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A Tzagoloff

Publications and source records attributed to A Tzagoloff.

At least 91 records · Page 5Linked to original sources
PubMed1985

A mutation in yeast mitochondrial DNA results in a precise excision of the terminal intron of the cytochrome b gene.

The yeast nuclear gene CBP2 was previously proposed to code for a protein necessary for processing of the terminal intron in the cytochrome b pre-mRNA (McGraw, P., and Tzagoloff, A. (1983) J. Biol. Chem. 258, 9459-9468). In the present study we describe a mitochondrial mutation capable of suppressing the respiratory deficiency of cbp2 mutants. The mitochondrial suppressor mutation has been shown to be the result of a precise excision of the last intervening sequence from the cytochrome b gene. Strains with the altered mitochondrial DNA have normal levels of mature cytochrome b mRNA and of cytochrome b and exhibit wild type growth on glycerol. These results confirm that CBP2 codes for a protein specifically required for splicing of the cytochrome b intron and further suggest that absence of the intervening sequence does not noticeably affect the expression of respiratory function in mitochondria.

Base Sequence↗
PubMed1985

Assembly of the mitochondrial membrane system. CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b.

A new gene function involved in the expression of mitochondrial cytochrome b is described. E158 is a respiratory deficient strain of Saccharomyces cerevisiae with a recessive mutation in nuclear DNA. The block in mitochondrial respiration is a consequence of the mutant's inability to synthesize cytochrome b. This has been confirmed by analysis of the mitochondrial translation products in E158. Since the mutant has wild-type concentrations of mature cytochrome b mRNA, the absence of the cytochrome b protein cannot be due to a transcriptional or RNA-processing defect. The wild-type gene (CBP6) responsible for the observed phenotype has been cloned by transformation of E158 with a genomic library of yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a basic protein with a molecular weight of 18,657. Both deletion and disruption of the CBP6 coding sequence in chromosomal DNA of wild-type yeast lead to respiratory deficiency with a concomitant loss of cytochrome b. Part of the CBP6 gene has been fused to the trpE gene on a high expression Escherichia coli vector. The hybrid protein encoded by the trpE/CBP6 fusion has been purified from E. coli and used as an antigen for antibody production. Antibodies to the hybrid protein cross-react with an 18-kDa protein present in yeast mitochondria.

Base Sequence↗
PubMed1985

Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae.

The genes of Saccharomyces cerevisiae coding for the mitochondrial threonine and tryptophan tRNA synthetases and for a putative mitochondrial ribosomal protein have been cloned. These, and the previously cloned gene for a mitochondrial elongation factor, were used to disrupt or partially delete the wild-type chromosomal copies of the genes in the respiratory-competent strain W303. In each case, inactivation of a gene whose product is required for mitochondrial protein synthesis causes an instability in mitochondrial DNA. Although intact mitochondrial genomes are rapidly and quantitatively eliminated in the protein synthesis defective strains, specific rho- genomes can be maintained stably over many generations. These results indicate that mitochondrial protein synthesis is required for the propagation of wild-type mitochondrial DNA in yeast.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Assembly of the mitochondrial membrane system. Nucleotide sequence of a yeast nuclear gene (CBP1) involved in 5' end processing of cytochrome b pre-mRNA.

A nuclear gene (CBP1) of Saccharomyces cerevisiae involved in 5' end processing of the cytochrome b pre-mRNA has been cloned and sequenced. The gene was originally selected by transformation of cbp1 mutants with a recombinant plasmid bank of random fragments of yeast nuclear DNA ligated to a plasmid vector with autonomous replicative function in yeast. The recombinant plasmid pG60/T10 with a nuclear DNA insert of 6.7 kilobase pairs (kb) was used to construct a new plasmid pool with the CBP1 gene on smaller fragments of nuclear DNA. A number of subclones have been isolated with plasmids carrying inserts of 2.4 to 6.1 kb. The plasmid designated pG60/T31 confers respiratory competency to cbp1 mutants and restores their ability to synthesize mature cytochrome b mRNA. The pG60/T31 plasmid has a nuclear DNA insert of 2.4 kb. The sequence of the cloned fragment reveals only one open reading frame capable of coding for a protein. The reading frame is 1962 nucleotides long and codes for a basic polypeptide with a molecular weight of 76,140. A transcript of a size commensurate with the length of the gene has been detected in wild type yeast.

Amino Acid Sequence↗
PubMed1984

Assembly of the mitochondrial membrane system. CBP1, a yeast nuclear gene involved in 5' end processing of cytochrome b pre-mRNA.

Noncomplementing mutations in a nuclear gene (CBP1) of Saccharomyces cerevisiae D273-10B specifically affect the synthesis of cytochrome b, a mitochondrially encoded carrier of the respiratory chain. The nuclear mutants have been shown to have lowered levels of cytochrome b-specific transcripts. This phenotype is attributed to the inability of the mutant strains to process the 5' end of the cytochrome b pre-mRNA. Impairment of the processing function encoded by the CBP1 gene introduces an instability in the transcripts and promotes nucleolytic degradation. Mutations in CBP1 can be suppressed by a p- genome in which the 5' untranslated leader of the oli1 gene (subunit 9 of the ATPase) is fused near the 5' side of the cytochrome b coding sequence. The rearranged genome allows the cytochrome b gene to be transcribed from the oli1 promoter and results in novel cytochrome b transcripts with the 5' leader sequence of the oli1 mRNA. The presence of the oli1 leader sequence confers stability to the RNA and circumvents the CBP1 processing function.

Base Sequence↗
PubMed1983

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.

Adenosine Triphosphatases↗
PubMed1983

Assembly of the mitochondrial membrane system. Characterization of a yeast nuclear gene involved in the processing of the cytochrome b pre-mRNA.

The cytochrome b gene of Saccharomyces cerevisiae D273-10B was previously shown to be composed of three exons and two introns (Nobrega, F.G., and Tzagoloff, A. (1980) J. Biol. Chem. 255, 9828-9837). In the present study nuclear respiratory deficient mutants of this strain have been screened for defects in processing of the cytochrome b pre-mRNA. Fifteen independently isolated mutants lacking cytochrome b have been assigned to a single genetic complementation group (G36). Members of this complementation group are blocked in the excision of the second intervening sequence of cytochrome b and consequently are unable to produce the mature mRNA. The wild type gene defined by this class of mutants has been named CBP2. A recombinant plasmid with the CBP2 gene has been selected from a library of wild type nuclear DNA and further subcloned by transformation of a cbp2 mutant to respiratory competency. The smallest plasmid (pG36/T5) capable of complementing cbp2 mutants and of restoring their ability to complete processing of the cytochrome b pre-mRNA has a nuclear DNA fragment of 2.6 kilobase pairs inserted at the BamHI site of the yeast vector YEp13. The sequence of the cloned DNA fragment has revealed an 1890-nucleotide-long reading frame encoding a basic protein with a molecular weight of 74,000. Deletion analysis confirms that the entire reading frame is required for complementation of cbp2 mutants. This reading frame is proposed to code for the CBP2 gene product.

Base Sequence↗
PubMed1983

Assembly of the mitochondrial membrane system. Characterization of the oxi2 transcript and localization of its promoter in Saccharomyces cerevisiae D273-10B.

The oxi2 gene of yeast mitochondrial DNA was previously shown to code for subunit 3 of cytochrome oxidase (Thalenfeld, B.E., and Tzagoloff, A. (1980) J. Biol. Chem. 255, 6173-6180). In Saccharomyces cerevisiae D273-10B, a 3.6-kilobase (kb) transcript has been mapped to the oxi2 region of mitochondrial DNA. This transcript, presumed to be the messenger RNA of subunit 3, has been characterized by Northern hybridization analysis and by S1 nuclease mapping. The 3.6-kb transcript has a 5' untranslated leader of 490 nucleotides followed by a 807-nucleotide long coding sequence and a 3' extension of approximately 2450 nucleotides. The nucleotide sequence of the coding region in the 3.6-kb transcript is identical with the gene sequence, thus excluding the presence of introns in the oxi2 gene. Analysis of mitochondrial RNA in cytoplasmic petite mutants containing the oxi2 gene, but with varying lengths of flanking sequences, suggest the presence of a common promoter for oxi2 and the upstream valine tRNA. The promoter has been mapped to a 400-nucleotide long region located on the 5' side of the tRNA gene. Generation of the mature subunit 3 mRNA must, therefore, involve the excision of the tRNA from the primary transcript.

Base Sequence↗
PubMed1983

A rapid method for detecting specific RNA transcripts by hybridization to DNA probes in solution.

A method is described for detecting specific transcripts in crude mixtures of RNA. The method employs hybridization of single-stranded or double-stranded radioactive DNA probes in solution, followed by electrophoretic separation of the hybrid and probe on agarose and visualization by radioautography. The procedure offers the advantages of decreased preparation time and increased sensitivity over currently used methods.

Autoradiography↗
PubMed1982

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.

Base Sequence↗
PubMed1982

Identification and cloning of a yeast nuclear gene (CBP1) involved in expression of mitochondrial cytochrome b.

Nuclear pet mutants of Saccharomyces cerevisiae deficient in mitochondrial respiration have been studied genetically and biochemically. Seven noncomplementing mutations leading to a deficiency of mitochondrial cytochrome b have been assigned to a single complementation group (group 60). Examination of mitochondrial RNA by blot hybridization on diazobenzyloxymethyl-paper has revealed that group 60 mutants produce a large number of novel apocytochrome b transcripts not detected in wild-type yeast. The product of the gene affected in the mutants, therefore, appears to be required either for correct transcription or for processing of apocytochrome b premessenger RNA. The gene has been designated CBP1. A representative mutant from complementation group 60 (N5-26) has been transformed to respiratory competency with a recombinant plasmid pool consisting of random fragments of wild-type yeast nuclear DNA inserted into a vector capable of replicating in yeast and Escherichia coli. The complementation of the N5-26 mutation has been shown for a number of independent transformants to be due to the presence of plasmid DNA. The plasmid pG60/T10 was further characterized to have a nuclear DNA insert of 6.7 kilobase pairs. This plasmid complements the mutations of all group 60 mutants, thus confirming that it contains the CBP1 gene.

Cytochrome b Group↗
PubMed1981

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.

Amino Acid Sequence↗
PubMed1980

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.

DNA Restriction Enzymes↗
PubMed1980

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.

Amino Acid Sequence↗
PubMed1980

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.

Base Sequence↗