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.     

Some Effects of Pentamidine Di-isethionate on Crithidia fasciculata

SYNOPSIS. Growth-inhibitory concentrations of pentamidine inhibit to a similar extent net synthesis of DNA, RNA, protein and phospholipid by washed cell suspensions of Crithidia fasciculata. The incorporation of ³H-thymidine into DNA, ¹⁴C-adenine into DNA and RNA and ¹⁴C-lysine into protein is similarly inhibited. The same concentrations of drug have little or no effect on viability, motility, 1-C metabolism, respiration or K⁺ content of organisms, altho they do cause increased amounts of intracellular ATP. Lysine uptake (in the absence of arginine) is, however, inhibited. At higher concentrations of drug respiration is inhibited and organisms lose K⁺. The mode of action of pentamidine is considered in the light of these observations and a mechanism of uptake of pentamidine into organisms is suggested.     

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.     

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.     

Minicircle-encoded guide RNAs from Crithidia fasciculata.

Although the mitochondrial uridine insertion/deletion, guide RNA (gRNA)-mediated type of RNA editing has been described in Crithidia fasciculata, no evidence for the encoding of gRNAs in the kinetoplast minicircle DNA has been presented. There has also been a question as to the capacity of the minicircle DNA in this species to encode the required variety of gRNAs, because the kinetoplast DNA from the C1 strain has been reported as essentially containing a single minicircle sequence class. To address this problem, the genomic and mature edited sequences of the MURF4 and RPS12 cryptogenes were determined and a gRNA library was constructed from mitochondrial RNA. Five specific gRNAs were identified, two of which edit blocks within the MURF4 mRNA, and three of which edit blocks within the RPS12 mRNA. The genes for these gRNAs are all localized with identical polarity within one of the two variable regions of specific minicircle molecules, approximately 60 bp from the "bend" region. These minicircles were found to represent minor sequence classes representing approximately 2% of the minicircle DNA population in the network. The major minicircle sequence class also encodes a gRNA at the same relative genomic location, but the editing role of this gRNA was not determined. These results confirm that kinetoplast minicircle DNA molecules in this species encode gRNAs, as is the case in other trypanosomatids, and suggest that the copy number of specific minicircle sequence classes can vary dramatically without an overall effect on the RNA editing system.     

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.     

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.     

The transport of alpha-aminoisobutyrate into Crithidia fasciculata.

The transport of alpha-aminoisobutyrate into Crithidia fasciculata was characterized under aerobic and anaerobic conditions. Kinetic data for alpha-aminoisobutyrate transport were consistent with the operation of a single system of broad specificity that showed no marked dependence on Na+. Under anaerobic conditions alpha-aminoisobutyrate transport was inhibited by uncouplers such as 2,4-dinitrophenol, lipophilic cations such as methyltriphenylphosphonium ion and adenosine triphosphatase inhibitors such as dicyclohexylcarbodi-imide and NaN3. A working model in which alpha-aminoisobutyrate enters this organism by an H+-symport mechanism, the electrochemical gradient of protons being maintained by an H+-translocating adenosine triphosphatase on the cytoplasmic membrane, is proposed.     

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.     

A DNase from the trypanosomatid Crithidia fasciculata.

We have purified to homogeneity a DNase from a Crithidia fasciculata crude mitochondrial lysate. The enzyme is present in two forms, either as a 32 kDa polypeptide or as a multimer containing the 32 kDa polypeptide in association with a 56 kDa polypeptide. Native molecular weight measurements indicate that these forms are a monomer and possibly an alpha 2 beta 2 tetramer, respectively. The monomeric and multimeric forms of the enzyme are similar in their catalytic activities. Both digest double-stranded DNA about twice as efficiently as single-stranded DNA. They introduce single-strand breaks into a supercoiled plasmid but do not efficiently make double-strand breaks. They degrade a linearized plasmid more efficiently than a nickel plasmid. Both enzymes degrade a 5'-32P-labeled double-stranded oligonucleotide to completion, with the 5'-terminal nucleotide ultimately being released as a 5'-mononucleotide. One difference between the monomeric and multimeric forms of the enzyme, demonstrated by a band shift assay, is that the multimeric form binds tightly to double-stranded DNA, possibly aggregating it.     

Cell surface charge and sugar residues of Crithidia fasciculata and Crithidia luciliae.

The surface charge of Crithidia fasciculata and Crithidia luciliae was analysed by measurement of the zeta-potential and labelling of the protozoan surface with cationized ferritin particles. Both trypanosomatids have a net negative surface charge, with a zeta-potential of -10.39 mV and -11.12 mV for C. luciliae and C. fasciculata, respectively. Enzyme treatment showed that phosphate groups, but not sialic acid, significantly contributed to the negative surface charge. Lectin-induced agglutination was used to analyse the presence of surface-exposed carbohydrates in C. fasciculata and C. luciliae. The cells did not agglutinate when incubated in the presence of lectins which recognized L-fucose, N-acetyl-D-glucosamine and sialic acid. However, lectins which bind to N-acetyl-D-galactosamine, D-galactose and D-mannose agglutinated both protozoa.     

Biosynthesis of phosphatidylinositol in Crithidia fasciculata

Microsomal preparations from the protozoan (Crithidia fasciculata were shown to incorporate myo-[2-3H]inositol into phosphatidylinositol by both the CDPdiacylglycerol:myo-inositol phosphatidyltransferase reaction and by a myo-inositol exchange reaction. Non-ionic detergent and Mg2+ were necessary for the measurement of transferase activity. Untreated preparations could not be saturated with Mg2+, even at very high concentrations (50-75 mM). However, low concentrations of EGTA (75 micro M) both stimulated the activity 3-fold and reduced the Mg2+ required for saturation to 15-20 mM. EGTA also increased the apparent Km for CDPdiacylglycerol while increasing the sensitivity to substrate inhibition above 1 mM. The transferase activity was inhibited by relatively low concentrations of Ca2+ (50 micro M). This and the EGTA effect suggest a possible role for Ca2+ in the modulation of phosphatidylinositol synthesis. The myo-inositol exchange activity required Mn2+, was insensitive to Ca2+ inhibition and was only slightly stimulated by detergents and EGTA. This activity was preferentially inactivated by heating at 50 degrees C in the presence of Triton X-100. In a detergent solubilized preparation the exchange activity but not the transferase exhibited a non-specific requirement for phospholipid. The differences in properties of the two activities suggest the presence of a separate exchange enzyme.     

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.     

Characterization of a novel endonuclease from Crithidia fasciculata.

A new endonuclease activity has been identified in whole cell lysates of the trypanosomatid Crithidia fasciculata. This activity, termed endonuclease A (Endo A), introduces single-strand breaks at highly preferred sites in double stranded DNA substrates Physical analysis of this enzyme indicates that it has a sedimentation coefficient S20,W of 4.9 and a Stokes radius of 59A and thus, a native molecular weight of 125,000 and a frictional coefficient of 1.8. A monomeric structure is suggested for the enzyme based on the recovery of Endo A activity associated with a polypeptide with a molecular weight of 116,000-120,000, following electrophoresis on sodium dodecyl sulfate polyacrylamide gels. Endo A shows an absolute requirement for Mg2+ or Mn2+ and exhibits activity over a broad pH and temperature range, with optimal conditions for activity at pH 8.0 and 30 degrees C.     

Free minicircles of kinetoplast DNA in Crithidia fasciculata.

The major form of kinetoplast DNA in Crithidia fasciculata is a network which contains thousands of minicircles linked together in a two-dimensional array. This paper reports the existence of free minicircles in Crithidia which by several criteria are identical to those in networks. They are the same size (about 2500 base pairs), and they yield the same products upon digestion with restriction enzymes. About 0.4% of the minicircles in exponentially growing nonsynchronized cells are free and the remainder are in networks. After a 5-min pulse with [3H]thymidine, above 10% of all of the incorporated radioactivity in the cell is in free minicircles, and the minicircles have a higher specific radioactivity than the average of other DNAs in the cell. Three-branched structures, which resemble Cairns-type replication intermediates, are occasionally observed by electron microscopy. Kinetic studies of the incorporation of [3H]thymidine into free minicircles indicate that they turn over, and this turnover was confirmed by a pulse-chase experiment. These properties of free minicircles suggest that they may be intermediates in the replication of network minicircles.