A Plastid of Probable Green Algal Origin in Apicomplexan Parasites

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The complete sequence of the T. gondii 35-kb DNA has been deposited in GenBank, with accession number U87145. The map of this genome is virtually identical to that of Plasmodium (3).

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The apicomplexan tufA genes are 70 to 80% A-T (∼95% at third-codon positions), versus 48% (43%) for bacterial tufA genes and 66% (84%) for plastid tufAs. Biases in base composition can influence phylogenetic analysis [P. J. Lockhart, M. A. Steel, M. D. Hendy, D. Penny, Mol. Biol. Evol. 11, 605 (1994)], but bias-tolerant methods such as transversion and LogDet analyses consistently place the 35-kb element well within the plastids. Although apicomplexan tufAs are highly divergent (Plasmodium in particular) and therefore may be vulnerable to “long branch” effects [J. Felsenstein, Syst. Zool. 27, 401 (1978); D. M. Hillis, J. P. Huelsenbeck, C. W. Cunningham, Science 264, 671 (1994); J. H. Kim, Syst. Biol. 45, 363 (1996)], the Apicomplexa remain within the plastids even in the presence of the rapidly evolving mitochondrial and Mycoplasma genes. Only the association with Coleochaete in parsimony analyses is a probable example of long-branch effects.

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We prepared nick-translated DNA probes covering 10.5 kb of the T. gondii 35-kb circle by incubating 1 to 2 μg of template DNA for 2 hours at 14°C in a 50-μl reaction mixture containing 50 mM tris (pH 7.8), 0.1 mM digoxigenin-11-deoxyuridine 5′-triphosphate (dUTP) (Boehringer-Mannheim), 0.4 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP, 5 mM MgCl₂, 10 mM dithiothreitol, 2.5 μg of nuclease-free bovine serum albumin, 10 U of DNA Polymerase I, and DNase I at concentrations titrated to produce labeled fragments with an average length of ∼150 bp. RH-strain T. gondii tachyzoites were cultured in vitro in primary human fibroblasts [D. S. Roos, R. G. K. Donald, N. S. Morrissette, A. L. C. Moulton, Methods Cell Biol. 45, 27 (1994)], resuspended in phosphate-buffered saline (PBS) at ∼5 × 10⁷parasites/ml, attached to silane-coated glass slides, and fixed for 10 min at 25°C in a solution of 4% formaldehyde, 65% methanol, and 25% glacial acetic acid, followed by two 5-min fixations in methanol and glacial acetic acid (3:1). Slides were rinsed twice for 5 min in 100% ethanol, rehydrated, and permeabilized for 10 min at 25°C in Proteinase K at concentrations from 0.1 to 1.0 μg/ml (optimal concentrations varied from batch to batch) in 10 mM tris (pH 8.0) containing 5 mM EDTA. Specimens were then fixed for 5 min on ice in PBS-buffered 4% formaldehyde, rinsed twice in PBS, and incubated for 5 min in 2× standard saline citrate (SSC). Intracellular RNA was removed by digestion for 1 to 2 hours in a 200-μg/ml solution of DNase-free RNase A (in 2× SSC), followed by dehydration through an ethanol series. Parasite DNA was denatured for 5 min at 70°C in 70% formamide (in 2× SSC), chilled in ice-cold 70% ethanol, and dehydrated. Hybridization was carried out for 12 hours at 37°C in a 15-μl volume [10 ng of heat-denatured probe, 1 μg of yeast tRNA, and 1 to 2 μg of heat-denatured calf thymus DNA per microliter of 50% formamide, 10 mM tris (pH 7.4), 300 mM NaCl, 1 mM EDTA (pH 8), 10% dextran sulfate, and 1× Denhardt's solution]. After hybridization, the slides were rinsed in 4× SSC and twice washed for 10 min at 25°C in 4× SSC, twice for 3 min at 37°C in 50% formamide (in 2× SSC), twice for 5 min at 37°C in 2× SSC, once for 2 min at 25°C in 2× SSC, and twice for 5 min at 25°C in 4× SSC. We visualized the hybrids by incubating the specimens for 40 min at 25°C in 4× SSC containing rhodamine-conjugated polyclonal sheep anti-digoxigenin (Boehringer) and 0.5% nuclease-free blocking reagent. Control hybridizations with labeled pGEM-3 vector DNA showed no signal. Nuclear DNA was stained for 20 min at 25°C with 2.5 nM YOYO-1 (Molecular Probes) in 1× SSC. In Fig. 1, F through H, extranuclear DNA was stained with a monoclonal antibody raised against double-stranded DNA (Boehringer), followed by a secondary fluorescein isothiocyanate (FITC)-conjugated rabbit-antimouse antibody (Pierce). Both YOYO and FITC were visualized with a fluorescein filter set. Specimens were mounted in Aqua-Poly/Mount (Polysciences) and analyzed with a Leitz scanning confocal microscope equipped with a Kr-Ar laser, FITC and tetramethyl rhodamine isothiocyanate filter sets, and a transmitted-light detector.

Antisense- and sense-RNA probes were prepared by standard procedures, with the use of the T7 and Sp6 promoters in pGEM-3, flanking a 0.7-kb cloned fragment from the T. gondii 35-kb genome predicted to encode rps4 (5). Freshly harvested parasites were fixed for 5 min at 25°C in 4% PBS-buffered formaldehyde, washed in PBS, attached to silane-coated slides, fixed for 5 min on ice in 4% formaldehyde, briefly washed in PBS, and treated with Proteinase K. After another 5 min of fixation on ice in 4% formaldehyde, the slides were washed in PBS, dehydrated, and incubated with 15 μl of hybridization solution (26), at probe concentrations of 1 to 2 ng/μl. Nuclear DNA was counterstained with YOYO-1.

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Extracellular tachyzoites were fixed for 1 hour on ice in 4% PBS-buffered formaldehyde and then for 12 hours at 4°C in 8% PBS-buffered formaldehyde. The cell suspension was embedded in 10% gelatin, incubated for 2 hours at 4°C in PBS containing 2.3 M sucrose, and frozen in liquid nitrogen. Ultrathin sections of the frozen samples were freshly prepared before each hybridization experiment. Cryosections were transferred to grids and digested for 40 min at 37°C in 2× SSC containing 200 μg/ml of DNase-free RNase A. Cellular DNAs were denatured for 5 min at 70°C in 70% formamide (in 2× SSC), chilled on ice, transferred to 50% formamide (in 2× SSC), and incubated briefly at 25°C. Sections were hybridized for 12 hours at 37°C in a humidified chamber in 5 μl of hybridization mix containing 10 to 20 ng/μl of DNA probe (26), washed three times for 5 min at 25°C in 4× SSC, twice for 3 min at 37°C in 50% formamide (in 2× SSC), twice for 5 min at 25°C in 2× SSC, and kept in 4× SSC at 25°C before staining. Hybridized probe was detected with polyclonal sheep anti-digoxigenin, followed by a secondary rabbit antibody directed against sheep immunoglobulin G (Pierce), and Protein A conjugated to 10-nm particles of gold. Immunogold-labeled sections were blocked for 20 min at 25°C in 4× SSC containing 0.5% blocking reagent and were incubated with a monoclonal antibody against DNA, followed by a rabbit anti-mouse secondary antibody and protein A conjugated to 5-nm gold particles. To improve the contrast of membranous structures, we counterstained hybridized cryosections on ice for 10 min in 0.3% aqueous uranyl acetate plus 2% methylcellulose. Grids were air-dried on loops and examined with a Phillips EM400 microscope.

The antibody directed against DNA used in Fig. 2A probably recognizes both endogenous DNA and the digoxigenin-labeled probe. Similarly, the 5-nm gold-protein A conjugate used to visualize this antibody (by means of a secondary rabbit antibody) is potentially able to recognize any anti-digoxigenin that remained unblocked. Comparable staining with antibody against DNA was observed even in the absence of a DNA probe, however (Fig. 2C), or when control plasmid was used as a probe. Cryosections labeled with antibody to DNA before the application of anti-digoxigenin also showed co-localization of large and small gold particles. The apparent clustering of label in Fig. 2, A and B, may be an artifact of in situ hybridization conditions, because antibody directed against DNA labels the organelle uniformly (Fig. 2C).

Infected cultures were fixed for 45 min in freshly prepared 50 mM phosphate buffer (pH 6.3) containing 1% glutaraldehyde and 1% OsO₄, rinsed in distilled water, stained in 0.5% uranyl acetate overnight, dehydrated, and embedded in Epon. Ultrathin sections were picked up on uncoated grids, stained with uranyl acetate and lead citrate, and examined with a Phillips 200 electron microscope.

A total of 65 sequences, including nearly all available bacterial sequences and representative plastid sequences, were aligned using PILEUP [Genetics Computer Group, Madison, WI (1991)], with manual refinement on the basis of secondary structural information. Maximum likelihood analysis was performed with fastDNAml v1.0.6 [G. J. Olsen, H. Matsuda, R. Hagstrom, R. Overbeek, CABIOS 10, 41 (1994)], compiled as parallel code running on an Intel Paragon 64-node partition. Three random addition sequences and global swapping were used, but it cannot be guaranteed that the tree found is the highest likelihood tree possible. Bootstrap data sets and consensus trees were generated using PHYLIP tools SEQBOOT and CONSENSE [J. Felsenstein, University of Washington, Seattle, WA (1993)]. Bootstrap replicates were analyzed with fastDNAml using a single random addition sequence and local branch swapping only. LogDet, parsimony, and constraint analyses were performed with PAUP*4.0d48 [D. L. Swofford; Smithsonian Institution, Washington, DC (1996)] using nucleotide data from the first and second codon positions, and bootstrapping was carried out using 100&nbs.