RESUMO
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.
Assuntos
DNA Circular/química , DNA de Protozoário/química , DNA Super-Helicoidal/química , Entamoeba histolytica/genética , Animais , DNA Circular/isolamento & purificação , DNA Fúngico/isolamento & purificação , DNA de Protozoário/isolamento & purificação , DNA Super-Helicoidal/isolamento & purificação , Eletroforese em Gel de Ágar/métodos , Etídio , Genoma de Protozoário , Cariotipagem , Análise de Regressão , Saccharomyces cerevisiae/genética , SoftwareRESUMO
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
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
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.