Partial Phylogeny of the Unicellular Eukaryotes Based on Rapid

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Partial Phylogeny of the Unicellular Eukaryotes Based on Rapid Proc. Nail. Acad. Sci. USA Vol. 85, pp. 3474-3478, May 1988 Evolution Partial phylogeny of the unicellular eukaryotes based on rapid sequencing of a portion of 28S ribosomal RNA (large subunit rRNA/ribosome/protists/evolution) ANNE BAROIN*, ROLAND PERASSO*, LIANG-HU Qut, GuY BRUGEROLLEf, JEAN-PIERRE BACHELLERIEt, AND ANDRE ADOUTTE* *Laboratoire de Biologie Cellulaire 4 (Centre National de la Recherche Scientifique, Unite Associde 1134), Batiment 444, Universit6 Paris-Sud, 91405 Orsay Cedex, France; tCentre de Recherche de Biochimie et de Gdndtique Cellulaires (Centre National de la Recherche Scientifique, LP 8201), 118, Route de Narbonne, 31062 Toulouse Cedex, France; tLaboratoire de Zoologie et Protistologie (Centre National de la Recherche Scientifique, Unite Associee 40138), Universitd de Clermont, B.P. 45, 63170 Aubibre, France Communicated by Pierre Joliot, December 7, 1987 (received for review August 19, 1987) ABSTRACT Using a rapid rRNA sequencing technique, sequencing of informational macromolecules would there- we have determined the sequence ofthe 400 nucleotides located fore appear to constitute the method of choice to derive at the 5' end of the large subunit rRNA molecule from eight classification schemes and phylogenetic inferences on these species of unicellular eukaryotes (protists). This region con- organisms. Data on 5S RNA have been obtained in many tains a pair of conservative domains well-suited for long-range species during the last few years (8, 9). This molecule suffers, phylogenetic evaluations among eukaryotes, due both to their however, from a number of limitations when used for very substantial length and to their intrinsic rate of sequence distant species (discussed in ref. 9). variation during evolution. It also comprises a central more The development of a technique for rapidly and easily rapidly evolving portion, which allows for a rme tuning of sequencing large stretches of 18S or 28S rRNA, which has distance evaluation between closely related species. Molecular been reported (10), opened the way for systematic exploita- distances were computed between the aligned nucleotides of all tion of the remarkable properties of these molecules as presently available protist sequences and were used to derive a phylogenetic indicators (11). In addition to having a large tentative dendrogram. Within the limitations inherent to this information content, they present the advantage of having a approach, a number of interesting observations emerge: The characteristic mosaic structure, with an interspersion of various protist groups appear to have separated very early adjacent domains evolving at widely different rates (12). One from each other. The most deeply divergent protists belong to can therefore use fast evolving portions for comparing a number oforders offlagellates (mastigotes), suggesting a very species that are expected to be closely related and slowly ancient origin for organelles containing a 9 + 2 microtubular evolving regions for distantly related species. The technique arrangement. Ciliates emerged late among eukaryotes, sug- involves neither DNA cloning nor RNA end-labeling and is gesting that their peculiar genetic code was derived second- carried out directly on total cellular RNA (10). The general arily. Moreover, a dinoflagellate clusters with ciliates, thus applicability of this method has recently been confirmed by making it likely that the unusual features of nuclear organi- Lane et al. (13) by using partial sequence determinations of zation and mitosis of this group are not primitive but derived small subunit rRNA. In this paper, we report on the use of characters. Finally, within groups, taxonomic and evolution- this method for sequencing the 400 5'-terminal nucleotides of ary inferences appear to be feasible using this portion of the the 28S RNA of eight species of protists.§ rRNA. Using a distance matrix method, we derive a tentative dendrogram and discuss a number of interesting biological Unicellular eukaryotes (protists or protoctists; see refs. 1 and conclusions that emerge from the sequence comparisons of 2) have probably played an essential role in the history oflife all presently available large subunit rRNA sequences. The as intermediates between prokaryotes and eukaryotes on one conclusions are in excellent agreement with those based on hand and single-celled eukaryotes and multicellular plants complete small ribosomal subunit sequences (14). and animals on the other hand. Many of the present day protists presumably correspond to descendants of some of the groups that proliferated during these two major evolu- MATERIALS AND METHODS tionary transitions. Their analysis may therefore shed light on Sources of Protist Cultures. Fresh cultures ofBlepharisma the path followed during early cellular evolution. A prelim- japonicum, Stentor coeruleus, and Amoeba proteus (grown inary requirement for the reconstruction of possible evolu- on bacterized rice medium, Chlorogonium, and Chilomonas, tionary scenarios is the availability of a general outline of the respectively) were generously supplied by G. Fryd-Versavel chronological emergence of the various protistan groups. A (Laboratoire de Zoologie 2, Universitd Paris-Sud, Orsay) and number of attempts have been made to this end by using all were harvested after disappearance of the food organism. the information that was available (mostly ultrastructural Crithidia fasciculata (ATCC no. 117) was grown on and, to a more limited extent, biochemical), and some explicit brain/heart extract (Difco) supplemented with rabbit blood. phylogenies have been cautiously suggested (see, for exam- Trichomonas vaginalis (human isolate) was grown on ple, refs. 2-6). The difficulties in deriving phylogenies of the trypticase/yeast extract/maltose (TYM) medium. Parame- protists have been stressed repeatedly: (i) the organisms are cium caudatum (unreferenced wild-type strain) was grown in extremely diverse and may belong to as many as 45 phyla (1), bacterized wheat grass powder medium. Total RNA from (ii) they have left little easily identifiable fossil record (7), and Paramecium primaurelia (strain 156) and Tetrahymena py- (iii) in many groups biochemical data are fragmentary. Direct riformis was kindly provided by Francois Caron (Centre de The publication costs of this article were defrayed in part by page charge §The sequences reported in this paper are being deposited in the payment. This article must therefore be hereby marked "advertisement" EMBL/GenBank data base (Intelligenetics, Mountain View, CA, in accordance with 18 U.S.C. §1734 solely to indicate this fact. and Eur. Mol. Biol. Lab., Heidelberg) (accession no. J03648). 3474 Downloaded by guest on September 28, 2021 Evolution: Baroin et al. Proc. Natl. Acad. Sci. USA 85 (1988) 3475 Genetique Moleculaire, Centre National de la Recherche with some modifications described in the Phylips documen- Scientifique, Gif-sur-Yvette) and J.-J. Curgy (Biologie Ani- tation. male, Universite de Lille 1), respectively. RNA Isolation and Sequencing. The starting material usu- RESULTS ally consisted of 0.5-1.0 ml of washed packed cells that were either fresh (all the ciliates) or had been frozen in liquid Strategy. The presence of invariant (or almost invariant) nitrogen (C. fasciculata and T. vaginalis). Total RNA was tracts in rRNA allows for the selection of "universal" DNA extracted from cells homogenized either in guanidium thio- primers, which can hybridize to rRNA of any species and cyanate medium at 60'C (4 M guanidium thiocyanate/50 mM thus be used for sequencing large portions of the molecule by Tris Cl, pH 7.6/4 mM EDTA/2% N-lauryl-sarcosinate/1% reverse transcriptase elongation in the presence of dideoxy- 2-mercaptoethanol) or in NaDodSO4 medium at room tem- nucleotides (10, 13, 15). We have recently shown (15) that a perature (5% NaDodSO4/50 mM Tris Cl, pH 7.4/0.15 M region of the eukaryotic large subunit rRNA, which extends NaCl/5 mM EDTA). All subsequent steps were carried out over about 400 nucleotides from the 5' end, contains two as described elsewhere (10, 15). Briefly, RNA sequencing conservative domains. These domains appear ideally suited by reverse transcriptase elongation of 32P 5' for evaluating distances among very distant eukaryotes since was carried out substitutions do not reach saturation levels within them end-labeled synthetic DNA primers in the presence of chain (unlike the situation with 5S RNA) but remain, nevertheless, terminating dideoxynucleotides. Three oligonucleotides, sufficiently numerous to allow meaningful calculations. A complementary to the evolutionarily conserved 28S rRNA few "hot spots" (denoted by short vertical lines in Fig. 1) segments 84-106, 278-302, and 382-404 [numbers referring have been detected within these two domains by comparison to mouse sequence coordinates (16)], respectively, were used of all the presently available sequences. These noninforma- systematically as primers. tive sites have been excluded from the homology measure- Sequence Comparisons. Alignment of sequences, compu- ments. As for the D1 domain (see Fig. 1 for delineation), tation of the observed as well as the corrected number of which corresponds to one of the rapidly evolving areas ofthe nucleotide differences [using Kimura's Knuc correction 28S RNA molecule (12), changes become saturating for even (16)], and derivation of the resulting distance matrix were medium-range comparisons among eukaryotes (15). This carried out exactly as described (15, 17). Dendrograms were domain
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