Evolution of Life

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S. Osawa

T. Honjo (Eds.)

Evolution of Life Fossils, Molecules, and Culture

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3.7 Archaebacteria Vs Metabacteria: Phylogenetic Tree of Organisms Indicated by Comparison of 5S Ribosomal RNA Sequences HIROSHI HORI, YUKIO SATOW,i ISAO INouE

and,

i\llITSUO CHIHARA2

Summary. Over 550 complete nucleotide sequences of 5S ribosomal RNAs from cytoplasm, chloroplasts, and mitochondria arc now available. A phylogcnctic tree deduced from them suggests that all extant organisms arc classified into three major lineages: cubactcria, mctabactcria (archacbactcria)' and cukaryotcs. In the cubactcrial branch, several groups, such as Gram-negative bacteria, cyanobactcfia, Gram-positive bacteria, and actinobactcria, diverged during the early stage of evolution. !vletabactcria and cukaryotcs separated after the emergence and divergence of cubactcria. By using 16S rRNA sequences, many trees are also now available [1-4]. However, there are some instances where 5S and 16S rRNA sequences give discrepancies larger than those expected from statistical error [5J. The explanation is that the discrepancy, especially that of me tabacteri a versus archaebacteria, is due to a drastic change of substitution rates in 16S rRNAs. Although metabacteria form a very unique group in bacteria, the 5S rRNA tree supports the view that these bacteria still belong to the prokaryotic domain together with eubacteria. Therefore, life on this planet is seen as comprising two domains, the eukaryotic domain and the prokaryotic domain (the latter includes the metabactcrial and eubacterial kingdoms), but not comprising the three domains of Archaea, Bacteria, and Eukarya by '-\foese et al. [4].

Introduction Because of its universal occurrence and conservative rate of evolution, ribosomal RNA, especially 5S ribosomal RNA (5S rRNA) seems to be onc of the most useful and ideal molecules for the resolution of phylogenctic relationships at high hierarchial level [6]. The 5S rRNA sequences from over 550 different organisms: cyto-

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Department of Genetics, Gen-Iken, Hiroshima University, Kasumi, Hiroshima, 734Japan of Bio!ogy, Tsukuba University, Tsukuba, 305Japan

2 Department

325

326

H. Hori et al.

plasm (cyt), chloroplast (chi), and mitochondria (mt) have bccn reported. To estimate the evolutionary relationship ofcyt, chi, and mt more precisely, wc thought it important to sequence the 5S rRNA from each of the three cell components of one organism. We therefore have determined the nucleotide sequence of cyt, chI, and mt 5S rRNAs from a primitive unicellular green alga, lliamiella sp., from which it was technically easy to isolate the three types. Using these sequences, we constructed a phylogenetic trec. The tree indicates that after the emergence of eubacteria, Sulfolobus, methanogens, and extreme halophiles, which collectively we [7,8] called metabacteria (archaebacteria, according to \Voese and Fox [1] and Pace et al. [3]) and eukaryotes separated each other from their common ancestor.

Materials and Methods A prasinophyte green alga, 111amiella sp. TKB8052, isolated in Hachinohe harbor, northern Japan, was cultured in ES:tvI medium [9]. Crude RNA was directly extracted from 8.9 g of wet cells by the phenol method and purified by means of 7M urea-12% acrylamide gel electrophoresis [10). RNA was labelled enzymatically with ([32P)) phosphate at its 3' or 5' terminus and purified again by using long urca-acrylamide gel electrophoresis (80 X 20 X 0.1 cm). Three RNA bands corresponding to cyt, chI, and mt 5S rRNAs were sequenced by the chemical and enzymatic methods [11,12). The 558 5S rRNA sequences from various organisms available as of February 1990 have been compiled in a database IRIS together with their taxonomic information and details of the alignment [13]. The evolutionary distance, Kll11C, between two sequences and its SE were calculated as previously shown [6J. Briefly, these were calculated by means of the equations described by Kimura [14]. Knuc estimates the number of base substitutions per nucleotide site that have occurred since the separation of the two sequences.

Kllue = - (1/2) loge[ (I - 21' - Q) (I - 2Q)'/2) where P and Qare the fractions of nucleotide sites between two sequences showing transition- and transversion-type differences respectively. A phylogenetic tree was constructed by the simplified unweighted pair group average method [6).

Results Sequence Data and Secondary Structure The nucleotide sequences of cyt, chI and mt 5S rRNAs from Alamiella sp. arc shown in Fig. la-c. Generally, the secondary structure model of5S rRNAs is the same for all organisms, but there exists partial specificity in each group; secondary structures may be classified into four types: the eukaryotic type, metabacterial type, eubacterial type (including chloroplasts), and mitochondrial type (sce [6,!5] for

3.7. Archaebacteria Vs ?vletabacteria

327

details). Eukaryotic 5S rRNA diU-ers from all the eubacterial 5S rRNAs by having the eukaryote-specific E-E' base-pairing and the loop (Fig. la; sce Fig. I of [15]). The metabacterial type has the nonconserved "eukaryotic" E-E' helix, a bulge of the D-D' region and the conserved loop between D and E (eLd region) as in the eukaryotic 5S rRNA, supporting the view that metabacteria arc more closely related to eukaryotes than to eubacteria. The third type, to which all eubacterial 5S rRNAs (and chloroplast 5S rRNAs) belong, may be called the cubactcrial type [15]. They have highly conserved regions corresponding to less conserved E-E' regions in eukaryotic 5S rRNAs. The cyt and chI 5S rRNAs from ,.ylmniella sp. determined in this paper obey these rules, revealing typical eukaryotic and eubacterial type sequences respectively (Fig. la-b). The secondary structure of mitochondrial 5S rRNA from land plants is quite different from that of other rRNAs, having insertions or deletions in the alignment (Fig. Id), and it is not possible to estimate the exact divergence point (6). However, the mt 5S rRNA from klamiella sp. shows a secondary structure more similar to the eubacterial type (Fig. lc) than is that of wheat mt 5S rRNAs. On the basis of the 5S rRNA alignment (not shown), percentage similarities of all possible pairs of sequences were calculated and summarized in a homology triangle of 500 representative sequences (Fig. 2). This schema does not show as detail as a dendrogram, but groups with high homologies can be recognized distinctly. Figure 2 clearly indicates the low homology of metabacterial 5S rRNA sequences to those of eukaryotes and eubacteria. In spite of these low homology, metabacterial sequences reveal slight homology to those from actinobacteria, representing their evolutionary relatedness.

Outline of the Phylogenetic Tree A phylogenetic tree deduced from 558 5S rRNA sequences has been constructed, and an outline of it including most of the major groups of photosynthetic organisms is represented in Fig. 3. As we pointed out previously [6,7], the tree shows that eubaeteria first separated from the metabacteria/eukaryotes branch. IVlctabacteria form a unique group that is phylogenetically closer to eukaryotes than to eubacteria. Among eukaryotes, cyt 5S rRNA from iHamiella Sf). indicates that all green plants examined herein belong to the same green plant branch in accordance with the classical view. Three major types of photosynthetic eukaryotes-rcd algae (chlorophyll a + phycobilins group), green plants (a + b group) and chromophytc algae (a + c group)-are more remotely related to onc another. It is plausible that a single symbiosis of photosynthetic prokaryotes with eukaryotes (emergence of photosynthetic cukaryotes) took place before the divergence of the different algae at the time in the tree marked E in Fig. 3. In organelle evolution, chloroplasts have a typical eubacterial 5S rRNA (Fig. 1b) and their phylogenetic position in the tree is very close to cyanobacteria and the cyanelle of C)1allophora paradoxa, suggesting the symbiotic origin of chloroplast (point D in Fig. 3).

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