Circular DNA of Entamoeba histolytica encodes ribosomal RNA

The presence of repeated DNA sequences encoding RNA in Entamoeba histolytica has been reported. In the present study we demonstrate by agarose gel electrophoresis. DNase digestion and electron microscopic analysis that these genes are located on extrachromosomal circular DNA molecules with an approximate size of 26 kb. Detection of replication intermediates suggests the episomal nature of these molecules. Amplified, extrachromosomal rRNA genes appear to be a common feature among the lower eukaryotes, occurring more commonly as linear molecules and less commonly as circles. Entamoeba histolytica is 1 of the few organisms studied in which rRNA genes are located predominantly on extrachromosomal circles.     

The ribosomal DNA plasmids of entamoeba

In most organisms, the nuclear ribosomal RNA (rRNA) genes are highly repetitive and arranged as tandem repeats on one or more chromosomes. In Entamoeba, however, these genes are located almost exclusively on extrachromosomal circular DNA molecules with no clear evidence so far of a chromosomal copy. Such an uncommon location of rRNA genes may be a direct consequence of cellular physiology, as suggested by studies with Saccharomyces cerevisiae mutants in which the rDNA is extrachromosomal. In this review, Sudha Bhattacharya, Indrani Som and Alok Bhattacharya summarize current knowledge on the structural organization and replication of the Entamoeba rDNA plasmids. Other than the rRNAs encoded by these molecules, no protein-coding genes (including ribosomal protein genes) are found on any of them. They are unique among plasmids in that they do not initiate replication from a fixed origin but use multiple sites dispersed throughout the molecule. Further studies should establish the unique biochemical features of Entamoeba that lead to extrachromosomal rDNA.     

Inter- and intra-strain variation in the 5.8S ribosomal RNA and internal transcribed spacer sequences of Entamoeba histolytica and comparison with Entamoeba dispar, Entamoeba moshkovskii and Entamoeba invadens

The ribosomal RNA genes in Entamoeba histolytica are located on circular DNA molecules in about 200 copies per genome equivalent. Nucleotide sequence analysis of the 5.8S rRNA gene and the flanking internal transcribed spacers was carried out to determine the degree of sequence divergence in the multiple rRNA gene copies of a given strain; amongst three different E. histolytica strains (HM-1:IMSS, Rahman and HK-9); and amongst four species of Entamoeba (Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii and Entamoeba invadens). The results show that all rRNA gene copies of a given strain are identical. Few nucleotide positions varied between strains of a species but the differences were very pronounced amongst species. In general, the internal transcribed spacer 2 sequence was more variable and may be useful for strain- and species-identification. The 5.8S rRNA gene and the internal transcribed spacer 2 of E. invadens were unusually small in size.     

The hemolytic properties of the membrane modifying peptide antibiotics alamethicin, suzukacillin and trichotoxin (author's transl)

1. Using hybridisation techniques nuclei from both amoebae and plasmodia of Physarum polycephalum were found to contain 275 genes each coding for 5.8-S, 19-S and 26-S rRNA, 685 genes for 5-S rRNA and 1050 genes for tRNA. 2. Hybridisation of these RNA species to both amoebal and plasmodial DNA fractionated on CsCl gradients reveal that the 5.8-S, 19-S and 26-S rRNA genes are located at a satellite position (formula: see text) with respect to the main band of DNA, whereas 4-S RNA genes are located exclusively in the main band of DNA (formula: see text). 3. This result was confirmed by demonstrating that only the 5.8-S, 19-S and 26-S rRNA species hybridise to purified plasmodial ribosomal DNA. 4. The 19-S and 26-S rRNA genes of amoebae are located on extrachromosomal DNA molecules of a discrete size (Mr = 38 X 10(6)) with identical properties to plasmodial ribosomal DNA.     

Extrachromosomal circular ribosomal DNA in the yeast Saccharomyces carlsbergensis.

Purified ribosomal DNA from Saccharomyces carlsbergensis contains a small proportion of circular DNA molecules with a contour length of 3 micron or integral multiples thereof. Hybridization of yeast ribosomal DNA with 26 S rRNA, using the R-loop technique, reveals that these circular molecules contain sequences complementary to yeast ribosomal RNA. We suggest that these extrachromosomal rRNA genes may be intermediates in the amplification of rRNA genes in yeast.     

Metabolic stability of the extrachromosomal ribosomal RNA genes in the slime mould Physarum polycephalum

The rRNA genes of the slime mould Physarum polycephalum are located on free, linear DNA molecules of a discrete size, Mr=38X10(6). Using an isotope dilution technique we have examined the metabolic stability of these extrachromosomal genes during active, balanced growth. Microplasmodia, prelabelled with [3H]thymidine, were used to prepare synchronous surface plasmodial cultures which were subsequently grown on unlabelled medium. The gross synthesis of ribosomal DNA was then determined over three consecutive mitotic divisions from the ratio of 3H to 14C in a hybrid formed between the extracted ribosomal [3H]DNA and a [14C]rRNA probe. It was found that ribosomal DNA, like chromosomal DNA, is completely stable during active growth.     

Entamoeba histolytica extrachromosomal circular ribosomal DNA: analysis of clonal variation in a hypervariable region.

The ribosomal RNA genes of the protozoan parasite Entamoeba histolytica are highly repeated and display restriction fragment length polymorphism. Using a set of four DNA probes spanning the coding region and part of the flanking region of the E. histolytica ribosomal RNA genes, an analysis of the DNA bands generated by EcoRI digestion of Entamoeba DNA is presented. This analysis included five strains of E. histolytica, four strains of E. moshkovskii, and one strain each of E. invadens and E. terrapinae. No common bands were observed between E. histolytica and the other Entamoeba. Within E. histolytica, two bands were conserved in all strains while the others were polymorphic. Detailed analysis of DNA from independently isolated clones of the strain HM-1:IMSS of E. histolytica showed two bands to be highly polymorphic. Of these, the 4.4-kb band of clone 6 was further analyzed. Polymorphism in this band could even be demonstrated in cells of the same clone. Restriction enzyme analysis of this DNA band from two clones of HM-1:IMSS showed that the polymorphism may be due to variable numbers of DraI repeat units present in this DNA stretch.     

Entamoeba histolytica: rapid identification and differentiation of Indian isolates by riboprinting

Entamoeba histolytica infection still remains one of the major public health problem for developing countries like India. A rapid and accurate detection of this parasite is essential for prevention and control of amoebiasis. In this study, using the method of 'riboprinting' (PCR-RFLP of rRNA genes from amoeba) we have analysed 15 stool samples from symptomatic patients of amoebiasis. All 15 patients of clinical amoebiasis had E. histolytica in their stool and two of the samples also showed mixed infection of E. dispar. Apart from the known restriction enzyme sites within the amoeba SSU-rRNA genes, a new Sau3A site having a discriminatory value is identified in these E. histolytica isolates from India. Hence, it is possible to rapidly identify E. histolytica DNA and differentiating it from E. dispar using minute amounts of clinical stool samples, thus eliminating the laborious parasite culturing process. Thus, riboprinting is advantageous for clearcut identification of E. histolytica in order to decide an effective antiamoebic therapy.     

Autonomous replication sequences in an extrachromosomal element of a pathogenic Entamoeba histolytica.

Entamoeba histolytica possesses a 24.5 kilobase plasmid-like molecule which encodes for the organism's ribosomal RNAs. Sequence analysis of this extrachromosomal element revealed the presence of AT rich sequences which show homology to the origin of replication of other lower eucaryotes. An 802 bp fragment containing these sequences was cloned into a yeast shuttle vector lacking the origin of replication and the construct tested for its ability to replicate autonomously in yeast. Mitotic stability tests as well as evidence for plasmid maintenance indicate that the transformed cells contained self-replicating episomes and not stably integrated molecules. The nucleotide sequence of this ARS-containing fragment is presented.     

Extrachromosomal DNA of pea-root (Pisum sativum) has repeated sequences and ribosomal genes

Summary Restriction endonuclease digestion and Southern blotting procedure were used to determine differences between extrachromosomal, nuclear, plastid, and mitochondrial DNAs from meristematic cells of cultured pea roots.Extrachromosomal and nuclear DNA are highly methylated and neither DNA is homologous to plastid or mitochondrial DNA. Hybridization of extrachromosomal DNA to nuclear DNA indicated that extrachromosomal DNA differed quantitatively from total nuclear DNA in repetitive sequences. Cloned rDNA showed that extrachromosomal DNA contains rRNA genes but the hybridization signal indicated that the copy number was less than that expected if the molecules were amplified. These and cytological findings suggest that extrachromosomal DNA is involved in or a product of genomic changes associated with the onset of differentiation by precursor cells of vascular parenchyma and the root cap.     

Characterisation of a repetitive DNA family from Entamoeba histolytica containing Saccharomyces cerevisiae ARS consensus sequences

Several repetitive DNA families were identified in Entamoeba histolytica DNA digested with Sau3AI. Characterisation of one of these repetitive DNA families showed the presence of multiple copies of Saccharomyces cerevisiae autonomously replicating sequence (ARS) core consensus sequences. The E. histolytica ARS consensus sequences allowed a yeast-integrating plasmid, YIP5, to replicate autonomously in S. cerevisiae. A 'bent DNA' fragment was located in one member of this E. histolytica repetitive DNA family.     

Entamoeba histolytica: DNA carrier vesicles in nuclei and kinetoplast-like organelles (EhkOs)

Entamoeba histolytica, the protozoan responsible for human amoebiasis, has a complex genome, whose linear chromosomes and DNA circles have so far eluded detailed analysis. We report the detection by transmission electron microscopy of nuclear vesicles (0.05-0.3 microm in diameter) carrying DNA in E. histolytica trophozoites. In late anaphase many of these nuclear vesicles were found to be organized in structures of approximately 2.5 x 1 microm, in association with chromosomes and microtubules. In glutaraldehyde-fixed and detergent-treated trophozoites, nuclear vesicles displayed a non-membranous envelope. Binding of phosphotungstate stain and recognition by serum from patients with systemic lupus erythematosus indicated that these vesicles contain DNA. Similar DNA carrier vesicles were found in the cytoplasm and in the E. histolytica kinetoplast-like organelle (EhkO). By Feulgen staining, we detected DNA carrier vesicles entering or leaving the nuclei, suggesting a structural relationship between the nuclear vesicles and the vesicles present in the EhkOs.     

The Entamoeba histolytica rDNA episome: nuclear localization, DNAase I sensitivity map, and specific DNA-protein interactions

Structural and functional features of the extrachromosomal DNA element that contains the ribosomal RNA genes of Entamoeba histolytica were studied using a variety of techniques. Using in situ hybridization, the element was found to be distributed along the inner phase of the nuclear membrane in the trophozoite stage; it appears to be part of the so-called peripheral chromatin. DNAase I-sensitive regions on the episome were mapped and found to correspond to the borders of the ribosomal RNA coding region. Other DNAase I-sensitive regions were found to correspond to DNA containing a 145bp sequence that exists in the episome as tandem repeats. Electrophoretic shift assays and footprinting experiments demonstrate the existence of specific nuclear factors that bind specifically to the 145bp repeat. Preliminary analysis of the binding factors showed that a 28 kDa polypeptide is a likely candidate for a specific DNA:protein interaction involving the repetitive element. These results suggest that a protein-binding domain within the 145bp repeat may have a specific function in the episome.     

Lack of a chromosomal copy of the circular rDNA plasmid of Entamoeba histolytica

A number of small circular DNAs constitute a part of the genome of Entamoeba histolytica. Among them, the 24.5 kb circular DNA encoding rRNA (EhR1) is the most abundant. Pulsed field gel electrophoresis was used to determine if a chromosomal copy of EhR1 exists and what fraction of the total genome is circular. The results show that the chromosomes of E. histolytica are linear, and that no copy of EhR1 could be detected in any of the linear chromosomes.     

DNA amplification in arsenite-resistant Leishmania

Arsenite-resistant variants of a trypanosomatid protozoan, Leishmania mexicana amazonensis, were selected in vitro by stepwise increases of sodium arsenite concentrations up to 30 microM in the culture medium. These variants were found to contain amplified DNA as extrachromosomal supercoiled molecules of about 69 kb. They originate from a single chromosome in the wild-type cells. There is evidence of chromosomal changes in these cells associated with the selection for arsenite resistance. The apparent absence of these circular molecules in the wild type and their loss from the drug-sensitive revertants suggest amplification of chromosomal DNA into these extrachromosomal circles as the mechanism of arsenite resistance.     

Genomic DNA microarrays for Entamoeba histolytica: applications for use in expression profiling and strain genotyping

The parasite Entamoeba histolytica is a causative agent of dysentery and liver abscesses. Found predominantly in developing countries, this parasitic infection is responsible for significant morbidity and mortality. We have developed a genomic DNA microarray for E. histolytica. The array composed of 11,328 clones contains >2000 unique genes and was utilized for expression profiling and comparative genomic hybridizations of Entamoeba strains. We present a synopsis of our results to date and potential future applications of microarray technology for the study of Entamoeba biology.     

Comparative genomic hybridizations of Entamoeba strains reveal unique genetic fingerprints that correlate with virulence

Variable phenotypes have been identified for Entamoeba species. Entamoeba histolytica is invasive and causes colitis and liver abscesses but only in approximately 10% of infected individuals; 90% remain asymptomatically colonized. Entamoeba dispar, a closely related species, is avirulent. To determine the extent of genetic diversity among Entamoeba isolates and potential genotype-phenotype correlations, we have developed an E. histolytica genomic DNA microarray and used it to genotype strains of E. histolytica and E. dispar. On the basis of the identification of divergent genetic loci, all strains had unique genetic fingerprints. Comparison of divergent genetic regions allowed us to distinguish between E. histolytica and E. dispar, identify novel genetic regions usable for strain and species typing, and identify a number of genes restricted to virulent strains. Among the four E. histolytica strains, a strain with attenuated virulence was the most divergent and phylogenetically distinct strain, raising the intriguing possibility that genetic subtypes of E. histolytica may be partially responsible for the observed variability in clinical outcomes. This microarray-based genotyping assay can readily be applied to the study of E. histolytica clinical isolates to determine genetic diversity and potential genotypic-phenotypic associations.     

Absence of rDNA amplification in the uninucleolate oocyte of the cockroach Blattella germanica (Oorthoptera: Blattidae)

Amplification of the genes coding for ribosomal RNA oocurs in the oocytes of a wide variety of organisms. In oocytes of various species of crickets (Orthoptera: Gryllidae) the amplified DNA is contained in a large extrachromosomal DNA body. Multiple nucleoli form about the periphery of the DNA body during the diplotene stage of meiosis I. In contrast to the general pattern of orthopteran oocytes, oocytes of the cockroach Blattella germanica demonstrate a single large nucleolus instead of many nucleoli. In order to determine whether the genes coding for rRNA are amplified in the oocytes of B. germanica, the relative amount of rDNA in oocytes was compared with the rDNA content of spermatocytes and somatic cells. An extrachromosomal DNA body similar to that present in crickets is not present in B. germanica. A satellite DNA band which contains nucleotide sequences complementary to rRNA accounts for approximately 3-5% of the total DNA in somatic and in male and female gametogenic tissues. Female cells contain approximately twice as much rDNA as do male cells. An XX-XO sex-determining mechanism is operative in B. germanica. In situ hybridization with rRNA indicates that the nucleolar organizer is located on one end of the X chromosome and that oocytes do not contain more than twice the amount of rDNA found in spermato cytes. The data indicate that rDNA is not amplified in the uninucleolate oocyte of B germanica.