The mitochondrial genome of Saccharomyces cerevisiae codes for 24 tRNAs. The nucleotide sequences of the tRNA genes suggest a unique set of rules that govern the decoding of the mitochondrial genetic code. The four codons of unmixed families are recognized by single tRNAs that always have a U in the wobble position of the anticodon. The codons of the mixed families are read by two different tRNAs. Codons terminating in a C or U are recognized by tRNAs with a G and codons terminating in a G or A are recognized by tRNAs with a U in the corresponding positions of the anticodons. There are two exceptions to these rules. In the AUN family for isoleucine and methionine, the isoleucine tRNA has a G and the methionine tRNA has a C in the wobble position. The tRNA for the arginine CGN family also has an A in the wobble position of the anticodon. It is of interest that the CGN codons have not been found in the mitochondrial genes sequenced to date. The simplified decoding system of yeast mitochondria allows all the codons to be recognized by only 24 tRNAs. Yeast mitochondria contain a complete set of tRNAs that function in the translation of a discrete number of mitochondrial messengers. During the past year, the sequences of most of the mitochondrial tRNA genes have been determined and virtually all of their anticodons are now known (1-4). Together with the codon usage in five mitochondrial genes, this information has permitted us to formulate the coding properties of this self-contained translational system.In this communication we present evidence that yeast mitochondria are capable of interpreting their genetic code with less than the 32 tRNAs required by the wobble hypothesis (5). The reduction in the number of tRNAs is achieved by the use of a single tRNA to recognize the four codons in each of the eight unmixed families.* These families are read by the "two out of three" method first proposed by Lagerkvist (6). Furthermore, because only two tRNAs are used to discriminate among the codons of the mixed families, the entire system including the formylmethionine initiator consists of 24 tRNAs. The 24 tRNAs represent the minimum number needed to interpret a degenerate code whose amino acid assignments follow the general outline of the universal code. Number of mitochondrial tRNAs in Saccharomyces cerevisiae Several approaches have been used to estimate the number of mitochondrial tRNAs in yeast. Separation of the aminoacylated tRNAs by reverse phase chromatography on RPC-5 has provided information about the number of isoaccepting species for each of the 20 amino acids. Such analyses indicate [31][32][33][34][35] chromatographically distinct tRNA species (7-9). This number includes species that arise from posttranscriptional modification and therefore is an overestimate of the actual number of tRNA genes. For example, Berlani et al. (3) have recently shown that a point mutation in mitochondrial DNA abolishes charging of the four histidine tRNAs, suggesting that all four isoacceptors are products of a single gen...

The DNA sequence of a 1.6-kilobase-pair SalI-KpnI Acinetobacter calcoaceticus restriction fragment carrying catA, the structural gene for catechol 1,2-dioxygenase I, was determined. The 933-nucleotide gene encodes a protein product with a deduced molecular weight of 34,351. The similarly sized Pseudomonas clcA gene encodes catechol 1,2-dioxygenase II, an enzyme with relatively broad substrate specificity and relatively low catalytic efficiency. Comparison of the catA and clcA sequences demonstrated their common ancestry and suggested that acquisitions of direct and inverted sequence repetitions of 6 to 10 base pairs were frequent events in their evolutionary divergence. The catechol 1,2-dioxygenases proved to be evolutionarily homologous with the alpha and beta subunits of Pseudomonas protocatechuate 3,4-dioxygenase, and analysis of conserved residues in the intradiol dioxygenases revealed conserved histidyl and tyrosyl residues that are probably involved in the ligation of ferric ion in their active sites.

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A cytoplasmic "petite" (rho-) clone of Saccharomyces cerevisiae has been isolated and found through DNA sequencing to contain the genes for cysteine, histidine, leucine, glutamine, lysine, arginine, and glycine tRNAs. This clone, designated DS502, has a tandemly repeated 3.5 kb segment of the wild type genome from 0.7 to 5.6 units. All the tRNA genes are transcribed from the same strand of DNA in the direction cap to oxil. The mitochondrial DNA segment of DS502 fills a sequence gap that existed between the histidine and lysine tRNAs. The new sequence data has made it possible to assign accurate map positions to all the tRNA genes in the cap-oxil span of the yeast mitochondrial genome. A detailed restriction map of the region from 0 to 17 map units along with the locations of 16 tRNA genes have been determined. The secondary structures of the leucine and glutamine tRNAs have been deduced from their gene sequences. The leucine tRNA exhibits 64% sequence homology to an E. coli leucine tRNA.

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