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C L Dieckmann

Publications and source records attributed to C L Dieckmann.

28 records · Page 2Linked to original sources

Overproduction of yeast viruslike particles by strains deficient in a mitochondrial nuclease.

Saccharomyces cerevisiae strains are often host to several types of cytoplasmic double-stranded RNA (dsRNA) genomes, some of which are encapsidated by the L-A dsRNA product, an 86,000-dalton coat protein. Here we present the finding that nuclear recessive mutations in the NUC1 gene, which encodes the major nonspecific nuclease of yeast mitochondria, resulted in at least a 10-fold increase in amounts of the L-A dsRNA and its encoded coat protein. The effect of nuc1 mutations on L-A abundance was completely suppressed in strains that also hosted the killer-toxin-encoding M dsRNA. Both NUC1 and nuc1 strains containing the L-A genome exhibited an increase in coat protein abundance and a concomitant increase in L-A dsRNA when the cells were grown on a nonfermentable carbon source rather than on glucose, an effect independent of the increase in coat protein due to nuc1 mutations or to the absence of M. The increase in L-A expression in nuc1 strains was similar to that observed in strains with mutations in the nuclear gene encoding the most abundant outer mitochondrial membrane protein, porin. nuc1 mutations did not affect the level of porin in the mitochondrial outer membrane. Since the effect of mutations in nuc1 was to alter the copy number of the L-A coat protein genome rather than to change the level of the M toxin genome (as do mak and ski mutations), these mutations define a new class of nuclear genes affecting yeast dsRNA abundance.

Amino Acid Sequence↗

Preferential recombination between GC clusters in yeast mitochondrial DNA.

Yeast mitochondrial DNA molecules have long, AT-rich intergenic spacers punctuated by short GC clusters. GC-rich elements have previously been characterized by others as preferred sites for intramolecular recombination leading to the formation of subgenomic petite molecules. In the present study we show that GC clusters are favored sites for intermolecular recombination between a petite and the wild-type grande genome. The petite studied retains 6.5 kb of mitochondrial DNA reiterated tandemly to form molecules consisting of repeated units. Genetic selection for integration of tandem 6.5 kb repeats of the petite into the grande genome yielded a novel recombination event. One of two crossovers in a double exchange event occurred as expected in the 6.5 kb of matching sequence between the genomes, whereas the second exchange involved a 44 bp GC cluster in the petite and another 44 bp GC cluster in the grande genome 700 bp proximal to the region of homology. Creation of a mitochondrial DNA molecule with a repetitive region led to secondary recombination events that generated a family of molecules with zero to several petite units. The finding that 44 bp GC clusters are preferred as sites for intermolecular exchange adds to the data on petite excision implicating these elements as recombinational hotspots in the yeast mitochondrial genome.

Base Composition↗

Nuclearly-encoded CBP1 interacts with the 5' end of mitochondrial cytochrome b pre-mRNA.

CBP1 is a nuclearly-encoded protein that is imported into mitochondria and confers stability on the mRNA for cytochrome b. Previous work has shown that CBP1 interacts with the cytochrome b transcript upstream of the coding sequence; a region encompassing some 1,100 nucleotides. The work presented here narrows the site of action of CBP1 to the distal third of this upstream sequence through analysis of mRNA produced from a novel recombinant gene containing segments of the gene for cytochrome b, cob, and the ATP synthase subunit 9 gene, olil. In a wild-type CBP1 strain, the cob-olil-cob gene produces stable, mature mRNA that is translated and contributes a portion of the cytochrome b necessary for optimal growth on non-fermentable medium.

Cell Nucleus↗

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↗

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↗

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↗

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↗

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↗