Syozo Osawa

Recent evidence for evolution of the genetic code.

The genetic code, formerly thought to be frozen, is now known to be in a state of evolution. This was first shown in 1979 by Barrell et al. (G. Barrell, A. T. Bankier, and J. Drouin, Nature [London] 282:189-194, 1979), who found that the universal codons AUA (isoleucine) and UGA (stop) coded for methionine and tryptophan, respectively, in human mitochondria. Subsequent studies have shown that UGA codes for tryptophan in Mycoplasma spp. and in all nonplant mitochondria that have been examined. Universal stop codons UAA and UAG code for glutamine in ciliated protozoa (except Euplotes octacarinatus) and in a green alga, Acetabularia. E. octacarinatus uses UAA for stop and UGA for cysteine. Candida species, which are yeasts, use CUG (leucine) for serine. Other departures from the universal code, all in nonplant mitochondria, are CUN (leucine) for threonine (in yeasts), AAA (lysine) for asparagine (in platyhelminths and echinoderms), UAA (stop) for tyrosine (in planaria), and AGR (arginine) for serine (in several animal orders) and for stop (in vertebrates). We propose that the changes are typically preceded by loss of a codon from all coding sequences in an organism or organelle, often as a result of directional mutation pressure, accompanied by loss of the tRNA that translates the codon. The codon reappears later by conversion of another codon and emergence of a tRNA that translates the reappeared codon with a different assignment. Changes in release factors also contribute to these revised assignments. We also discuss the use of UGA (stop) as a selenocysteine codon and the early history of the code.

Codon reassignment (codon capture) in evolution.

SummaryThe genetic code, once thought to be “frozen”, show variations from the universal code. Variations are found in mitochondria,Mycoplasma, and ciliated protozoa. The variations results from reassignment of codons, especially stop codons. The ressignments 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 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.

The ribosomal protein gene cluster of Mycoplasma capricolum

SummaryThe DNA sequence of the part of the Mycoplasma capricolum genome that contains the genes for 20 ribosomal proteins and two other proteins has been determined. The organization of the gene cluster is essentially the same as that in the S10 and spc operons of Escherichia coli. The deduced amino acid sequence of each protein is also well conserved in the two bacteria. The G+C content of the M. capricolum genes is 29%, which is much lower than that of E. coli (51%). The codon usage pattern of M. capricolum is different from that of E. coli and extremely biased to use of A and U(T): about 91% of codons have A or U in the third position. UGA, which is a stop codon in the “universal” code, is used more abundantly than UGG to dictate tryptophan.

Molecular and metabolic properties of messenger RNA from normal and T2-infected Escherichia coli*

1 A method has been developed for the preparation of “undegraded“ messenger RNA from normal and T2-phage infected E. coli cells. On methylated albumin columns the RNA was heterogeneous, and could be fractionated into four main components with sedimentation coefficients of 8 s (M-RNA-I), 10 to 12 s (M-RNA-II), 19 to 21 s plus 13 s (M-RNA-III) and 23 to 30 s (M-RNA-IV), respectively. 2 The time course of the incorporation of [ 14 C]uracil into M-RNA-II, M-RNA-III and M-RNA-IV, or of the disappearance of radioactivity from these RNAs prelabeled with the isotopes, has been investigated. The radioactivity in these three components increased or decreased in a parallel fashion, indicating that no precursor-product relationship exists among them. 3 Chloramphenicol inhibited the synthesis of M-RNA-IV without preventing synthesis of M-RNA-II and M-RNA-III. 4 The specific ability of M-RNA-IV to associate with 70 s ribosomes in vitro has been demonstrated. Neither M-RNA-II nor M-RNA-III has such an ability. The RNA-ribosome complex has a sedimentation coefficient about 10 to 15 s higher than the normal 70 s ribosome.

Nuclear and cytoplasmic ribonucleic acids of calf thymus

1. 1. It has been shown that there are at least two kinds of nuclear ribonucleic acid in calf thymus tissue, one of which is associated with a fraction soluble in neutral phosphate buffer and has the same nucleotide composition as that of cytoplasmic RNA. On the other hand the RNA associated with the nuclear residue has a different nucleotide composition. 2. 2. Electrophoretically homogeneous ribonucleoprotein samples were isolated from a nuclear fraction soluble in neutral phosphate buffer and from microsomal fractions. Both showed the same electrophoretic mobilities and RNA content. 3. 3. The experiment of 32P incorporation into nuclear RNA and cytoplasmic RNA indicated that not all the RNA in the cytoplasm can be derived from the nuclear RNA soluble in phosphate buffer, assuming that no exchange reaction of cytoplasmic RNA phosphorus taked place.

Evolution of the genetic code as affected by anticodon content

Abstract A new approach to understanding the evolution of the genetic code has come from information about mitochondrial codes, directional mutation pressure and stop codon capture. The codes for various classes of organisms and organelles are characterized by different patterns of anticodon content and usage.

The nucleotide composition of ribonucleic acids from subcellular components of yeast, Escherichia coli and rat liver, with special reference to the occurrence of pseudouridylic acid in soluble ribonucleic acid.

Abstract The analyses of the nucleotide composition of RNAs derived from various subcellular fractions of yeast and Escherichia coli B, revealed that the RNA found in the supernatant fraction has a unique nucleotide composition as compared with the RNA from the particle fractions. The supernatant RNA is characterized by the presence of a considerable amount of pseudouridylic acid and by higher cytidylic acid and lower adenylic and uridylic acid contents. Predominant localization of pseudouridylic acid in the RNA of the “pH 5 enzyme fraction” of rat liver was also observed. The relation between the ability to bind [ 14 C]leucine and the content of pseudouridylic acid in RNAs prepared from large granules, small granules, and some subfractions of the 105,000 × g supernatant of yeast cells has also been investigated. The RNA prepared from the supernatant fraction is the only active RNA, those prepared from particle fractions failing to incorporate [ 14 C]leucine. In five preparations of RNA derived from the acid-fractionated supernatant subfractions and the unfractionated supernatant, it was found that the ability to incorporate [ 14 C]leucine into different RNAs is directly proportional to their pseudouridylic acid content. These data suggest that this nucleotide is characteristic of the RNA which incorporates amino acids (at least leucine).

UAG is a sense codon in several chlorophycean mitochondria

Abstract The mitochondrial genetic code of those land plants and green algae that have been examined does not deviate from the universal one. A red alga, Chondrus crispus, is the sole reported example throughout the algae that uses a deviant (non-universal) mitochondrial genetic code (UGA=Trp). We have analyzed 366-bp DNA sequences of the gene for mitochondrial cytochrome oxidase subunit I (COXI) from ten chlorophyceaen algae, and detected 3–8 in-frame UAG codons in the sequences of five species. Comparisons of these sequences with those of other algae and land plants have shown that most of the UAG sites in Hydrodictyon reticulatum, Pediastrum boryanum and Tetraedron bitridens correspond to alanine, and those of Coelastrum microporum and Scenedesmus quadricauda to leucine. The three species in which UAG probably codes for alanine are characterized by zoospore formation in asexual reproduction and form a clade in the COXI phylogenetic tree. The two species in which UAG codes for leucine are known to form daughter coenobia and pair in the tree. This is the first report on a deviant mitochondrial genetic code in green algae. Mutational change(s) in the release factor corresponding to UAG would be involved in these code changes. No genetic code deviation has been found in five other species examined.

Evolutionary change in 5S rRNA secondary structure and a phylogenic tree of 352 5S rRNA species

The secondary structure models of 5S rRNA have been constructed from the primary structure of 352 5S rRNA species available at present. All the 5S rRNAs examined can take essentially the same secondary structure, however they reveal characteristic differences between eukaryotes, metabacteria (= archaebacteria) and eubacteria. These three types of models can be further subgrouped by minor but characteristic differences. A phylogenic tree of organisms has been constructed using these 5S rRNA sequences by the weighted pairing method (WPG method). The tree reveals that there exist several major groups of eubacteria which seem to have diverged into different directions in the early stages of bacterial evolution. After emergence of eubacteria, metabacteria and eukaryotes separated from each other from their common ancestor. In the eukaryotic evolution, red algae (Rhodophyta) emerged first, and thereafter, thraustocytrids-Proctista, Ascomycota, green plants (green algae and land plants), Basidiomycota, Chromophyta (brown algae, diatoms and golden-yellow algae), slime- and water molds, various protozoans, and animals emerged in this order.

Intermediary steps of ribosome formation in Escherichia coli

Abstract The formation of Escherichia coli ribosomes was investigated using [ 3 H]adenosine or [ 14 C]adenine as labelled nucleotide precursor under conditions in which little messenger RNA was synthesized. Several “intermediary” particles in the formation of ribosomes could be demonstrated by short pulse-labelling with [ 3 H]adenosine. Attempts were made to arrest the transformation of intermediary components at specific stages. It was found that chloramphenicol and 5-fluoro-uracil accumulated 18-S–25-S particles, and 28-S–32-S particles respectively. These particles were converted to the normal 30-S–50-S ribosomal sub-units upon removal of the inhibitors. The possible route of ribosome synthesis in E. coli cells has been discussed.