Characterization of ubiquitin genes and -transcripts and demonstration of a ubiquitin-conjugating system in Entamoeba histolytica.

We have investigated the genomic organization of Entamoeba histolytica ubiquitin and looked for the occurrence of a ubiquitin-conjugating system in this organism. Southern blots indicated the presence of > or = 5 ubiquitin-coding regions. One of these, EhUBI1, was cloned and sequenced and found to correspond to a monoubiquitin gene; as shown by a polymerase chain reaction, E. histolytica lacked polyubiquitin genes altogether. Blots of poly(A)+ RNA from exponentially-growing trophozoite cultures exhibited five ubiquitin transcripts, the most prominent and smallest of which corresponded to EhUBI1 mRNA. Expression of the ubiquitin genes was not influenced by heat shock. Although the predicted amino acid sequence of the ubiquitin from E. histolytica differs significantly (in 7-9 amino acid residues) from that of yeast and animals, expression of the coding sequence of EhUBI1 suppressed the heat-sensitive phenotype of a polyubiquitin gene-deficient yeast mutant. In correlation, trophozoite extract catalyzed an ATP-dependent conjugation of radioiodinated bovine ubiquitin to trophozoite proteins. The latter data indicate that E. histolytica contains a functional ubiquitin-conjugating system.

Evidence for the existence of a single ubiquitin gene in Giardia lamblia.

All eukaryotes investigated so far contain multiple copies of ubiquitin genes, most of which are arranged in fusions coding for either polyubiquitin or ubiquitin-ribosomal protein constructs; the former are normally under the control of a heat shock promoter. Giardia lamblia, an intestinal parasite, is the most primitive eukaryote known to date. We have investigated the arrangement and expression of ubiquitin genes in this organism by Southern and Northern blotting. Our data strongly suggest that G. lamblia contains just one ubiquitin gene, which consists of a single copy of the coding sequence and the expression of which is not enhanced by heat shock. By pulsed-field gel electrophoresis we localized this gene on the largest of the five giardial chromosomes. These data imply that the ubiquitin system in Giardia has probably been trapped at an original stage.

Entamoeba histolytica as a model for the primitive eukaryotic cell.

Entamoeba histolytica is a structurally simple eukaryote lacking mitochondria, peroxisomes and a well-developed Golgi apparatus, also in its biochemistry, it deviates substantially from the more complex eukoryotes. These features have alternatively been interpreted as archaic, ie. the ancestor of Entamoeba branched off before the primitive eukaryotic cell obtained proto-mitochondria, or as regressive, ie. Entamoeba has lost its mitochondria in the course of its adaptation to a parasitic life style. Tilly Bakker-Grunwald and Claudia Wöstmann favor the first interpretation and discuss in which respects E. histolytica may serve as a model for the primitive eukaryote.

Ubiquitin of Entamoeba histolytica deviates in six amino acid residues from the consensus of all other known ubiquitins.

The amino acid sequence of ubiquitin from Entamoeba histolytica, as deduced from a cDNA nucleotide sequence, deviated at six positions from the consensus of all other known ubiquitins (ranging from Trypanosoma cruzi to Homo sapiens). The corresponding residues were scattered over the primary sequence, but came close together on the surface of the folded protein structure. We conclude that (i) E. histolytica branched off very early from the main eukaryotic line, and (ii) this organism may yield clues as to the evolutionary development of the ubiquitin system.

The bactericidal action of streptomycin: membrane permeabilization caused by the insertion of mistranslated proteins into the cytoplasmic membrane of Escherichia coli and subsequent caging of the antibiotic inside the cells due to degradation of these proteins.

The mechanism by which the aminoglycoside antibiotic streptomycin permeabilizes the cytoplasmic membrane of Escherichia coli cells was reinvestigated. For this purpose, the extent of streptomycin-induced K+ loss from cells growing at low external K+ concentrations was taken as a measure of membrane permeabilization. Experiments with different K(+)-uptake mutants showed that the antibiotic specifically increased the passive permeability of the cell membrane to K+ and other ions. These permeability changes were small and the membrane potential of the treated cells remained high. The membrane permeabilization was not due to a direct interaction of the antibiotic with the cell membrane, since cells that carry an rpsL mutation and synthesize proteins in a streptomycin-insensitive way did not lose K+ after the addition of the antibiotic. Due to misreading and premature termination of translation the cells synthesized aberrant proteins under the conditions where membrane permeabilization occurred. Two conditions are described under which the cells both degraded these mistranslated proteins rapidly and reaccumulated K+, lending support to the hypothesis that membrane permeabilization is due to the presence of the mistranslated proteins in the cell membrane. Evidence is presented that the irreversibility of (dihydro)streptomycin uptake by cells washed free from the antibiotic might also be due to rapid degradation of the mistranslated proteins, leading to 'caging' of the antibiotic inside the cells.