All extant organisms are thought to be classified into three primary kingdoms, eubacteria, eukaryotes, and archaebacteria. The molecular evolutionary studies on the origin and evolution of archaebacteria to date have been carried out by inferring a molecular phylogenetic tree of the primary kingdoms based on comparison of a single molecule from a variety of extant species. From such comparison, it was not possible to derive the exact evolutionary relationship among the primary kingdoms, because the root of the tree could not be determined uniquely. To overcome this difficulty, we compared a pair of duplicated genes, elongation factors Tu and G, and the a and f3 subunits of ATPase, which are thought to have diverged by gene duplication before divergence of the primary kingdoms. Using each protein pair, we inferred a composite phylogenetic tree with two clusters corresponding to different proteins, from which the evolutionary relationship of the primary kingdoms is determined uniquely. The inferred composite trees reveal that archaebacteria are more closely related to eukaryotes than to eubacteria for all the cases. By bootstrap resamplings, this relationship is reproduced with probabilities of 0.96, 0.79, 1.0, and 1.0 for elongation factors Tu and G and for ATPase subunits a and ., respectively. There are also several lines of evidence for the close sequence similarity between archaebacteria and eukaryotes. Thus we propose that this tree topology represents the general evolutionary relationship among the three primary kingdoms.Based on comparison of the small rRNAs, Woese and colleagues (1)(2)(3) proposed that there are two fundamentally different groups of bacteria, eubacteria and archaebacteria, and that, with eukaryotes, they constitute the three primary kingdoms of life. Although the existence of the archaebacterial urkingdom is accepted by many biologists, the classification is still a matter of controversy: Lake and colleagues (4,5) argued that archaebacteria are paraphyletic; sulfobacteria (eocytes) are more closely related to eukaryotes than to other archaebacteria, whereas halobacteria are more closely related to eubacteria than to other archaebacteria. Phylogenetic trees based on the small and large rRNAs (2, 3, 6), 5S rRNA (7), and the RNA polymerases (8), however, support the monophyletic view of the archaebacteria originally proposed by Woese and colleagues (1-3).The evolutionary relationship of the three primary kingdoms is another crucial problem that remains unanswered.There are several reports that, in some RNA and protein species, archaebacteria are much more similar in sequence to eukaryotes than to eubacteria. These include 5S rRNA (7, 9, 10), elongation factors Tu (11) and G (12) (EF-Tu and EF-G), large subunit of DNA-dependent RNA polymerase (8, 13), and several ribosomal proteins (14,15). The a and /3 subunits of Sulfolobus ATPase (16, 17) also bear closer resemblance in sequence to eukaryotic counterparts than to eubacterial ones. However, a close similarity in sequence does not ne...

The genomic guanine and cytosine (G+C) content of eubacteria is related to their phylogeny. The G+C content of various parts of the genome (protein genes, stable RNA genes, and spacers) reveals a positive linear correlation with the G+C content of their genomic DNA. However, the plotted correlation slopes differ among various parts of the genome or among the first, second, and third positions of the codons depending on their functional importance.

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UGA is a nonsense or termination (opal) codon throughout prokaryotes and eukaryotes. However, mitochondria use not only UGG but also UGA as a tryptophan codon. Here, we show that UGA also codes for tryptophan in Mycoplasma capricolum, a wall-less bacterium having a genome only 20-25% the size of the Escherichia coil genome. This conclusion is based on the following evidence. First, the nucleotide sequence of the S3 and L16 ribosomal protein genes from M. capricolum includes UGA codons in the reading frames; they appear at positions corresponding to tryptophan in E. coli S3 and L16. Second, a tRNA"rP gene and its product tRNA found in M. capricolum have the anticodon sequence 5' U-C-A 3', which can form a complementary base-pairing interaction with UGA.We recently have sequenced a part of the Mycoplasma capricolum ribosomal-protein gene cluster that codes for polypeptides highly homologous to the Escherichia coli ribosomal proteins S3 and L16. The sequence contains four UGA codons in the reading frames; three appear at the sites corresponding to tryptophan, and one, at a site corresponding to arginine in the E. coli proteins. No "universal" UGG codon for tryptophan has so far been found. We have also isolated a clone containing a pair of M. capricolum tRNA genes, the sequence of both of which resembles that of tRNATrp of E. coli. The anticodon sequence of one of these tRNA genes is 5'-T-C-A-3', which can base-pair with both opal codon UGA and universal tryptophan codon UGG. That of the other is 5'-C-C-A-3', which may base-pair exclusively with UGG. These two tRNA genes are expressed in the cell. All these findings suggest strongly that, in M. capricolum, UGA codes for tryptophan using the opal tRNAUCA but not tRNACCA.RESULTS AND DISCUSSION UGA Codons in M. capricolum S3 and L16 Genes. As reported in a previous paper (1), we isolated the recombinant plasmid pMCB1088 containing a 9-kilobase-pair fragment of M. capricolum DNA. The fragment contains the genes for at least nine ribosomal proteins-S3, S5, S8, S14, S17, L5, L6, L16, and L18-as deduced from its encoded protein sequences being highly homologous with the corresponding E. coli ribosomal protein sequences (refs. 1 and 2; unpublished results). Fig. 1 shows the complete nucleotide sequence of a 629-base-pair (bp) HindIII fragment which is a part of the insert of pMCB1088 (see refs. 1 and 2). The DNA corresponds to the 3' half of the S3 gene and about 90% of the L16 gene from the 5' terminus. When the M. capricolum sequences are aligned with the E. coli protein sequences (3, 4) ( Fig. 1), four UGA (opal) codons are found within the reading frames. The possibility that these UGA codons are termination signals can be excluded by their occurrence in the regions having extensive sequence homologies with the E. coli proteins. More importantly, three out of the four UGA codons appear at the positions corresponding to tryptophan in the E. coli proteins. This suggests that UGA is a sense codon, probably for tryptophan, in M. capricolum. No UGG codon for tryptop...

The genetic code, once thought to be "frozen," shows variations from the universal code. Variations are found in mitochondria, Mycoplasma, and ciliated protozoa. The variations result from reassignment of codons, especially stop codons. The reassignments take place by disappearance of a codon from coding sequences, followed by its reappearance in a new role. Simultaneously, a changed anticodon must appear. We discuss the role of directional mutation pressure in the events, and we also describe the possibility that such events have taken place during early evolution of the genetic code and can occur during its present evolution.