Sub-Pellicular Microtubules in Crithidia fasciculata

SYNOPSIS. Crithidia fasciculata sectioned transversally and studied with the electron microscope had gaps among an otherwise uniform row of sub-pellicular microtubules. Cultures were submitted to various treatments known to affect the structure of microtubules in other cell types. Only those very drastic treatments that killed the cells affected the sub-pellicular microtubules. Others, altho affecting the structure of the cell, left the sub-pellicular gaps and microtubules unchanged.     

Methionine or Folate and Phosphoenolpyruvate in the Biosynthesis of Threonine in Crithidia fasciculata1

SYNOPSIS. Crithidia fasciculata can synthesize threonine but it lacks an aspartokinase. The carbons of threonine may be derived from methionine when it is present in the medium. However, methionine can be synthesized by the organism provided organic sulfur is present. When both methionine and threonine are omitted from the medium, growth will occur if cysteine and high levels of folate are present. The compound common to both methionine and threonine under these conditions, α-keto-γ-hydroxy-butyrate, is derived from phosphoenolpyruvate (PEP) and the β-carbon of serine. High levels of folate are required for this coupling reaction, which is carried out by what may be called phosphoenolpyruvate-tetrahydrofolate hydroxymethyltransferase. By the use of radioactive tracers, both in growth experiments and in cell-free preparations, virtually all of the intermediates in these two series of reactions were identified.     

Respiratory Pigments of Crithidia fasciculata

Mitochondria were isolated from the heme-requiring insect trypanosomatid, Crithidia fasciculata, which had respiratory activity, showed a P/O ratio with succinate of 0.5 to 1.0, and contained 40 to 50% of the heme a and heme c found in the intact cells. Cytochromes b, c(555), possibly c(1), cytochrome oxidase, a carbon monoxide-binding pigment, and flavoproteins were detectable in the spectra of both intact cells and mitochondria. Cytochrome c(555) is a basic protein that was extracted from cells and mitochondria with salt solutions. The molar ratio of heme c to heme a was approximately 2:1 in both cells and mitochondria. This organism could possibly serve as a model for studies of the respiratory activity of the pathogenic trypanosomes.     

Auxotrophic mutants of Crithidia fasciculata

In pursuance of genetic studies, after exposure to ethylmethanesulfonate, 11 auxotrophic mutants of Crithidia fasciculata were cloned. Three proved uracil dependent; 3 serine dependent; and the remainder have not had their auxotrophy defined.     

Effects of Hydroxyurea on Crithidia fasciculata

SYNOPSIS Hydroxyurea (HU) inhibits increase in cell number in cultures of Crithidia fasciculata. Complete inhibition is produced by 8 mM and higher concentrations. If HU is not removed, population growth resumes in 45–50 h: if HU is removed, partially synchronous growth occurs through 2 cycles. During HU inhibition, the rate of DNA synthesis is reduced to 1% of that in exponentially growing cultures; protein and RNA syntheses continue at slightly reduced rates. Mean cell size and protein and RNA contents per cell increase; rate of oxygen consumption per mg cell protein remains constant. The behavior of a culture upon addition of HU and upon its removal agrees with predictions based on the hypothesis that the only direct effect of HU is to block DNA synthesis. The synchrony produced by HU is judged satisfactory for investigations of kinetoplast and nuclear replication but not for biochemical characterization of other aspects of the cell cycle.     

The ribonucleic acids of Crithidia fasciculata

Crithidia fasciculata ribosomes were found to be 80S and to dissociate into 58 and 41S subunits; on 5 to 50% sucrose gradients, rRNA was separated into 25, 18, and 5S components. The molecular sizes of the heavier rRNA species, estimated by polyacrylamide gel electrophoresis were 1.24 and 0.84 M (X 10(6) daltons). The 25S RNA has a tendency to interact with the 18S RNA to give a complex that is difficult to separate by sucrose gradient centrifugation. The 25S RNA is also unstable and dissociates into 0.73 and 0.57 M components. The 18S RNA has molecular size (0.84 M) higher than the 0.7 M reported for most eukaryotes, but similar to that of Euglena and Amoeba. Ribosomal RNA hybridized 0.29% of the nuclear DNA. Mitochondrial RNA, extracted by a rapid procedure was resolved into 16 and 5S components in sucrose gradients.     

The purine phosphoribosyltransferases of Crithidia fasciculata

The purine phosphoribosyltransferases of Crithidia fasciculata were identified and some of their properties described. The organism possesses three separate enzymes for the production of AMP, IMP, and GMP. The evidence for this comes from the observed differences in elution patterns from gel filtration columns, differences in heat sensitivity, and especially the clear separation of hypoxanthine phosphoribosyltransferase from guanine phosphoribosyltransferase by affinity chromatography on GMP-agarose. APRTase is activated most efficiently by Zn++, whereas HPRTase and GPRTase are activated most effectively by Co++. In no case did the product mononucleotides produce strong inhibition of the transferase activities.     

The origin of unconjugated pteridines in Crithidia fasciculata

Summary By using folic acid-2-¹⁴C in the growth medium as the sole source of pteridine it was found that the kinetoplastid flagellate, Crithidia fasciculata, produced a total of 87 mg of labeled biopterin and 23 g of 2-amino-4-hydroxy-6-hydroxymethylpteridine from 2 mg of folic acid in 2 liters of medium. Eighty percent of the biopterin and 70% of the 6-hydroxymethylpteridine was isolated from the spent medium, the remainder from the cells. These results and the results whereby the total pteridine was supplied as a folic acid analog make it obvious that this flagellate does convert folate to biopterin and is incapable of de novo pteridine synthesis.Supported in part by Research grants AM 01005 and CA 02924 from the National Institutes of Health, United States Public Health Service, and by Deutsche Forschungsgemeinschaft.Dedicated to Professor C. B. Van Niel on the occasion of his 70th birthday.     

Crystallization of recombinant Crithidia fasciculata tryparedoxin.

Recombinant tryparedoxin, a thioredoxin homologue from Crithidia fasciculata, has been purified from an Escherichia coli expression system and used in crystallization trials. Orthorhombic needles in space group P212121, with unit cell dimensions of a = 38.63, b = 51. 47, and c = 73.41 A, have been obtained. The crystals present a monomer of approximate molecular mass 16 kDa in the asymmetric unit and diffract to 1.8-A resolution using synchrotron radiation. Structure determination will be carried out to further the understanding of the role tryparedoxin plays in regulating oxidative stress in parasitic trypanosomatids.     

The Ribonucleic Acids of Crithidia fasciculata*

SYNOPSIS. Crithidia fasciculata ribosomes were found to be 80S and to dissociate into 58 and 41S subunits; on 5 to 50% sucrose gradients, rRNA was separated into 25, 18, and 5S components. The molecular sizes of the heavier rRNA species, estimated by polyacrylamide gel electrophoresis were 1.24 and 0.84 M (×10⁶ daltons). The 25S RNA has a tendency to interact with the 18S RNA to give a complex that is difficult to separate by sucrose gradient centrifugation. The 25S RNA is also unstable and dissociates into 0.73 and 0.57 M components. The 18S RNA has molecular size (0.84 M) higher than the 0.7 M reported for most eukaryotes, but similar to that of Euglena and Amoeba. Ribosomal RNA hybridized 0.29% of the nuclear DNA. Mitochondrial RNA, extracted by a rapid procedure was resolved into 16 and 5S components in sucrose gradients.     

Effects of hydroxyurea on Crithidia fasciculata.

Hydroxyurea (HU) inhibits increase in cell number in cultures of Crithidia fasciculata. Complete inhibition is produced by 8 mM and higher concentrations. If HU is not removed, population growth resumes in 45-50 h; if HU is removed, partially synchronous growth occurs through 2 cycles. During HU inhibition, the rate of DNA synthesis is reduced to 1% of that in exponentially growing cultures; protein and RNA syntheses continue at slightly reduced rates. Mean cell size and protein and RNA contents per cell increase; rate of oxygen consumption per mg cell protein remains constant. The behavior of a culture upon addition of HU and upon its removal agrees with predictions based on the hypothesis that the only direct effect of HU is to block DNA synthesis. The synchrony produced by HU is judged satisfactory for investigations of kinetoplast and nuclear replication but not for biochemical characterization of other aspects of the cell cycle.     

Tubulin heterogeneity in the trypanosome Crithidia fasciculata.

The interphase cell of Crithidia fasciculata has three discrete tubulin populations: the subpellicular microtubules, the axonemal microtubules, and the nonpolymerized cytoplasmic pool protein. These three tubulin populations were independently and selectively purified, yielding, in each case, microtubule protein capable of self-assembly. All three preparations polymerized to form ribbons and sheets rather than the more usual microtubular structures. Analyses of the tubulin by two-dimensional polyacrylamide gel electrophoresis, isoelectric focusing, and peptide mapping indicated that the beta-tubulin complex remained constant regardless of source but that some heterogeneity was present in the alpha subunit. Cytoplasmic pool alpha tubulins (alpha 1/alpha 2) were the only alpha isotypes in the cytoplasm and also formed most of the alpha tubulin species in the pellicular fraction. Flagellar alpha tubulin (alpha 3) was the sole alpha isotype in the flagella; it appeared in small amounts in the pellicular fraction but was completely absent from the cytoplasm. In vitro translation products from polyadenylated RNA from C. fasciculata were also examined by two-dimensional polyacrylamide gel electrophoresis and possessed a protein corresponding to alpha 1/alpha 2 tubulin but lacked any alpha 3 tubulin. The alpha 3 polypeptide arose from a post-translational modification of a precursor polypeptide not identifiable by two-dimensional polyacrylamide gel electrophoresis as alpha 3. Peptide mapping data indicated that cytoplasmic alpha tubulin is the most likely precursor. These results demonstrate alpha-tubulin heterogeneity in this organism and also how close the relationship between flagellar and cytoskeletal tubulins can be among lower eucaryotes.     

Desmosomes and hemidesmosomes in the flagellate Crithidia fasciculata

Summary The flagellum of the trypanosomatid flagellate Crithidia fasciculata expands asymmetrically as it emerges from the reservoir. Where the flagellar memhrane approaches the membrane lining the reservoir, desmosomes are found. These structures are arranged in several slightly curved lines and have many features in common with vertebrate desmosomes.In cultures, the flagellates stick to each other by their flagella and form rosettes. In these bundles of cells, probable sites of adhesion between flagella, or between flagella and pieces of debris, are marked by a dense filamentous tract which passes posteriorly along the flagellum and by a thick band lying just below the flagellar membrane. It is suggested that similar adhesions are found in the insect host where the flagellate attaches itself to the gut wall.