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A glob is defined as a group of atoms in the crystal which can be chosen in various ways. Globs themselves can be used as scattering elements in the theory of structure determination, just as atoms are used at present. In this paper, amino-acid residues are chosen to form globs and empirical formulas for residue-based scattering factors have been developed.

Most eukaryotic organisms including protozoans like Crithidia, Leishmania, and Plasmodium encode a repertoire of equilibrative nucleoside transporters (ENTs). Using genomic sequencing data from Crithidia fasciculata, we discovered that this organism contains multiple ENT genes of highly similar sequence to the previously cloned and characterized adenosine transporter CfNT1: CfAT1 and CfNT3, and an allele of CfAT1, named CfAT1.2. Characterization of CfAT1 shows that it is an adenosine-only transporter, 87% identical to CfNT1 in protein sequence, with a 50-fold lower Km for adenosine. Site directed mutation of a key residue in transmembrane domain 4 (TM4) in both CfNT1 and CfAT1 shows that lysine at this position results in a high affinity phenotype, while threonine decreases adenosine affinity in both transporters. These results show that C. fasciculata has at least two adenosine transporters, and that as in other protozoan ENTs, a lysine residue in TM4 plays a key role in ligand affinity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

DNA is the biopolymer most studied by scanning probe methods, and it is now possible to obtain reliable and reproducible images of DNA using atomic force microscopy (AFM). AFM has been extensively used to elucidate morphological changes to DNA structure, such as the formation of knots, nicks, supercoiling and bends. The mitochondrial or kinetoplast DNA (kDNA) of trypanosomatids is the most unusual DNA found in nature, being unique in organization and replication. The kDNA is composed of thousands of topologically interlocked DNA circles that form a giant network. To understand the biological significance of the kinetoplast DNA, it is necessary to learn more about its structure. In the present work, we used two procedures to prepare kDNA networks of Crithidia fasciculata for observation by AFM. Because AFM allows for the examination of kDNA at high resolution, we were able to identify regions of overlapping kDNA molecules and sites where several molecules cross. This found support the earlier described kDNA structural organization as composed by interlocked circles. We also observed an intricate high-density height pattern around the periphery of the network of C. fasciculata, which appears to be a bundle of DNA fibers that organizes the border of the network. Our present data confirm that AFM is a powerful tool to study the structural organization of biological samples, including complex arrays of DNA such as kDNA, and can be useful in revealing new details of structures previously visualized by other means., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Equilibrative nucleoside transporters play essential roles in nutrient uptake, cardiovascular and renal function, and purine analog drug chemotherapies. Limited structural information is available for this family of transporters; however, residues in transmembrane domains 1, 2, 4, and 5 appear to be important for ligand and inhibitor binding. In order to identify regions of the transporter that are important for ligand specificity, a genetic selection for mutants of the inosine-guanosine-specific Crithidia fasciculata nucleoside transporter 2 (CfNT2) that had gained the ability to transport adenosine was carried out in the yeast Saccharomyces cerevisiae. Nearly all positive clones from the genetic selection carried mutations at lysine 155 in transmembrane domain 4, highlighting lysine 155 as a pivotal residue governing the ligand specificity of CfNT2. Mutation of lysine 155 to asparagine conferred affinity for adenosine on the mutant transporter at the expense of inosine and guanosine affinity due to weakened contacts to the purine ring of the ligand. Following systematic cysteine-scanning mutagenesis, thiol-specific modification of several positions within transmembrane domain 4 was found to interfere with inosine transport capability, indicating that this helix lines the water-filled ligand translocation channel. Additionally, the pattern of modification of transmembrane domain 4 suggested that it may deviate from helicity in the vicinity of residue 155. Position 155 was also protected from modification in the presence of ligand, suggesting that lysine 155 is in or near the ligand binding site. Transmembrane domain 4 and particularly lysine 155 appear to play key roles in ligand discrimination and translocation by CfNT2.

In contrast to earlier proposals, recent evidence suggests that trans-spliceosomes in trypanosomatid protozoa may contain a homolog of U1 small nuclear (sn) RNA (Schnare, M.N. and Gray, M.W. (1999) J. Biol. Chem. 274, 23,691-23,694). However, the candidate trypanosomatid U1 snRNA is unconventional because it lacks the highly conserved stem/loop II present in all other U1 snRNAs. Trypanosomatids also possess a unique spliced leader-associated (SLA) RNA of unknown function. We present the complete sequence of the SLA RNA from Crithidia fasciculata and propose that it may contribute a U1 snRNA-like stem/loop II to the trans-spliceosome.

Tryparedoxin-I is a recently discovered thiol-disulfide oxidoreductase involved in the regulation of oxidative stress in parasitic trypanosomatids. The crystal structure of recombinant Crithidia fasciculata tryparedoxin-I in the oxidized state has been determined using multi-wavelength anomalous dispersion methods applied to a selenomethionyl derivative. The model comprises residues 3 to 145 with 236 water molecules and has been refined using all data between a 19- and 1.4-A resolution to an R-factor and R-free of 19.1 and 22.3%, respectively. Despite sharing only about 20% sequence identity, tryparedoxin-I presents a five-stranded twisted beta-sheet and two elements of helical structure in the same type of fold as displayed by thioredoxin, the archetypal thiol-disulfide oxidoreductase. However, the relationship of secondary structure with the linear amino acid sequences is different for each protein, producing a distinctive topology. The beta-sheet core is extended in the trypanosomatid protein with an N-terminal beta-hairpin. There are also differences in the content and orientation of helical elements of secondary structure positioned at the surface of the proteins, which leads to different shapes and charge distributions between human thioredoxin and tryparedoxin-I. A right-handed redox-active disulfide is formed between Cys-40 and Cys-43 at the N-terminal region of a distorted alpha-helix (alpha1). Cys-40 is solvent-accessible, and Cys-43 is positioned in a hydrophilic cavity. Three C-H...O hydrogen bonds donated from two proline residues serve to stabilize the disulfide-carrying helix and support the correct alignment of active site residues. The accurate model for tryparedoxin-I allows for comparisons with the family of thiol-disulfide oxidoreductases and provides a template for the discovery or design of selective inhibitors of hydroperoxide metabolism in trypanosomes. Such inhibitors are sought as potential therapies against a range of human pathogens.

The cell-surface expression of sialoglycoconjugate structures in wild-type Crithidia fasciculata and its TFR(R1) drug-resistant mutant was analyzed with the aid of an influenza C virus strain, lectin, enzymatic treatment, and flow cytofluorimetry analysis probed with fluorescein isothiocyanate-labeled (FITC) lectins. 9-O-Acetyl-N-acetyl neuraminic acid (Neu5,9Ac2) structures mediate influenza C virus cell-binding. The SAalpha2,3Gal and SAalpha2,6Gal sequences are specifically recognized by Maackia amurensis (MAA) and Sambucus nigra (SNA) lectins, respectively. On the basis of these parameters the TFR(R1) mutant strain of C. fasciculata was found to contain exposed sialoglycoconjugates bearing Neu5,9Ac2 surface structures. After the removal of sialic acid residues by neuraminidase activity the marked increases in PNA (peanut agglutinin)-mediated agglutinating activity showed that those acidic units on C. fasciculata cells were glycosidically linked to D-galactose. The bond involves SAalpha2,6Gal and SAalpha2,3Gal linkages as suggested by the use of FITC-SNA and FITC-MAA lectins, respectively. Both SAalpha2,3Gal and SAalpha2,6Gal sequences were preferentially expressed by the TFR(R1) mutant. The SAalpha2,6 linkage markedly predominated. In the TFR(R1) mutant, but not in wild-type cells, two distinct populations of cells were distinguished by reactivity with FITC-SNA, one of which was enriched with surface SAalpha2,6Gal sequences. These diverse findings suggest that sialoglycoconjugate structures present on the flagellate surface may be associated with mutation and the cell growth cycle in C. fasciculata.

Tryparedoxin (TXN) has recently been discovered as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke et al. (1997) Biol. Chem. 378, 827-836] where it catalyzes the reduction of a peroxiredoxin-type peroxidase by trypanothione. Here we report on the full-length DNA sequence of the TXN previously isolated from C. fasciculata (TXN1). The deduced amino acid sequence comprises 147 residues and matches with all the peptide sequences of fragments obtained from TXN1. It shares a characteristic sequence motif YFSAxWCPPCR with some thioredoxin-related proteins of unknown function. This motif is homologous with the CXXC motif, which characterizes the thioredoxin superfamily of proteins and is known to catalyze disulfide reductions. Sequence conservations between TXNs and the typical thioredoxins are restricted to the intimate environment of the CXXC motif and three more remote residues presumed to contribute to the folding pattern of the thioredoxin-type proteins. The TXNs thus form a distinct molecular clade within the thioredoxin superfamily. TXN1 was expressed in Escherichia coli BL21 (DE3)pLysS as a C-terminally extended and His-tagged protein, isolated by chelate chromatography and characterized functionally. The recombinant product exhibited a kinetic pattern identical with, and kinetic parameters similar to those of the authentic enzyme in the trypanothione/peroxiredoxin oxidoreductase assay. The recombinant TXN1 can therefore be considered a valuable tool for the screening of specific inhibitors as potential trypanocidal agents.

The family of the RACK molecules (receptors for activated C kinases) are present in all the species studied so far. In the genus Leishmania, these molecules also induce a strong immune reaction against the infection. We have cloned and characterised the gene that encodes the RACK analogue from the parasite trypanosomatid Crithidia fasciculata (CACK). The molecule seems to be encoded by two genes. The sequence analysis of the cloned open reading frame indicates the existence of a high degree of conservation not only with other members of the Trypanosomatidae but also with mammalians. The study of the protein kinase C phosphorylation sites shows the presence of three of them, shared with the mammalian species, additional to those present in the other protozoa suggesting a certain phylogenetic distance between the protozoon Crithidia fasciculata and the rest of the Trypanosomatidae. The CACK-encoded polypeptide shows an additional sequence of four amino acids at the carboxy-terminal end, which produces a different folding of the fragment with the presence of an alpha-helix instead of the beta-sheet usual in all the other species studied. A similar result is elicited at the amino-terminal end by the change of three amino acid residues. The immunolocalisation experiments show that the CACK displays a pattern with a distribution mainly at the plasma membrane, different from that of the related Leishmania species used as control, that displays a distribution close to the nucleus. Altogether, the data suggest that the existence of the structural differences found may have functional consequences.

The Crithidia fasciculata KAP1 gene encodes a small basic protein (p21) associated with kinetoplast DNA. The p21 protein has a nine amino acid cleavable presequence closely related to those of several other proteins targeted to the kinetoplast and binds non-specifically to kinetoplast minicircle DNA. The p21 protein also has a calculated pI of 13 with two amino acids (lysine and alanine) accounting for more than 50% of the residues and with 25 out of 28 lysine residues contained in the C-terminal half of the protein. Immunolocalization of p21 shows that the protein is found exclusively in the kinetoplast with a localization distinctly different from the antipodal localization of kinetoplast DNA topoisomerase and DNA polymerase. The KAP11 gene is a single copy gene and the KAP1 mRNA is present at a constant level throughout the cell cycle. This highly basic protein may play a role in the condensation or segregation of the kinetoplast DNA.

Tryparedoxin, a thioredoxin-related protein from Crithidia fasciculata with a molecular mass of 16 kDa catalyses the reduction of a peroxiredoxin-type peroxidase, Cf21, at the expense of trypanothione [Nogoceke, E., Gommel, D. U., Kiess, M., Kalisz, H. M. & Flohé, L. E. (1997) Biol. Chem. Hoppe-Seyler 378, 827-836]. The kinetic analysis of tryparedoxin revealed an enzyme substitution mechanism. The corresponding molecular event was elucidated to be a reversible oxidoreduction of the disulfide bridge in the thioredoxin-related motif WCPPC. The amino-proximal cysteine residue of this active site was more reactive in S-alkylation experiments than the distal residue. The natural substrates of tryparedoxin, trypanothione and Cf21, could only be substituted by glutathione and glutathione disulfide with considerable loss in activity. The pronounced specificity of tryparedoxin is further accentuated by low limiting Km values for Cf21 and trypanothione (2.2 microM and 130 microM, respectively, as compared to 990 microM for gluthathione disulfide and an infinite value for glutathione). Tryparedoxin can therefore be classified as a trypanothione: peroxiredoxin oxidoreductase. The reduction of tryparedoxin by trypanothione appears to be the rate-limiting step in the trypanothione-dependent hydroperoxide reduction because(a) the regeneration of reduced tryparedoxin from the tryparedoxin-trypanothione complex is rate limiting (k[cat] 392 min[-1]), (b) the physiological trypanothione concentrations may not always saturate tryparedoxin, and (c) the rate constants for the net forward reaction of Cf21 are faster than those of the tryparedoxin reaction. The functional characteristics of tryparedoxin explain the limited capacity of trypanosomatids in coping with oxidative stress and qualify the enzyme as a potential target for the design of specific trypanocidal compounds.

Mitochondrial mRNAs encoding subunits of respiratory-chain complexes in kinetoplastids are post-transcriptionally edited by uridine insertion and deletion. In order to identify the proteins encoded by these mRNAs, we have analyzed respiratory-chain complexes from cultured cells of Crithidia fasciculata with the aid of 2D polyacrylamide gel electrophoresis (PAGE). The subunit composition of F0F1-ATPase (complex V), identified on the basis of its activity as an oligomycin-sensitive ATPase, is similar to that of bovine mitochondrial F0F1-ATPase. Amino acid sequence analysis, combined with binding studies using dicyclohexyldiimide and azido ATP allowed the identification of two F0 subunits (b and c) and all of the F1 subunits. The F0 b subunit has a low degree of similarity to subunit b from other organisms. The F1 alpha subunit is extremely small making the beta subunit the largest F1 subunit. Other respiratory-chain complexes were also analyzed. Interestingly, an NADH: ubiquinone oxidoreductase (complex I) appeared to be absent, as judged by electron paramagnetic resonance (EPR), enzyme activity and 2D PAGE analysis. Cytochrome c oxidase (complex IV) displayed a subunit pattern identical to that reported for the purified enzyme, whereas cytochrome c reductase (complex III) appeared to contain two extra subunits. A putative complex II was also identified. The amino acid sequences of the subunits of these complexes also show a very low degree of similarity (if any) to the corresponding sequences in other organisms. Remarkably, peptide sequences derived from mitochondrially encoded subunits were not found in spite of the fact that sequences were obtained of virtually all subunits of complex III, IV and V.

The Trypanosomatidae family is characterized by flagellated protozoa presenting a kinetoplast. Several genera of this family contain species that are pathogenic to man and domestic animals. Their chromatin is not condensed into chromosomes during cell division. As a contribution to the understanding of basic aspects of their genome organization, we present a systematic characterization of the histones from three genera of the Trypanosomatidae family. Crithidia fasciculata and Leishmania mexicana show core nucleosomal histones with electrophoretic mobilities both similar to and different from those of Trypanosoma cruzi and higher eukaryotes. Another protein is extracted from the chromatin of these organisms by procedures designed to purify histone H1. This protein presents elution profiles by HPLC and amino acid composition of histone H1. Considering these data and the high mobility of this protein in Triton-acetic acid-urea-polyacrylamide gel electrophoresis, as well as its position relative to the nucleosomal core histones in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we postulate that Crithidia and Leishmania possess a histone H1 shorter than that of higher eukaryotes as we have previously shown to be the case for T. cruzi. The possible presence of a shorter histone H1 in these trypanosomatids may explain the absence of chromatin condensation during cell division in these flagellates.

Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomatids, consists of thousands of minicircles and 20 to 30 maxicircles catenated into a single large network and exists in the cell as a highly organized compact disc structure. To investigate the role of kinetoplast-associated proteins in organizing and condensing kDNA networks into this disc structure, we have cloned three genes encoding kinetoplast-associated proteins. The KAP2, KAP3, and KAP4 genes encode proteins p18, p17, and p16, respectively. These proteins are small basic proteins rich in lysine and alanine residues and contain 9-amino-acid cleavable presequences. Proteins p17 and p18 are closely related to each other, with 48% identical residues and carboxyl tails containing almost exclusively lysine, alanine, and serine or threonine residues. These proteins have been expressed as Met-His6-tagged recombinant proteins and purified by metal chelate chromatography. Each of the recombinant proteins is capable of compacting kDNA networks in vitro and was shown to bind preferentially to a specific fragment of minicircle DNA. Expression of each of these proteins in an Escherichia coli mutant lacking the HU protein rescued a defect in chromosome condensation and segregation in the mutant cells and restored a near-normal morphological appearance. Proteins p16, p17, and p18 have been localized within the cell by immunofluorescence methods and appear to be present throughout the kDNA. Electron-microscopic immunolocalization of p16 shows that p16 is present both within the kDNA disc and in the mitochondrial matrix at opposite edges of the kDNA disc. Our results suggest that nucleus-encoded H1-like proteins may be involved in the organization and segregation of kDNA networks in trypanosomatids.

The monosaccharide D-arabinopyranose has only been found in glycoconjugates of the trypanasomatid parasites Leishmania major, Endotrypanum schaudinni and Crithidia fasciculata. The donor molecule for the relevant arabinosyltransferases is known to be GDP-alpha-D-Arap in L. major and C. fasciculata, and the latter organism is being used to study the biosynthesis of GDP-alpha-D-Arap. In this study, we describe the structure of the terminal product of arabinose metabolism in C. fasciculata, namely lipoarabinogalactan. This molecule was purified by hydrophobic-interaction chromatography and studied by a variety of techniques, including gas chromatography-mass spectrometry, electrospray mass spectrometry and chemical and enzymic digestions. These data show that lipoarabinogalactan contains a previously described D-arabino-D-galactan polysaccharide component covalently attached to a glycosylphosphatidylinositol type of membrane anchor that is similar to, but not identical with, that found in the lipophosphoglycans of the Leishmania.

The monoclonal antibody BS7, raised to bovine sperm flagellum cytoskeletal antigens in a previous study, is here reported to detect flagellum-associated structures in Trypanosoma brucei and Crithidia fasciculata. Immunoblotting showed that BS7 cross-reacts with several cytoskeletal T. brucei proteins but phosphatase treatment did not diminish this complex immunoblot reactivity. To characterize further the cross-reactive proteins recognized in T. brucei-cytoskeletons by BS7 each was excised from preparative gels and used as an immunogen for antiserum production. Two proteins, with apparent sizes around 43 and 47 kDa, produced antisera shown to be monospecific by immunoblotting total T. brucei flagellum preparations. Each of these detected the basal body-associated immunofluorescence in T. brucei. Identification of the smaller, 43 kDa, component as a basal body-associated product was supported by the behaviour of a second monoclonal antibody, BBA4, which was also shown to detect the T. brucei basal body complex by immunofluorescence and immunoblots the 43 kDa polypeptide. These observations reveal new components of the trypanosome cytoskeleton. Also, they provide a further example of an immunological approach for identification of interesting, rare components of the T. brucei cytoskeleton starting from a complex mixture of proteins.

Riboprotein particles containing the ribosomal RNA-like 9S and 12S RNAs in the trypanosome mitochondrion have never been isolated. Using the formaldehyde cross-linking procedure I show here that one or more of three proteins, q2 (16.5 kDa), r (15 kDa) and s (13 kDa), are closely associated in vivo with the 9S and 12S RNAs of the trypanosomatid parasite, Crithidia fasciculata. These proteins were also found to be associated with the parasite's cytosolic ribosomal RNA and to have strong immunological cross reactivity with riboprotein S11 of the bacterium Escherichia coli. These data provide the first evidence for the association of riboprotein-like proteins with the 9S and 12S mitochondrial RNA in a trypanosome, possibly as components of a mitochondrial ribosome.

Promastigotes of Leishmania major contain a ubiquinone which has a side chain made up of nine isoprene subunits (UQ9). Incorporation of radioactivity from [14C] acetate and [14C] mevalonate into ubiquinone as well as the identification of hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase), and mevalonate kinase indicate that the isoprenoid portion of the molecule is synthesized by the acetate-mevalonate pathway as in mammalian cells. Incorporation of [14C] tyrosine into ubiquinone is low, but [14C] parahydroxybenzoic acid is readily incorporated. Distribution of radioactivity from [14C] acetate indicates that about 60-80% is associated with the side chain and about 20% with the ring. Label from parahydroxybenzoic acid is, however, incorporated preferentially into the ring. L. major is capable of synthesizing the aromatic ring of ubiquinone from acetate, parahydroxybenzoate being an important intermediate. In this behaviour it resembles procaryotes. Ubiquinone biosynthetic pathway in L. major thus shares characteristics with mammalian and bacterial systems.

The cell-surface expression of sialic acids in wild-type Crithidia fasciculata and three drug-resistant mutants (FU(R)11, TR3, and TFRR1) was analyzed using fluorescein-labeled Limulus polyphemus agglutinin (LPA) binding, glycosidase of known sugar specificity, and thin-layer chromatography (TLC). Gas-liquid chromatography-mass spectrometry (GC-MS) analysis using both electron-impact (EI-MS) and chemical ionization (CI-MS) by isobutane with selected ion monitoring (SIM) was also used. The surface location of sialic acid was inferred from LPA binding to whole cells abrogated by previous treatment with neuraminidase. An exception occurred with the TFRR1 strain, which after incubation with neuraminidase showed increased reactivity with the fluorescent lectin. Both N-acetyl- and N-O-diacetyl-neuraminic acids were identified in the flagellates by TLC, with a clear predominance being noted for the former derivative. However, the content of N-O-diacetyl-neuraminic acid was preferentially found in the TFRR1 strain. The GC-MS analysis of the acidic component of the TFRR1 mutant strain confirmed the occurrence of N-acetyl-neuraminic acid (Neu5Ac) by the presence of the diagnostic ions (m/z values: 684 and 594 for CI-MS and 478, 298, and 317 for EI-MS) and also by comparison with the standard Neu5Ac retention time. GC-MS analysis also showed fragments (m/z values: 654 and 564 for CI-MS and 594, 478, 298, and 317 for EI-MS) expected for the 7-O- and 9-O-acetyl-N-acetyl-neuraminic acids (Neu5,7Ac2 and Neu 5,9Ac2, respectively).

An unidentified low-molecular-mass thiol, U23, previously detected as the 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin derivative in extracts of the trypanosome Crithidia fasciculata, was purified as the bimane derivative. Resonances attributable to U23 were discerned from those of the bimane label by comparison of the 1H- and 13C-NMR spectra of monobromobimane and U23-bimane. The complete 1H- and 13C-NMR spectra of U23-bimane were assigned by means of 1H-1H correlation spectroscopy, 1H-13C correlation spectroscopy and 13C multiplicity determinations. The results indicated identity of U23 with 1-N-methyl-4-mercaptohistidine (ovothiol A), previously isolated from marine sources. This assignment was confirmed by NOE difference experiments, fast-atom-bombardment mass spectrometry of U23-bimane and ultraviolet/visible spectrophotometry of U23, which was isolated as the disulfide. The isolation of ovothiol A from a parasitic protozoan suggest that the 4-mercaptohistidines may have a wider distribution and function as antioxidant thiols than was hitherto realized.

Methods for the qualitative and quantitative analysis of thiols by means of the fluorogenic reagent 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin are described, with particular reference to the trypanosomatid metabolites glutathionylspermidine (GSH-spermidine) and trypanothione. Second-order rate constants for the derivatization of seven different thiols under defined experimental conditions and at 21 degrees C varied between 619 +/- 34 and 10,560 +/- 236 M-1.s-1.T.l.c. of the thiols from Crithidia fasciculata was used to monitor the purification of trypanothione from this organism in three steps involving adsorption, ion-exchange and reversed-phase chromatography. The yield was approx. 50 mg of pure trypanothione from 100 g (wet wt.) of trypanosomatids. The method for the quantitative analysis of biological thiols is based on fluorometric detection after separation by reversed-phase or ion-paired chromatography on a phenyl-silica column. Analysis of the thiol composition of cell lysates prepared under nondenaturating conditions point to the rapid degradation of the GSH-spermidine conjugates. In addition to GSH, GSH-spermidine and trypanothione, at least one other prominent thiol was detected, and the contribution of this thiol to the total thiol content in the various growth phases of C. fasciculata was investigated.

The single mitochondrion of trypanosomatid protozoans such as Crithidia fasciculata has a large and complex network of AT-rich DNA called kDNA. Little is known about proteins involved in the packaging and morphogenesis of this network. I report the purification of a protein fraction from this parasite which preferentially retained kDNA in the gel slot during electrophoresis. The proteins had approximate molecular weights of 22, 21, 17.5, 16.5, 15, and 13 kDa. They copurified with mitochondrial DNA, parts of which they protected from nuclease attack. The proteins resembled histones but they also differed from histones in significant ways. Immunological evidence showed that the proteins were enriched in a mitochondrial fraction, implying their association with kDNA in vivo.

Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomes, is a highly condensed disc-shaped network of catenated DNA circles consisting of maxicircles, the equivalent of conventional mitochondrial DNA, and several thousand smaller circular DNAs termed minicircles. Upon cell lysis, kDNA expands, giving rise to a two-dimensional network of catenated circles with an overall diameter close to that of the whole cell. To identify proteins associated with the condensed form of kDNA in the cell, proteins were reversibly crosslinked to kDNA in whole cells of Crithidia fasciculata by formaldehyde treatment. Crosslinked networks were purified and found to retain a condensed structure which becomes fully expanded upon proteinase K treatment or reversal of the crosslinks by heating at 65 degrees C. Five low molecular weight proteins released from the kDNA by heat treatment were purified by polyacrylamide gel electrophoresis and their amino-terminal sequences were determined. PCR amplification and sequence analysis of cDNA sequences between these amino-terminal sequences and the miniexon (spliced leader) sequence present at the 5' end of all C. fasciculata mRNAs predicts the presence of 9-amino acid presequences with features characteristic of mitochondrial presequences on three of the proteins. Two of these proteins are lysine-rich basic proteins. These findings suggest that basic proteins may play a role in the condensation of kDNA in the kinetoplast and that these proteins are imported into the kinetoplast by a mechanism involving a cleavable presequence.

A gene (CRK) encoding a cdc2-related protein has been identified in the trypanosomatid Crithidia fasciculata. CRK has a high degree of sequence identity with the human cdc2 gene and contains the sixteen amino acid PSTAIR motif, characteristic of p34cdc2 protein-serine/threonine kinases, with four amino acid substitutions in the motif. In addition, two inserts of more than sixty amino acids have been found between conserved domains of this putative protein-serine/threonine kinase. CRK is a single copy gene and is expressed on a 3.8 kb mRNA. Anti-CRK antibodies detect a 53kDa protein in extracts of C.fasciculata in agreement with the size predicted from the nucleotide sequence of the cloned gene. These antibodies also recognize proteins of 48 and 60 kDa in extracts of the trypanosomatid Leishmania tarentolae. Antibodies against the human PSTAIR peptide detect the p34cdc2 protein in human nuclear extracts but fail to detect a 34 kDa protein in C.fasciculata extracts. These results suggest that novel higher molecular weight forms of the cdc2 protein family may be involved in cell cycle control in trypanosomes.

Kinetoplast DNA is a network of interlocked minicircles and maxicircles. In situ hybridization, using probes detected by digital fluorescence microscopy, has clarified the in vivo structure and replication mechanism of the network. The probe recognizes only nicked minicircles. Hybridization reveals prereplication kinetoplasts (with closed minicircles), donut-shaped replicating kinetoplasts (with nicked minicircles on the periphery and closed minicircles in the center), and postreplication kinetoplasts (with nicked minicircles). Replicating kinetoplasts are associated with two peripheral structures containing free minicircle replication intermediates and DNA polymerase. Replication may involve release of closed minicircles from the center of the kinetoplast and their migration to the peripheral structures, replication of the free minicircles therein, and then peripheral reattachment of the progeny minicircles to the kinetoplast.

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.