strain of L. tarentolae (Thiemann et al., 1994). LEM125 has the same 15 maxicircle gRNA genes but also has an estimated 80 total minicircle-encoded gRNAs, of which 30 have been cloned and sequenced and the remainder inferred to be present because of the existence of completely edited mRNA transcripts in this strain. These additional gRNAs mediate the editing of three components of complex I of the respiratory chain, ND3, ND8, and ND9, and also two unidentified genes, which were termed G-rich regions 3 and 4 (G3 and G4). It was proposed that multiple gRNA-encoding minicircle sequence classes had been lost from the UC strain probably because of a lack of a requirement for complex I activity in culture (Simpson and Maslov, 1994; Thiemann et al., 1994). The presence of productively edited ND8, ND9, G3 (=CR3), G4 (=CR4), and ND3 mRNAs in T. brucei and the presence of productively edited G3 mRNA in P. serpens (D.A.M., unpublished results) implies that the corresponding minicircle-encoded gRNAs also exist in these species, and this provides phylogenetic evidence for our hypothesis that the ancestral cell had a complete complement of minicircle classes. In addition, the presence of two minicircle-encoded gRNAs, gG4-III and gND3-IX, in the UC strain, which are remnants of the complete editing cascade of gRNAs for these two genes in LEM125, corroborates this evidence. To propose a loss of multiple minicircle classes from the UC strain is also more parsimonious than to propose a gain of multiple classes in the LEM125 strain. And finally, the existence of a 5′ terminal block of misedited sequence in the LEM125 ND3 mRNA (Thiemann et al., 1994) is indicative that this gene originally was completely edited and has lost the 5′ terminal gRNA.
Minicircles from L. tarentolae and other members of the Leishmania-Crithidia clade contain a single gRNA gene situated at a constant distance from the origin of replication (Sturm and Simpson, 1990b; Yasuhira and Simpson, 1995). Minicircles from T. brucei, however, also have a single origin of replication but contain three gRNA genes situated between 18-mer inverted repeats (Pollard et al., 1990), and minicircles from T. cruzi contain four gRNA genes situated within each of the four variable regions between four origins of replication (Avila and Simpson, 1995). The total number of different minicircle sequence classes in T. brucei is estimated to be 200–300 (Stuart, 1979), which would yield a total of 600–900 gRNAs. Although only 72 gRNAs have been identified so far in T. brucei (Souza et al., 1997), it is clear that there are extensive redundant gRNAs, which are gRNAs of different sequence but possessing the identical editing information because of the allowed G:U base pairing (Corell et al., 1993). In fact, 28 of the 72 identified gRNAs are redundant over the entire length of the gRNA. Only a single redundant gRNA pair has been observed in L. tarentolae (Thiemann et al., 1994). T. brucei also contains gRNAs with sev-