metabolismo , Animais , Cisteína Endopeptidases/metabolismo , Entamoeba/efeitos dos fármacos , Entamoeba/crescimento & desenvolvimento , Dodecilsulfato de Sódio/farmacologia , Especificidade da EspécieRESUMO
Different electrophoretic conditions were used to improve the molecular karyotype of Entamoeba histolytica clone L6 derived from the heterogeneous strain HM1:IMSS. Eleven to 17 bands ranging between 0.3 and over 3 megabases (Mb) were resolved by transverse alternating field electrophoresis (TAFE). Amebic chromosomes presented similar pattern when they were TAFE separated at 72, 90 or 120 h running time, but resolution of the bands was increased at 90 h, and the best electrophoretic pattern was obtained at 120 h. Bal 31 digestion of DNA in the plugs suggested that most of the bands are lineal molecules and that pMD, an amebic DNA fragment, hybridizes with both lineal and nonlineal DNA molecules.
Assuntos
DNA de Protozoário/genética , Entamoeba histolytica/genética , Animais , DNA Circular/genética , DNA Complementar/genética , DNA Ribossômico/genética , Eletroforese em Gel de ÁgarRESUMO
We report a study on the DNA organization in Entamoeba histolytica using a ribosomal DNA probe. The rDNA genes were found forming mers which were separated in a typical ladder pattern by pulse field electrophoresis. DNA rosette structures were visualized through electron microscopy in DNA eluted from bands recognized by the ribosomal probe. The in situ hybridization experiments using a DNA probe suggested that the rDNA genes are portioned between the nucleus and a cytoplasmic structure. These findings provide new data on DNA organization in E. histolytica and open the question concerning the presence of a novel organelle in this eukaryotic parasite.
Assuntos
Citoplasma/química , DNA de Protozoário/genética , DNA Ribossômico/genética , Entamoeba histolytica/genética , Animais , Núcleo Celular/química , DNA de Protozoário/ultraestrutura , DNA Ribossômico/ultraestrutura , Eletroforese em Gel de Campo Pulsado , Entamoeba histolytica/ultraestrutura , Hibridização In Situ , Microscopia Confocal , Microscopia Eletrônica , OrganelasRESUMO
We identified some gene linkage groups in Entamoeba histolytica using a 4-M urea improved transversal alternating field electrophoresis (TAFE) method. Complex rosette-structured DNA molecules were found trapped along the gel lanes, explaining the fuzziness of the patterns. Using several episomal probes, including 16 S, 5.8 S, and 25 S ribosomal (r)Dna genes, an autonomous replication sequence (ARS), and EhVR1, we identified a complete ribosomal episome linkage group (CELG) at the 1.2-Mb position. Three other incomplete groups were found: IELG-1, formed by EhVR1, 16 S, 5.8 S, and 25 S genes; IELG-2 formed by EhVR1, 16 S and 25 S; and IELG-3 formed only by 5.8 S. Ehadh3, Ehpfo, and Ehredox genes migrated at the 1.8-Mb position, forming the non-ribosomal linkage group, NRLG-1.8, while the Ehenl-1 gene migrated at 1.6 Mb forming the NRLG-1.6 group. Ehhk was located at 1.2, 0.8, and 0.17 Mb in three different groups: NRLG-1.2, IELG-3-0.8, and NRLG-0.17. Putative lineal chromosomes were also identified using an heterologous telomeric probe. By in situ hybridization experiments, the rDNA and Ehhk genes were located in both nucleus and cytoplasm, while the Ehpfo and Ehredox genes were found mainly in the nucleus. We propose a model hypothezising that the 16 S and 25 S genes are in a linear molecule, duplicated in two inverted repeats, which may be looped out of the linear DNA to form an episome probably lacking or not the 5.8 S sequence, which could be added later by recombination.
Assuntos
Entamoeba histolytica/genética , Ligação Genética , Animais , Mapeamento Cromossômico , Sondas de DNA , DNA de Protozoário/genética , DNA Ribossômico/genética , Eletroforese em Gel de Ágar , Genes de Protozoários , Hexoquinase/genética , Cariotipagem , Cetona Oxirredutases/genética , Dados de Sequência Molecular , Oxirredutases/genética , Piruvato Sintase , Ribonucleases , UreiaRESUMO
We show here data suggesting that Entamoeba histolytica, the protozoan responsible for human amebiasis, presents DNA amplification in a fashion similar to that described for transformed mammalian cells. By transmission electron microscopy (TEM), we found linear, circular and concentric circular DNA molecules exhibiting the main events of the unscheduled DNA amplification process. Loops were formed after the recombination of two nonadjacent DNA regions, and bubbles appeared from the recombinant strands without involving the looped-out sequences. Bubbles grew up and underwent further replication rounds to produce a nested set of partially replicated circles. Multicircle complexes were also formed from putative replication origin without recombination of distant DNA regions. Clones derived from the strain HM1:IMSS exhibited different DNA contents, suggesting DNA amplification. The parental clone A and its daughter clone C2 differed in rDNA gene copy numbers, but this was observed only when total DNA was separated by pulse field gel electrophoresis, and no significant differences were detected in nuclear DNA. The dissection of the events observed by TEM led us to propose an onion skin model for gene amplification in E. histolytica.
Assuntos
DNA de Protozoário/genética , Entamoeba histolytica/genética , Amplificação de Genes , Animais , Replicação do DNA , DNA Circular/análise , DNA Circular/genética , DNA Circular/ultraestrutura , DNA de Protozoário/análise , DNA de Protozoário/ultraestrutura , Entamoeba histolytica/metabolismo , Microscopia EletrônicaRESUMO
Entamoeba histolytica genome was analysed by pulsed field gel electrophoresis under conditions to separate linear chromosomes in the 170-1400 kb range. We identified linear DNA molecules of 227, 366, 631, 850, 1112 and 1361 kb (mean sizes obtained by three different methods) and we estimated their reorientation times and migration velocities at various experimental conditions. DNA shift mobility assays, using ethidium bromide, suggested that bands migrating at 227 and 631 kb contain linear and circular DNA, whereas a band at 436 kb has only circular DNA. We obtained a regression equation relating sizes of supercoiled DNA molecules with their migration velocities during a pulse at constant electric field and temperature. We also developed a computer program (EHPATTERNS) that predicts the migration per pulse and the resolution order of circular and linear E. histolytica DNA at different pulse times and constant driving and frictional forces. The simulation showed that linear DNA molecules frequently co-migrate with circular molecules, but circular molecules change when the pulse time varies. This molecular mixture generates broad bands and difficulties in the interpretation of the molecular karyotype of E. histolytica.