What is a genus in the Trypanosomatidae family? Phylogenetic analysis of two small rRNA sequences.

The family Trypanosomatidae of the order Kinetoplastida is characterized by flagellated protozoa that present a kinetoplast. Most of the recognized genera are parasitic protozoa and, depending on host range, have been classified as either monogenetic trypanosomatids (parasitic in a single invertebrate) or digenetic trypanosomatids (parasitic in both invertebrate and vertebrate hosts). Trypanosomatids are unusual eukaryotes in that their nuclear large-subunit t-RNA genes reveal a complex organization with the occurrence of internal transcribed spacers separating the coding regions for two large (a and B) and five small (Sl-S4 and S6) rRNA molecules (White et al. 1986; Spencer et al. 1987; Hemindez et al. 1988 ) . The small rRNA molecule S3 corresponds to the characteristic eukaryotic 5.8s rRNA (Hasan et al. 1984). We have previously published the molecular cloning of rRNA genes from Trypanosoma cruzi (Hemandez et al. 1988) and determined its small-subunit (SSU) rRNA coding region ( Hemlndez et al. 1990). We report here the nucleotide sequences of T. cruzi S3 (or 5.8s) and S 1 rRNA coding regions and compare them evolutionarily with their published homologues from the monogenetic species Crithidia fasciculata (Spencer et al. 1987) and the digenetic species T. brucei (Campbell et al. 1987). In addition, our previous SSU rRNA sequence (Hemandez et al. 1990) was further analyzed with the inclusion of six of nine variable regions and with Dictyostelium discoideum (Sogin et al. 1986) as a second outgroup. Appropriate restriction fragments from our genomic rDNA clone pRTC42 (Hernandez et al. 1988) were subcloned into the polylinker region of M 13mp18 and M13mp19 vectors (Yanisch-Perron et al. 1985). Nucleotide sequencing was carried out from single-stranded templates by the dideoxynucleotide chain termination method with modified T7 DNA polymerase (Tabor and Richardson 1987) by using the SequenaseM kit (U.S. Biochemical Corp.) and (u-~~P) dATP ( Amersham). The 5’ and 3’ ends of Sl and S3 rRNA coding regions were assigned by comparison with published sequences from C. fasciculata (Spencer et al. 1987) and from T. brucei (Campbell et al. 1987) (fig. 1). Sl rRNA, whose coding region did not exhibit 5’ terminal identity with the homologous regions of C. fasciculata and T. brucei, was partially sequenced from 5 ‘kinased RNA molecules (‘Y-~~P) ATP (New England Nuclear), after the partial enzymatic digestion technique using the Bethesda Research Laboratories RNA sequencing kit and protocol. An analogous genome sequence has been recently published from a Brazilian T. cruzi strain (Vieira de Arruda et al. 1990). Our direct rRNA sequence places the S 1 rRNA 5 ’ terminal coding region two nucleotides downstream from its indirect comparative assignment. The rest of the sequence is identical to ours, with the exception of an extra G in a run of five G’s that we find in positions 128132. This addition could be a natural polymorphism among strains. The trypanosomatid S3 and S 1 rRNA molecules correspond, respectively, to the rRNA 5.8s and to the central region of domain IV of the large-subunit rRNA from other eukaryotes (Hasan et al. 1984; Lanversin and Jacq 1989). To root the phylo-