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(Kilejian, 1975) and Plasmodium berghei (Dore et al., 1983). These molecules matched the size range and conformation expected for mitochondrial genomes of unicellular eukaryotes, and so were immediately labeled as such. It was, of course, the logical conclusion. It was also wrong. Who would have suspected these were remnant chloroplast genomes? The only clue was the cruciform structure, typical of chloroplast but not mitochondrial genomes. Certainly no one connected the circular genomes with the multi-membraned organelle.

Research on organelle DNA in apicomplexans was initially pursued exclusively in Plasmodium. Williamson, Wilson, and colleagues identified three bands in isopycnic sucrose density gradients of Plasmodium knowlesi (Williamson et al., 1985) and P. falciparum (Gardner et al., 1988) lysates. One was lighter than the main band of nuclear DNA, as is usually the case for mitochondrial genomes. It proved to be a ~35 kb circular DNA (Williamson et al., 1985; Gardner et al., 1988), and when spread for electron microscopy it demonstrated a cruciform structure (Williamson et al., 1985). It thus displayed the characteristics of the previously reported 'mitochondrial' DNAs of apicomplexans.

The lowest band on the P. falciparum gradient, described as 'diffuse and weakly fluorescent' (Feagin et al., 1992), migrated just below the nuclear DNA band and proved to contain tandem repeats of a 6 kb DNA sequence. A similar repeated sequence had been identified in P. yoelii (Vaidya and Arasu, 1987; Suplick et al., 1988; Vaidya et al., 1989) and P. gallinaceum (Aldritt et al., 1989; Joseph, 1990). Upon sequencing, the '6 kb element' in all three species was found to encode classic mitochondrial proteins (apocy-tochrome b, cob; cytochrome c oxidase subunit I, cox1; and subunit III, cox3), and small, fragmented rRNAs (Aldritt et al., 1989; Vaidya et al., 1989; Joseph, 1990; Feagin, 1992; Feagin et al., 1992) (see section 9.3.2). Despite its minute size, this repeated element has the requisite minimum of genes invariably expected in mitochondrial genomes (Gillham, 1994). But if the 6 kb element was the mitochondrial genome, what was the 35 kb DNA?

Analysis of the 35 kb DNA revealed that it contains a large inverted repeat composed of two copies of a small subunit (SSU) rRNA and large subunit (LSU) rRNA, arranged tail to tail (Gardner et al., 1988, 1991a, 1993). The rRNAs are similar to those of prokaryotes, as expected for both mitochondrial and plastid rRNAs. However, mitochon-drial genomes do not typically have duplicated rRNAs, while those of chloroplasts do (Gillham, 1994). Further sequencing showed that the 35 kb DNA also encodes subunits of a eubacterial-like RNA polymerase (Gardner et al., 1991b). This is unequivocally a plastid characteristic; all plastid genomes studied thus far encode and are transcribed by such RNA polymerases. Some plastids import one or more additional RNA polymerases from the cytoplasm. In contrast, almost all mitochondria employ a single subunit RNA polymerase most closely related to phage RNA polymerases (Gray and Lang, 1998). Further analysis from the P. falciparum 35 kb DNA identified components of an organelle translation system, but no photosynthesis-related genes.

Serendipitously, the chloroplast genomes of Epifagus virginiana, a non-green plant, and Astasia longis, a non-green alga, were under analysis at the same time. These are both much reduced in size compared to those of green plants. Plastid-encoded genes related to photosynthesis were missing but those needed for expression of the organellar genome were retained, including rRNAs duplicated as an inverted repeat (reviewed in dePamphilis and Palmer, 1989). The parallels with the P. falciparum 35 kb DNA are striking (Figure 9.2). With the accumulating data, the formerly implausible explanation that the 35 kb DNA was derived from chloroplast DNA became increasingly believable (Wilson et al., 1991; Palmer, 1992). It is now well established that apicomplexans have ancestors in the plant kingdom, though many questions remain about the details (see section 9.2.2).

The T. gondii plastid genome was sequenced by 1997 (GenBank accession U87145, RefSeq NC-001799). It is strikingly similar to its P. falciparum

FIGURE 9.2 Plastid genome structure. Schematic depiction of the plastid genomes of Nicotiana tabacum (Nt), a green plant; Epifagus virginiana (Ev), a non-green plant, and P. falciparum (Pf). Genome sizes are indicated. The salient feature of each genome is an inverted repeat, producing two IR regions per genome (thickened lines). The IRs include rRNAs, tRNAs, and except for P. falciparum, some protein-coding genes. IR size for N. tabacum, E. virginiana, and P. falciparum: 25 kb, 23 kb, and 5 kb, respectively.

FIGURE 9.2 Plastid genome structure. Schematic depiction of the plastid genomes of Nicotiana tabacum (Nt), a green plant; Epifagus virginiana (Ev), a non-green plant, and P. falciparum (Pf). Genome sizes are indicated. The salient feature of each genome is an inverted repeat, producing two IR regions per genome (thickened lines). The IRs include rRNAs, tRNAs, and except for P. falciparum, some protein-coding genes. IR size for N. tabacum, E. virginiana, and P. falciparum: 25 kb, 23 kb, and 5 kb, respectively.

counterpart in gene content and organization (see section 9.2.3). Complete apicoplast genome sequences are currently available for three more apicomplexans - the chicken pathogen Eimeria tenella (Cai et al., 2003), and the bovine pathogens Theileria parva (Gardner et al., 2005) and Babesia bovis (O.T. Lau, E.H. Roalson, K.A. Brayton, V.M. Nene, D.P Knowles, and T.F. McElwain, personal communication) - and partial sequences are available for numerous other species (Lang-Unnasch and Aiello, 1999; Obornik et al., 2002a). Genome sequencing projects by the Pathogen Sequencing Group at The Sanger Institute provide apicoplast gene sequences for additional apicom-plexans, including Theileria annulata, Babesia bigemina, and multiple Plasmodium species (http://www.sanger.ac.uk/Projects/Protozoa/). Conservation of gene content and genome organization is strong; the principal difference is that the piroplasms Babesia and Theileria have only one copy of the rDNA transcription unit

(Gardner et al., 2005; Lau et al., personal communication). This high degree of genome similarity is matched by functional conservation that is largely dependent on the import of nuclearly encoded proteins (see sections 9.2.7 and 9.2.9). This makes Toxoplasma an excellent model for study of the apicomplexan plastid.

A lingering question was the subcellular location of the 35 kb genome. It did not co-localize with the mitochondrial genome in sucrose gradients of P. falciparum lysates (Wilson et al., 1992), so a mito-chondrial location appeared unlikely. As a remnant chloroplast genome, it should reside in an organelle with more than one bounding membrane. The 35 kb DNA was a genome without a home, and the spherical body was an organelle without a role -might they intersect? The well-defined subcellular structure of T. gondii makes it more amenable for cell biological studies than P. falciparum, so it is unsurprising that the localization question was first answered for T. gondii. In the mid-1990s, in situ hybridization studies using probes derived from the T. gondii 35 kb DNA showed that this genome resides in an organelle located just apical to the nucleus, the hitherto mysterious multi-membraned organelle (McFadden et al., 1996; Köhler et al., 1997). The P falciparum 35 kb DNA has also been demonstrated to reside in the corresponding organelle (Wilson et al., 1996). The plethora of names for the organelle have been replaced with a single term: the apicoplast, for apicomplexan plastid. Solving the initial mysteries of the homeless genome and the unexplained organelle has generated a number of fascinating questions: How does a group of obligate intracellular parasites get a plastid? Why has it been maintained in non-photosyn-thetic organisms? What role does it play for the cell? What possibilities for disease intervention result from the presence of 'plant' genes in protozoan pathogens?

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