Ubiquinone-9 of the flagellates Crithidia oncopelti and Astasia longa

The content of ubiquinones (Co Q) of the Astasia longa and Crithidia oncopelti protozoa was studied. The protozoa were grown on an artificial nutrient broth. The cells were separated, washed, freeze-dried, and refluxed with KOH and pyrogallol in ethanol media. The hydrolyzate was concentrated. The residue was stored at -20 degrees and filtered. An ubiquinone fraction was isolated from the filtrate by TLC on silica gel. Identification of the ubiquinone homologues was carried out by reverse phase TLC and mass spectrometry. Ubiquinones were quantitated with respect to the difference in the density between the oxidized and reduced forms of Co Q at 275 nm. The A. longa and C. oncopelti flagellates were shown to contain ubiquinone-9 (Co Q9) at a concentration of 0.48 and 1.14 mumole/g dry cells, respectively. The higher Co Q level in zooflagellates as compared to that in phytoflagellates is discussed.     

The trypanocidal action of fluorouracil on Crithidia oncopelti in the presence of lipid metabolic modifiers]

Effect of fluorouracil in combination with ascorbate and L-valin, modifiers of lipid metabolism was studied in cultured protozoa Crithidia oncopelti. The inhibitory effect of preparation in a concentration of 100 mcg/ml gradually decreased in the course of cultivation. Its readdition to a 96-hour culture did not increase its effect on protozoa cells. Combined addition of fluorouracil and modifiers (100 mcg/ml each) resulted in insignificant decrease of the cell accumulation in the culture as compared with the effect of fluorouracil alone. When fluorouracil and modifiers were readded to the 96-hour culture, the trypanostatic effect of preparation was 2.5 times enhanced. This enhancement was confirmed by destructive alterations in cell morphology and by the culture lysis by 192 h of protozoa cultivation.     

DNA-dependent RNA polymerase activity of isolated zooflagellates (Crithidia oncopelti) nucleoli

A fraction of nucleoli is isolated from zooflagellates (Crithidia oncopelti) nuclei, its DNA-dependent RNA polymerase activity is studied at different temperature, ionic strength and Mg2+, Mn2+ and antibiotic concentrations. The effect of some factors and alpha-amantine on RNA polymerase activity of exonucleolar chromatin was studied as a control. A comparison of heat denaturation of nucleoli and chromatin RNA polymerase activities within the temperature range 30--55 degrees C has revealed a higher thermosensitivity of nucleoli RNA polymerase. Substitution of Mg2+ with equivalent amount of Mn2+ results in a considerable decrease of rRNA synthesis in nucleoli. Nucleoli RNA polymerase activity in the presence of Mg2+ is sensitive to the elevation of ionic strength from 0.12 to 1.30 u; chromatin RNA polymerase activity in the presence of Mn2+ is maximal at high ionic strength (1.30 mu). alpha-Amantine and cycloheximide at high concentrations (10 and 200 mkg/ml) practically do not affect RNA polymerase activity of nucleoli. Nucleoli RNA polymerase of zooflagellates (Crithidia oncopelti) is similar to the A-form of the enzyme in higher eukaryotes.     

Presence of genes of ribosomal and transfer RNAs in kinetoplast DNA from two Crithidia species

The coding properties of kinetoplast DNA from two respresentatives of the order Kinetoplastidae--Crithidia oncopelti and C. fasciculata--were studied. Experiments on hybridization of the whole network and fraction of minicircles with labelled 23S and 16S rRNA and with tRNA isolated from kinetoplasts of C. oncopelti clearly demonstrated the presence of the genes for these RNAs in the whole network and their absence in the minicircles. It may be thus concluded that the genes of ribosomal and transfer RNAs are localized in the maxicircular molecules. Similar efficiency of hybridization of rRNAs from C. oncopelti with kDNA from C. fasciculata and C. oncopelti revealed significant conservativity of ribosomal genes in the protozoa under study.     

Evidence for the amino-acid sequence of Crithidia fasciculata Cytochrome c555.

Evidence is presented which indicates that the amino acid sequence of cytochrome c555 from Crithidia fasciculata differs at sixteen positions from that of cytochrome c557 from Crithidia oncopelti. 101 residues were identified by dansyl-Edman degradation, carboxypeptidase digestion or considerations of the specificity of trypsin and of these, thirteen were found to be different from C. oncopelti cytochrome c557. The remaining 11 residues found in the amino acid composition of the trypic peptides were aligned on the basis of homology with cytochrome c557 and three further differences are proposed. The total of sixteen amino acid differences is surprising in view of the morphological and biochemical similarities of these organisms, and illustrates the problem of taxonomy of morphologically simple organisms. In both cytochromes only one cysteine residue is involved in the attachment of the protein to the prosthetic group.     

Kinetoplast DNA of Crithidia oncopelti. Recognition of the principles of structural organization of minicircular DNA molecules

The heterogeneity of the kinetoplast minicircular DNAs from Crithidia oncopelti was studied by electron microscopy and restriction analysis. The associate of kinetoplast DNA comprises five main classes of minicircles sizing 0.42, 0.51, 0.62, 0.78 and 0.83 microns. Examination of cleavage patterns revealed that each class of the minicircles is heterogeneous in base sequence, but there are restriction fragments of the same size common for all these classes. A model of structural arrangement of minicircles of Crithidia oncopelti was proposed. This model is based on the block structure of minicircular molecules. Each minicircle is composed of one variable block (V-block), 810 base pairs, and from 1 to 4 more conservative blocks (C-blocks) each containing 445 base pairs.     

Isolation of ribonucleic acids possessing template activity from Crithidia oncopelti mitochondria

Three RNA fractions (12SE, 9SE and 8SE) were isolated from highly purified mitochondrial preparations of Crithidia oncopelti. Using inhibitory analysis and competitive hybridization, it was shown that the mitochondrial fractions under study are synthesized in the kinetoplasts and do not belong to ribosomal RNA. It was demonstrated that these fractions possess template activity, since they stimulate protein synthesis in a cell-free system of E. coli. Each fraction is subjected to translation to form one predominant polypeptide with molecular weight of about 20 000, 10 000 and 7000, respectively.     

The Sterol Content of Some Protozoa

SYNOPSIS. The sterols of a number of protozoa grown in axenic culture have been examined. Ergosterol is present in Polytoma uvella, Astasia ocellata, Haematococcus pluvialis, Crithidia oncopelti, Prototheca zopfii, Chilomonas paramoecium and Trypanosoma mega , all grown on sterol-free media. Ergosterol is also present in the culture form of Trypanosoma rhodesiense and occurs together with cholesterol and an unidentified sterol in Peranema trichophorum grown in the presence of cholesterol. P. uvella, A. ocellata, H. pluvialis and C. oncopelti also contain a sterol which is either spinasterol or its isomer chondrillasterol. The main sterol of Ochromonas malhamensis is probably poriferasterol, which is also present in C. paramoecium. Cholesterol, probably from the environment, is the only sterol in the blood form of T. rhodesiense. Tetrahymena pyriformis and Hartmannella rhysodes do not synthesize sterols.     

Isolation of intracellular symbiotes by immune lysis of flagellate protozoa and characterization of their DNA

A new method dependent on immune lysis is described for the isolation of intracellular symbiotes from two species of flagellate protozoa Blastocrithidia culicis and Crithidia oncopelti. The symbiote- containing flagellates are exposed to complement and antisera prepared in rabbits against symbiote-free organisms. The immune lysis seems to weaken the plasma membranes of the flagellates so that subsequent application of gentle shearing force liberates the intracellular entities in an undamaged condition. The symbiotes are then separated from other cellular components by DNAse digestion and differential centrifugation. The average recovery of symbiotes isolated by this method is 20%. Light and electron microscopy establishes the structural integrity and numerical abundance of isolated symbiotes in the final fractions. Integrity of symbiotes is further indicated by the high activity of a marker enzyme, uroporphyrinogen I synthetase. The DNA's of symbiote-containing and symbiote-free flagellates, and of isolated symbiotes were purified and compared after isopycnic centrifugation. The comparison establishes the presence of DNA's in symbiotes of both species. The guanine-cytosine (G-C) content of symbiote DNA differs from that of host DNA's in C. oncopelti, but resembles that of kinetoplast DNA in B. culicis. The latter observation was further shown by heat denaturation study. Renaturation kinetics indicate that the genome complexity of symbiote DNA in B. culicis is similar to that of bacteria.     

DNA-dependent RNA-polymerase activity of isolated kinetoplasts of crithidia oncopelti]

DNA-dependent RNA-polymerase activity was found in the kinetoplasts of Crithidia oncopelti. Kinetic patterns of 14C-UTP incorporation into the acid-insoluble fraction of isolated kinetoplasts at 25 degrees, 30 degrees and 35 degrees C were estimated. The effects of different antibiotics and intercalating agents on RNA synthesis in kinetoplasts were studied. alpha-amanitin, a specific inhibitor of the nuclear enzyme, does not affect the RNA-polymerase activity of the kinetoplasts. Streptolidigin and rifampicin, inhibitors of bacterial RNA-polymerase, have little effect on RNA synthesis in the kinetoplasts even at high concentrations. Kinetoplasts preincubation in the phosphate buffer increases the permeability of their membranes for rifampicin. Intercalating agents, acriflavin and ethidium bromide, strongly inhibit the kinetoplast RNA synthesis. Similar specific effects of some antibiotics and intercalating agents on RNA synthesis in kinetoplasts and typical mitochondria may be indicative of similarity of functional properties of RNA-polymerases in those organelles.     

Heliomycin suppression of RNA synthesis in a cell-free system]

Heliomycin inhibited in vitro the RNA-polymerase reaction catalyzed by the preparation of DNA-dependent RNA-polymerase from E. coli. The blocking effect increased with a rise in the antibiotic concentration. The inhibitory effect of heliomycin decreased, when the amount of RNA-polymerase in the system increased. Yet, it did not depend on the content of DNA and the nature of the DNA preparation. Preincubation of RNA-polymerase with DNA resulting in formation of the enzyme-matrix complex did not prevent blocking RNA synthesis by heliomycin. Suppression of the RNA-polymerase reaction did not depend on the time of the antibiotic addition to the polymerizing system. Heliomycin had a significant activity not only with respect to the bacterial RNA-polymerase, but also in the system containing the enzyme isolated from the cells of Crithidia oncopelti.     

An Electron Microscopic Study of the Bipolar Bodies in Crithidia oncopelti

SYNOPSIS. An electron microscopic study of the structure of the flagellate Crithidia oncopelti was carried out with particular reference to the nature of the bipolar body which occurs in the organism. Apart from the bipolar body, the fine structure of C. oncopelti is similar to that of the related flagellate, C. fasciculata. The bipolar body is typically a sausage-shaped organelle limited by 2 unit membranes. Outpouchings of these membranes into the cytoplasm of C. oncopelti were found, along with a constant absence of (a) ribosomes on the outer aspect of the external of the 2 membranes, (b) structures analogous to nuclear pores and (c) an internal structure analogous to a nucleolus. It is concluded that the balance of structural and biochemical evidence favors the idea that the bipolar body is a bacterial protoplast rather than a 2nd nucleus.     

Composition and immunological activity of membrane-bound surface glycoprotein from Crithidia oncopelti

A membrane-bound glycoprotein with a molecular weight of 10000-12000 was isolated from Crithidia oncopelti and purified. The glycoprotein contained peptide, carbohydrate and lipid fragments and phosphorus. The peptide fragment was represented by 10 amino acids. The carbohydrate fragment was represented by 7 monosaccharides. The lipid part was mainly represented by stearic acid. The glycoprotein showed immunostimulating properties. It had a comitogenic effect on murine spleen cells in vitro and induced tumoricidal activity in murine peritoneal macrophages in vitro and in vivo.     

Physicochemical properties of kinetoplast DNA from Crithidia acanthocephali. Crithidia luciliae, and Trypanosoma lewisi

The protozoa Crithidia and Trypanosoma contain within a mitochondrion a mass of DNA known as kinetoplast DNA (kDNA) which consists mainly of an association of thousands of small circular molecules of similar size held together by topological interlocking. Using kDNA from Crithidia acanthocephali, Crithidia luciliae, and Trypanosoma lewisi, physicochemical studies have been carried out with intact associations and with fractions of covalently closed single circular molecules, and of open single circular and unit length linear molecules obtained from kDNA associations by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium centrifugation. Buoyant density analyses failed to provide evidence for base composition heterogeneity among kDNA molecules within a species. The complementary nucleotide strands of kDNA molecules of all three species had distinct buoyant densities in both alkaline and neutral cesium chloride. For C. acanthocephali kDNA, these buoyant density differences were shown to be a reflection of differences in base composition between the complementary nucleotide strands. The molar ratios of adenine: thymine:guanine:cytosine, obtained from deoxyribonucleotide analyses were 16.8:41.0:28.1:14.1 for the heavy strand and 41.6:16.6:12.8:29.0 for the light strand. Covalently closed single circular molecules of C. acanthocephali (as well as intact kDNA associations of C. acanthocephali and T. lewisi) formed a single band in alkaline cesium chloride gradients, indicating their component nucleotide strands to be alkaline insensitive. Data from buoyant density, base composition, and thermal melting analyses suggested that minor bases are either rare or absent in Crithidia kDNA. The kinetics of renaturation of 32P labeled C. acanthocephali kDNA measured using hydroxyapatite chromatography were consistent with at least 70% of the circular molecules of this DNA having the same nucleotide sequence. Evidence for sequence homologies among the kDNAs of all three species was obtained from buoyant density analyses of DNA in annealed mixtures containing one component kDNA strand from each of two species.     

Mini-exon Gene Sequences Define Six Groups within the Genus Crithidia

ABSTRACT. To develop molecular markers for lower trypanosmatids, we have examined the mini-exon gene repeats of 17 isolates that were classified as Crithidia by traditional methods. Representative repeats were amplified by polymerase chain reaction and the amplification products were cloned and used as hybridization probes against genomic DNA. Six hybridization groups of Crithidia were defined on the basis of the DNA blotting experiments. The three endosymbiont-bearing species ( C. deanei, C. desouzai and C. oncopelti ) and C. acanthocephali each belonged to single-member hybridization groups, while the C. fasciculata group contained additional named and undesignated species. The Crithidia lucilae thermophila probe hybridized to multiple undesignated isolates. The DNA sequence of the cloned products revealed that the specificity of the hybridization probes was due to substantial differences in the intron and the non-transcribed spacer regions. These data indicate substantial heterogeneity within the mini-exon gene locus of the taxon Crithidia .     

A cytochrome c methyltransferase from Crithidia oncopelti.

The mitochondrial cytochrome c-557 of Crithidia oncopelti contains two lysine residues and an N-terminal proline residue that are methylated in vivo by the methyl group of methionine. The purified cytochrome can act as a methyl acceptor for a methyltransferase activity in the cell extract that uses S-adenosylmethionine as methyl donor. Crithidia cytochrome c-557 is by far the best substrate for this methyltransferase of those tested, in spite of the fact that methylation sites are already almost fully occupied. The radioactive uptake of [14C]methyl groups from S-adenosylmethionine occurred only at a lysine residue (-8) and the N-terminal proline residue. This methyltransferase appears to differ from that of Neurospora and yeast [Durban, Nochumson, Kim, Paik & Chan (1978) J. Biol. Chem. 253, 1427-1435; DiMaria, Polastro, DeLange, Kim & Paik (1979) J. Biol. Chem. 254, 4645-4652] in that lysine-72 of horse cytochrome c is a poor acceptor. Also, the Crithidia methyltransferase appears to be stable to carry lysine methylation much further to completion than do the enzymes from yeast and Neurospora, which produce very low degrees of methylation in native cytochromes c.     

The membrane-bound surface glycoprotein of Crithidia oncopelti

Membrane-associated surface glycoprotein from Crithidia oncopelti (MAG) has been isolated and studied. Its molecular weight constitutes 10-12 kDa. MAG contains 12% protein, 29% carbohydrates, 1.2% phosphate and a lipid fragment. Amino acid components of MAG include glutamine, asparagine, alanine, proline, valine, leucine, serine, threonine, lysine, glycine. Ethanolamine and glycerophosphate have also been found. MAG contains stearic and small amounts of palmitic, oleic and myristic fatty acids. Carbohydrate fragment of MAG consists of seven monomers.     

Trypanosomatid protozoa: survey of acetylornithinase and ornithine acetyltransferase

Trypanosomatid protozoa (Crithidia deanei, C. deanei aposymbiotic, C. oncopelti, C. fasciculata, C. acanthocephali, Leptomonas seymouri, L. collosoma, L. samueli, Herpetomonas samuelpessoai, H. sp., H. megaseliae, H. muscarum muscarum, Leishmania donovani, L. braziliensis, Trypanosoma cruzi, T. conorhini and T. mega) were examined for the presence of acetylornithinase (EC 3.5.1.16) and ornithine acetyltransferase (EC 2.3.1.35). As a rule, species of the genus Crithidia presented one of the two enzymes for the conversion of acetylornithine into ornithine. Crithidia fasciculata and C. acanthocephali presented acetylornithinase, while C. deanei and C. oncopelti, species harboring symbionts, presented ornithine acetyltransferase. The enzyme was absent in the aposymbiotic strain of C. deanei, which suggests that the enzyme belongs to the symbiont. Among the other trypanosomatids examined only Herpetomonas samuelpessoai presented acetylomithinase. The participation of acetylornithinase and ornithine acetyltransferase in the metabolism of trypanosomatids is discussed in the light of their nutritional requirements and possession of enzymes of the arginineornithine metabolism.     

A giant phosphoprotein localized at the spongiome region of Crithidia luciliae thermophila

A giant protein with an apparent molecular mass of 2,300-kDa was identified in the Triton X-100 soluble fraction of Crithidia luciliae thermophila. Polyclonal antibody raised against this protein reacted by immunoblot analysis with proteins of similar molecular mass in Crithidia fasciculata and Crithidia oncopelti. In addition, the antibody immunoprecipitates the protein either after in vivo phosphorylation with [32P]orthophosphoric acid or after metabolically labeling with [35S]methionine. Indirect immunofluorescence microscopy analysis performed either with fixed or with live parasites showed a single fluorescent spot at the level of the flagellar pocket region. Immunogold electron microscopy of thin sections of the parasite revealed that the antigen is localized at a restricted area of the spongiome, between the contractile vacuole and the flagellar pocket. Furthermore, Triton X-114 phase separation of whole cell membrane proteins, metabolically labeled with [35S]methionine, demonstrated that the giant protein remains in the aqueous phase. These results indicate that this phosphoprotein behaves as a peripheral membrane protein localized at the spongiome region, suggesting that it might be involved in the osmoregulatory process.