oli1 Transcripts in wild type and in a cytoplasmic "petite" mutant of yeast.

Subunit 9 of ATPase is known to be encoded in the oli1 gene of yeast mitochondrial DNA. The oli1 transcripts of wild type and of a cytoplasmic "petite" mutant have been analyzed by hybridization of mitochondrial RNA to various DNA fragments from the internal and flanking regions of the gene and by S1 nuclease mapping of the 5' and 3' ends. The results of such studies indicate that the ATPase gene is co-transcribed with the downstream serine tRNA gene. The oli1 message and tRNA are generated by post-transcriptional processing. Two of the nucleolytic processing steps are blocked in the cytoplasmic petite mutant, resulting in the accumulation of several different intermediate transcripts containing both genes. Processing of the 3' ends occurs near a common seven-nucleotide sequence (5'-ATTCTTA-3') also found in the 3' regions of other mitochondrial genes. This sequence is proposed to be part of a signal necessary for either termination of transcription or RNA processing.

Assembly of the mitochondrial membrane system. Processing of the apocytochrome b precursor RNAs in Saccharomyces cerevisiae D273-10B.

The DNA sequence of the apocytochrome b gene in Saccharomyces cerevisiae D273-10B contains two intervening sequences (Nobrega, F. G., and Tzagoloff, A. (1980) J. Biol. Chem. 255, 9828-9837). The exon-intron boundaries of the gene have been determined in this study from the sequence of the DNA which was copied from the mRNA. A protein of 385 amino acid residues is predicted from the 1155-nucleotide long coding regions. Northern blot analysis of total mitochondrial RNA, probed with restriction fragments from both exon and intron regions of the gene, reveals a 4.3-kilobase (kb) transcript containing both introns and two partially spliced intermediates, one (2.9 kb) lacking the first intron and the other (3.6 kb) lacking the second intron. The most abundant transcript (2.1 kb) hybridizes only to exon probes and is presumed to the fully spliced mRNA. S1 nuclease mapping of the purified mRNA indicates existence of two separate RNAs with identical 3' termini but differing by approximately 217 nucleotides at their 5' ends. The larger transcript has a 950-nucleotide nontranslated leader. Analyses of the RNA species present in various rho- and mit- mutants indicate that: 1) exon mutants process both introns, albeit not as efficiently as wild type, 2) intron mutants blocked in the excision of the first or second intron are capable of processing the alternate intron, suggesting a non-obligatory order of excision of the two intervening sequences, and 3) excision of the second intron occurs in rho- mutants and therefore does not require a mitochondrial translation product.

Assembly of the mitochondrial membrane system. Analysis of the nucleotide sequence and transcripts in the oxi1 region of yeast mitochondrial DNA.

The region of yeast mitochondrial DNA between 10.7 and 17.9 map units has been characterized by restriction analysis and DNA sequencing. The DNA sequence was obtained from the partially overlapping genomes of the two rho- mutants DS200/A1 and DS302. Two tRNA genes have been found in the sequence upstream of the oxi1 gene. The deduced secondary structures indicate that the genes code for the methionine (5'-CAU-3') and the asparagine (5'-GUU-3') tRNAs of yeast mitochondria. The region between 10.7 and 17.9 units contains two reading frames. One of these corresponds to the oxi1 gene previously shown to code for subunit 2 of cytochrome oxidase (Coruzzi, G., and Tzagoloff, A. (1979) J. Biol. Chem. 254,. 9324-9330; Fox, T. D. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6534-6538). The second reading frame can potentially code for a basic protein with 386 amino acid residues. It is not known at present if this putative gene is translated in vivo. Northern blots of wild type mitochondrial RNA were hybridized to single-stranded probes from the oxi1 gene and flanking regions. The results of these analyses indicate that the primary transcript of the oxi1 region is a high molecular weight RNA (larger than 3 kilobase pairs) which is processed in discrete steps to a mature 850-nucleotide messenger. The 5' leader of the messenger has been established to be 54 nucleotides long and to have a sequence identical with that of the genomic DNA immediately upstream of the oxi1 gene.

Assembly of the mitochondrial membrane system. Physical map of the Oxi3 locus of yeast mitochondrial DNA.

The oxi3 locus of yeast mitochondrial DNA is currently thought to code for Subunit 1 of cytochrome oxidase (Tzagoloff, A., Macino, G., and Sebald, W. (1979) Annu. Rev. Biochem. 48, 419-441). The respiratory competent strain of Saccharomyces cerevisiae D273-10B/A48 was used to obtain cytoplasmic "petite" clones enriched for genetic markers in the oci3 locus. The most complex clone studied (DS6) was ascertained to have a mitochondrial genome with a tandemly repeated segment of mtDNA 16.5 kilobases in length. The oxi3 locus was dissected by mutagenesis of DS6 with ethidium bromide and selection of new clones having less complex genotypes. Six derivative clones with genome sizes ranging from 2.3 to 6.1 kilobases have been extensively analyzed. Most of the restriction sites present in the segments of mtDNA retained by the clones have been mapped, thereby providing a detailed restriction map of the oxi3 gene. Based on the physical locations of the most distal oxi3 mutations, the gene spans approximately 10,000 nucleotides and occupies the region of wild type mtDNA from 44 to 58 map units.

Assembly of the mitochondrial membrane system. Structure and nucleotide sequence of the gene coding for subunit 1 of yeast cytochrme oxidase.

the oxi3 locus of yeast mitochondrial DNA has been sequenced in Saccharomyces cerevisiae D273-10B. The sequence was obtained from the mitochondrial genomes of a series of cytoplasmic "petite" mutants selected for the retention of genetic markers in the oxi3 locus. The oxi3 locus has been ascertained to code for Subunit 1 of cytochrome oxidase. The Subunit 1 gene is 9,979 nucleotides long, consisting of seven to eight exons that account for only 16% of the gene sequence. The coding sequences have been identified on the basis of protein sequence homology with Subunit 1 of human cytochrome oxidase. The yeast Subunit 1 is 510 amino acid residues long and has a molecular weight of 56,000. In addition to the exon sequences, the Subunit I gene contains six to seven introns. The first four introns have long reading frames that are continuous with the exon coding sequences. These reading frames are potentially capable of coding for basic proteins with molecular weights ranging from 30,000 to 80,000. The first two introns of the gene have a sequence homology of 50%, while the reading frame of the fourth intron is 70% homologous with an intron of the apocytochrome b gene. At least five stable transcripts have been found by Northern blot hybridizations with single-stranded DNA probes containing either exon or intron sequences. A 1.9-kolobase transcript hybridizes only with probes from the exon regions of the gene. This RNA species has been tentatively identified as the fully processed messenger of Subunit 1. Other transcripts are detected with intron probes. Three transcripts with sizes of 2.5, 2.4, and 0.85 kilobases appear to be stable excision products from the first, second, and fifth introns.

Transfer RNA genes in the cap-oxil region of yeast mitochondrial DNA.

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.

Assembly of the mitochondrial membrane system. Sequences of yeast mitochondrial tRNA genes.

Two cytoplasmic "petite" (rho-) clones of Saccharomyces cerevisiae have been selected for the retention of the aspartic acid tRNA gene. The two clones, designated DS200/A102 and DS200/A5, have tandemly repeated segments of mitochondrial DNA (mtDNA) with unit lengths of 1,000 and 6,400 base pairs, respectively. The DS200/A102 genome has a single tRNA gene with a 3'-CUG-5' anticodon capable of recognizing the 5'-GAC-3' and 5'-GAU-3' codons for aspartic acid. The mtDNA segment of DS200/A102 has been determined to represent the wild type sequence from 5.3 to 6.8 map units. The genome of DS200/A5 is more complex encompassing the region of wild type mtDNA from 3.5 to 12.7 units. A continuous sequence has been obtained from 3.5 to 8.6 units. In addition to the aspartic acid tRNA, this region codes for the tRNAUGCAla,tRNAUCUArg, tRNAACGArg, tRNAGCUSer,tRNAUCCGly and tRNAUUULys. The DNA sequence of the DS200/A5 genome has allowed us to deduce the secondary structures of the seven tRNAs and to assign precise map positions for their genes. All the tRNAs except tRNA GUCAsp exhibit most of the invariant features of prokaryotic and eukaryotic tRNAs. The aspartic acid tRNA has unusual D and T psi C loops. The structure of this tRNA is similar to the mitochondrial initiator tRNA of Neurospora crassa (Heckman, J.E., Hecker, L.I., Shwartzbach, S.D., Barnett, W.E., Baumstark, B., and RajBhandary, U.L. Cell 13, 83-95).

Codon recognition rules in yeast mitochondria.

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 fmilies 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.