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The contribution of DNA slippage to eukaryotic nuclear 18S rRNA evolution.

Six of 204 eukaryotic nuclear small-subunit ribosomal RNA sequences analyzed show a highly significant degree of clustering of short sequence motifs that indicates the fixation of products of replication slippage within them in their recent evolutionary history. A further 72 sequences show weaker indications of sequence repetition. Repetitive sequences in SSU rRNAs are preferentially located in variable regions and in particular in V4 and V7. The conserved region immediately 5' to V7 (C7) is also consistently repetitive. Whereas variable regions vary in length and appear to have evolved by the fixation of slippage products, C7 shows no indication of length variation. Repetition within C7 is therefore either not a consequence of slippage or reflects very ancient slippage events. The phylogenetic distribution of sequence simplicity in small-subunit rRNAs is patchy, being largely confined to the Mammalia, Apicomplexa, Tetrahymenidae, and Trypanosomatidae. The regions of the molecule associated with sequence simplicity vary with taxonomic grouping as do the sequence motifs undergoing slippage. Comparison of rates of insertion and substitution in a lineage within the genus Plasmodium confirms that both rates are higher in variable regions than in conserved regions. The insertion rate in variable regions is substantially lower than the substitution rate, suggesting that selection acts more strongly on slippage products than on point mutations in these regions. Patterns of coevolution between variable regions may reflect the consequences of selection acting on the incorporation of slippage-derived sequences across the gene.

Animals↗

The evolutionary origin of the 35 kb circular DNA of Plasmodium falciparum: new evidence supports a possible rhodophyte ancestry.

In common with other Apicomplexan parasites, Plasmodium falciparum carries two extrachromosomal DNAs, one of which, the 6 kb element, is undoubtedly mitochondrial. The second, generally referred to as the 35 kb circle, is of unknown provenance, but the nature and organization of its genetic content makes a mitochondrial association unlikely and the molecule has features reminiscent of plastid genomes. We now report the occurrence on the circle of an open reading frame specifying a predicted 470 amino acid protein that shares more than 50% identity with a gene currently known only on the plastome of red algae. This high degree of conservation confirms the 35 kb circle's plastid ancestry, and we speculate that it may have originated from the rhodoplast of an ancient red algal endosymbiont in the progenitor of the Apicomplexa.

Amino Acid Sequence↗

Genetic comparison of Neospora caninum with Toxoplasma and Sarcocystis by random amplified polymorphic DNA-polymerase chain reaction.

To determine the relationship of Neospora caninum to protozoa classified in the family Sarcocystidae of the phylum Apicomplexa, the genomes of N. caninum, three Toxoplasma gondii strains (RHa, CEP, TPR) and three Sarcocystis species (S. tenella, S. muris, S. gigantea) that were thought to be closely related coccidia were compared by the random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) technique. The genomic DNAs were amplified by the use of seven 10-mer arbitrary sequence primers to generate polymorphic DNA. Significant DNA polymorphisms were observed among Neospora, Toxoplasma and Sarcocystis. It appears that one primer tested may have value in a diagnostic RAPD-PCR to differentiate T. gondii from other closely related protozoa. The high level of genetic divergence of N. caninum from T. gondii strains and several Sarcocystis species observed in this study is consistent with the hypothesis that N. caninum is indeed an independent species of protozoan parasite. As compared with the Sarcocystis species tested, a closer genetic relationship of N. caninum to T. gondii was not observed. By contrast, a closer genetic relationship of S. muris to T. gondii was revealed in this study.

Animals↗

The SAG2 antigen of Toxoplasma gondii and the 31-kDa surface antigen of Sarcocystis muris share similar sequence features.

In the search for similar cysteine distribution patterns among surface antigens of members of the phylum of Apicomplexa, we found a resemblance between TgSAG2, a 22-kDa surface antigen of Toxoplasma gondii, and the C-terminal half of a 31-kDa surface antigen of Sarcocystis muris, SmSAG1. The overall degree of similarity is low, corresponding to 28% of identity in the 146 amino acids aligned. However, the conservative spacing between the cysteine residues suggest that this similarity might have functional implications. The described amino acid similarity provides a new clue to the molecular relationship between two closely related coccidian parasites.

Amino Acid Sequence↗

The fine structure of Garnia gonadati and its association with the host cell.

Most of the studies on the fine structure of protozoa of the Apicomplexa group have been carried out with members of the ToxoPlasma, Eimeria, and Plasmodium genera. In the present study we analyzed the fine structure of Garnia gonadoti parasitizing the red blood cells of the Amazonian reptile Gonatodes humeralis (Reptilia; Lacertilia). Transmission electron microscopy of thin sections showed that G. gonadoti presented all structures characteristic of the group, including the apicoplast. However, four special features were observed: (1) absence of the hemozoin (malarial) pigment; (2) a group of microtubules associated with the mitochondrion; (3) a vacuole containing electron-dense material, which resembled the acidocalcisome described in trypanosomatids; and (4) a special array of the host-cell endoplasmic reticulum around the parasitophorous vacuole.

Animals↗

Lateral transfer and recompartmentalization of Calvin cycle enzymes of plants and algae.

Certain Calvin cycle enzymes also function in glycolysis or gluconeogenisis, thus photosynthetic eukaryotes would be predicted to have ancestrally possessed cytosolic homologues of these enzymes derived from the eukaryotic host and plastid homologues from the cyanobacterial endosymbiont. In practice, the evolutionary histories of these enzymes are often more complex. Focusing on eukaryotes with secondary plastids, we have examined the evolution of four such genes: class I and II fructose bisphosphate aldolase (FBA), sedoheptulose bisphosphatase (SBPase), and fructose bisphosphatase (FBPase). We show that previously observed distributions of plastid and cytosolic homologues are not always found in algae with secondary plastids: there is evidence for multiple events of both lateral gene transfer and retargeting to a new cellular compartment for both cytosolic and plastid enzymes of plants and algae. In particular, we show that a clade of class II FBAs spans a greater diversity of eukaryotes that previously recognized and contains both plastid-targeted (Phaeodactylum, Odontella) and cytosolic (ascomycetes, oomycetes, Euglena, and Bigelowiella) forms. Lateral transfer events also gave rise to a subset of plant cytosolic FBA, as well as cytosolic FBPase in Toxoplasma and other coccidian apicomplexa. In contrast, it has recently been suggested that the Trypanosoma FBA and SBPase are derived from a plastid, however, greater taxonomic sampling shows that these enzymes provide no evidence for a plastid-containing ancestor of Trypanosoma. Altogether, the evolutionary histories of the FBA and SBPase/FBPase gene families are complex, including extensive paralogy, lateral transfer, and retargeting between cellular compartments.

Eukaryota↗

Notes on coccidian phylogeny, based on the apicoplast small subunit ribosomal DNA.

We performed a phylogenetic analysis, based on the partial small subunit rRNA gene (SSU rRNA) sequences from 13 apicoplasts (including new sequences of Sarcocystis muris and Hyaloklossia lieberkuehni) and 16 other plastids, with cyanobacteria as an outgroup. The apicoplast sequences formed a highly supported monophyletic clade with two distinct clades, representing coccidia and haemosporidia, with coccidia divided into Eimeriidae and Sarcocystidae subclades. We confirmed the phylogenetic position of H. lieberkuehni within the Sarcocistidae, as a sister to the Toxoplasma/Neospora group. The coccidian plastid sequences appear to evolve slowly, while their homologues from haemosporidians are more rapidly evolving. We suggest that the higher evolutionary rate is reflected by the increase in the AT content and the possible reduction of the outer apicoplast membrane in some haemosporidians. Since the apicoplast SSU rRNA gene sequences, when compared with their nuclear homologues, offer a higher number of informative positions, they can be used for phylogenetic inference within the Apicomplexa.

Animals↗

Some observations on the fine structure of trophozoites of the haemogregarine Cyrilia lignieresi (Adeleina: Haemogregarinidae) in erythrocytes of the fish Synbranchus marmoratus (Synbranchidae).

This communication describes the fine structure of trophozoites of the haemogregarine Cyrilia lignieresi (Laveran, 1906) found in erythrocytes of the fresh-water fish Synbranchus marmoratus from Belém, Pará, North Brazil. The parasite possesses the usual structures, such as conoid, rhoptries and micronemes, seen in members of the phylum Apicomplexa. Three structures, however, appear to be characteristic features of this parasite. The parasitophorous vacuole is unusual in containing a large number of spherical bodies. Secondly, some of the dense bodies, which are usually spherical organelles, may appear as elongated structures. Thirdly, peculiar invaginations of the inner membrane appear to divide the parasite into compartments.

Animals↗

Phylogeny and sequence variability of the Sarcocystis singaporensis Zaman and Colley, (1975) 1976 ssrDNA.

The coccidium Sarcocystis singaporensis (Apicomplexa: Sarcocystidae) is a cyst-forming parasite with potential as a biological agent for the control of wild populations of rodents in non-native environments. Phylogenetic analysis based on the ssrDNA supports S. singaporensisisolates as a sister species to sarcosporidians transmitted between snakes and rodents but an association with the carnivore-ruminant Sarcocystis spp. could not be rejected by likelihood ratio tests. Four complete and six partial ssrDNA sequences representing this species are monophyletic in any tree reconstruction method; however, they possess very high pairwise distances of up to 0.053. The obtained sequences suggest the probable existence of at least two divergent paralogous ssrDNAs. Moreover, our results support the co-evolution of lsrDNA and ssrDNA in S. singaporensis. The utility of coccidian lsrDNA and ssrDNA for evolutionary studies and their abundance in the primary nucleotide databases is discussed.

Animals↗

Molecular cloning and characterization of an SRCAP chromatin remodeling homologue in Toxoplasma gondii.

We have identified and mapped a gene in Toxoplasma gondii that encodes a homologue of SRCAP (Snf2-related CBP activator protein), a member of the SNF/SWI family of chromatin remodeling factors. The genomic locus (TgSRCAP) is present as a single copy and contains 16 introns. The predicted cDNA contains an open reading frame of 8,775 bp and encodes a protein of 2,924 amino acids. We have identified additional SRCAP-like sequences in Apicomplexa for comparison by screening genomic databases. An analysis of SRCAP homologues between species reveals signature features that may be indicative of SRCAP members. Expression of mRNA encoding TgSRCAP is upregulated when tachyzoite (invasive form) parasites are induced to differentiate into bradyzoites (encysted form) in vitro. Recombinant TgSRCAP protein is functionally equivalent to the human homologue, being capable of increasing transcription mediated by CREB.

Adenosine Triphosphatases↗

Microneme antigens of Eimeria bovis recognized by two monoclonal antibodies.

Two IgG1 monoclonal antibodies (mAbs 8-23F9 and 9-21G9) were developed after immunization of mice with homogenates of Eimeria bovis first-generation merozoites. Both mAbs reacted with antigens in the apical two-thirds of the parasites and immune electron microscopy determined the micronemes as targets. When tested by immunoblotting, mAb 8-23F9 failed to react with antigens separated under reducing conditions; under nonreducing conditions it recognized two components of >200 kDa. mAb 9-21G9 bound to antigens of 135 and 180 kDa after electrophoresis under reducing conditions and to a series of components when separated without reduction. The epitope of mAb 8-23F9 was destroyed by treatment of the antigen with endoglycosidase H and removal of phosphocholine (PC) by phospholipase C. Since mAb 8-23F9 does not recognize cytidine-linked PC, the data suggest that PC in combination with N-linked sugars and/or N-glycans is part of its epitope. In the case of mAb 9-21G9, endoglycosidase H did not alter the epitope. When E. bovis merozoite antigen was treated with phospholipase C the number of mAb 9-21G9-reactive constituents increased, suggesting that PC may otherwise mask the epitope. mAb 8-23F9 also bound to the apical area and the surface of E. bovis sporozoites and recognized a >200-kDa sporozoite component. When sporozoites invaded Vero cells in vitro, epitope-bearing components were released onto the host cell surface and became part of the early parasitophorous vacuole wall. At day 5 the binding of the mAb was again confined to the intracellular parasite. mAb 9-21G9 did not react with sporozoites but recognized the apical area of intra-cellular trophozoites on day 5 after invasion of host cells in vitro. When testing was done against a variety of other Apicomplexa in various assays, the only cross-reaction observed occurred with mAb 8-23F9, which bound to a conformationally determined 180-kDa component of Toxoplasma gondii cystozoites.

Animals↗

The severe combined immunodeficient mouse as a definitive host for Sarcocystis muris.

Peroral and intraperitoneal inoculation of severe combined immunodeficient (SCID) mice with cystozoites of three coccidia of the genus Sarcocystis (Protozoa, Apicomplexa; S. dispersa, Sarcocystis sp., and S. muris) revealed that after peroral administration, only S. muris could develop in the immunodeficient mouse host. The cystozoites of S. muris transformed into gamonts and, after fertilization, performed sporulation with the production of infectious sporocysts in the small intestine of the SCID mice. Impaired immunity is probably responsible for the unusual behavior of S. muris (which is normally the heteroxenous mouse-cat parasite) in the SCID mice. We hypothesize that the phylogenetic distance between the intermediate and final hosts is the reason why cystozoites of the two other Sarcocystis species tested (S. dispersa with a mouse-owl cycle and Sarcocystis sp. with a murine rodent-snake cycle) could not develop when inoculated into SCID mice.

Animals↗

Characterization of a monoclonal antibody reacting with antigen-4 domain of gp900 in Cryptosporidium parvum invasive stages.

Cryptosporidium parvum (Protozoa, Apicomplexa) infects the apical surface of intestinal epithelial cells, where it grows and divides within a membrane-bound parasitophorous vacuole. gp900, an abundant glycoprotein of C. parvum merozoites and sporozoites, is localized in micronemes and at the surface of invasive stages and participates in the invasion process. Here, we describe a new monoclonal antibody (mAb) against gp900. As shown by immunofluorescence of excysted parasites and immunoelectron microscopy of infected tissues, the mAb reacted with micronemes present in the apical pole of invasive stages. In immunoprecipitation experiments, the mAb was shown to react with a high molecular weight antigen co-migrating with gp900. Finally, three reactive clones were selected upon screening of a C. parvum genomic expression library with the mAb; and sequencing of the insert from one of them showed a 596 bp sequence identical to the DNA region encoding a domain of gp900 identified as antigen 4.

Animals↗

A light and electron microscope study of Sarcocystis mitrani (sp. nov.) infecting the skink Scincus mitranus in the central region of Saudi Arabia.

The prevalence of Sarcocystis infection among skinks, Scincus mitranus, was studied for the first time. Grossly macroscopic sarcocysts were found to infect the skeletal muscles of the skink (infection rate: 4.16%). Fecal examination for the presence of sporocysts was negative in this study. Sarcocysts were studied using light and transmission electron microscopes. Mature sarcocysts measuring 0.05-0.3 x 0.5-1.8 mm (mean 0.1 5x 1.2 mm) were observed. The characteristic primary cyst wall, with long, finger-like, non-branched and non-stalked protrusions, is described. The ground substance gives rise to numerous thick septa dividing the interior of the cyst into chamber-like compartments. Zoites, including metrocytes and merozoites, were found to have the main architecture of Apicomplexa. Peculiarities of these elements and the importance of the primary cyst-wall ultrastructure for identification and specification of Sarcocystis are discussed. Secondary cyst wall was completely absent. Alterations in the infected host cell were observed.

Animals↗

The Duffy receptor family of Plasmodium knowlesi is located within the micronemes of invasive malaria merozoites.

Plasmodium vivax and Plasmodium knowlesi merozoites invade human erythrocytes that express Duffy blood group surface determinants. A soluble parasite protein of 135 kd binds specifically to a human Duffy antigen. Using antisera affinity purified on the 135 kd protein, we cloned a gene that encodes a member of a P. knowlesi family of erythrocyte binding proteins. The gene is a member of a family that includes three homologous genes located on separate chromosomes. Two genes are expressed as major membrane-bound products that give rise to soluble erythrocyte binding proteins: the 135 kd Duffy binding protein and a 138 kd protein that binds only rhesus erythrocytes. These different erythrocyte binding specificities may result from sequence divergence of the homologous genes. The Duffy receptor family is localized in micronemes, an organelle found in all organisms of the phylum Apicomplexa.

Amino Acid Sequence↗

Ribosomal DNA sequence comparison of Babesia and Theileria.

Previous studies on the taxonomy of Babesia spp. (phylum Apicomplexa) using morphological and life cycle characteristics have resulted in their classification into 3 subgenera, with the genus Theileria being most closely related to them. Using a strategy based on the direct sequence analysis of products derived by asymmetric PCR to determine the nucleotide sequences, we have tested the validity of this classification by sequencing the small subunit ribosomal RNA genes amplified from 2 Babesia species, namely Babesia bovis and Babesia rodhaini, and comparing these with previously published sequences of Theileria annulata and Babesia bigemina using Plasmodium falciparum as an outgroup. The results of this phylogenetic analysis support the recognition of at least 2 genera in Babesia--one to include B. bigemina and B. bovis, the other to include B. rodhaini.

Animals↗

Characterization of iron-dependent endogenous superoxide dismutase of Plasmodium falciparum.

Two main superoxide dismutase activities at isoelectric points (pI) 6.2 and 6.8 and two minor at pI 5.6 and 6.4 were found in crude extracts of Plasmodium falciparum. These activities were cyanide-resistant and hydrogen peroxide-sensitive and represented 20-30% of the total SOD activity found in the crude extract. A fragment of 424 bp, amplified from genomic DNA from P. falciparum, was cloned and sequenced. The deduced amino acid sequence identified this fragment as a coding region of an SOD gene. A cDNA corresponding to SOD was then isolated from a P. falciparum cDNA library and sequenced. The deduced amino acid sequence of SOD (197 aa) was compared with 32 known Feor Mn-SODs by the 'DARWIN' system. This analysis showed that the parasitic enzyme was related to typical Fe-SODs. The SOD subunit was purified and the N-terminal sequence, determined up to 29 residues, corresponded to that of cDNA isolated. The iron-dependent SOD activity found in Plasmodium falciparum represents the first level of the antioxidant defence system of the parasite. It is also the first SOD characterized in the parasitic Apicomplexa phylum whose sequence can be compared to equivalent iron-dependent enzymes known in other protozoa and bacteria.

Amino Acid Sequence↗

Use of proteinase K in the excystation of Sarcocystis cruzi sporocysts for in vitro culture and DNA extraction.

Proteinase K was used for the cleaning of Sarcocystis cruzi (Apicomplexa) sporocysts prior to excystation. Bovine pulmonary endothelial cell cultures inoculated with the excysted sporozoites remained free of bacterial contamination for the duration of the experiment and had high yields of merozoites. The excysted sporozoites also yielded genomic DNA that could be labelled efficiently with 32P dATP by the random priming method.

Animals↗