Search PubMed⌕ Search

Biomedical subjects

N C Martin

Publications and source records attributed to N C Martin.

At least 73 records · Page 4Linked to original sources
PubMed1986

Sequence and expression of four mutant aspartic acid tRNA genes from the mitochondria of Saccharomyces cerevisiae.

Expression of the mitochondrial tRNAAsp gene of Saccharomyces cerevisiae has been examined in five syn- mutants known to affect tRNAAsp function, and in a rho- mutant which accumulates precursor tRNAs. By comparison of wild-type versus mutant DNA sequence, the lesion in each syn- mutant has been identified as a single base change within the mitochondrial tRNAAsp structural gene. The mutant tRNAAsp genes are transcribed, and the transcripts can be processed to mature 4S-size tRNAAsp. The steady-state level of each mutant tRNAAsp is lower than that of wild-type tRNAAsp. The RNA from two of the syn- mutants contained a second, slow-migrating form of mitochondrial tRNAAsp which is correctly processed at the 5' end. We conclude that the lesions in the syn- mitochondrial tRNAAsp genes block neither transcription of these genes, nor 5'-end processing of the transcripts. The effect of each point mutation must be manifested at the level of 3'-end processing, or at a functional level.

Base Sequence↗
PubMed1986

Isolation and characterization of the TRM1 locus, a gene essential for the N2,N2-dimethylguanosine modification of both mitochondrial and cytoplasmic tRNA in Saccharomyces cerevisiae.

The trm1 mutation of Saccharomyces cerevisiae is a single nuclear mutation that affects a specific base modification of both cytoplasmic and mitochondrial tRNA. Transfer RNA isolated from trm1 cells lacks the modified base N2,N2-dimethylguanosine, and extracts from these cells do not have detectable N2,N2-dimethylguanosine-specific tRNA methyltransferase activity. As part of our efforts to determine how this mutation affects enzyme activities in two different cellular compartments we have isolated the TRM1 locus by genetic complementation. The TRM1 locus restores the N2,N2-dimethylguanosine modification to both cytoplasmic and mitochondrial tRNA in trm1 cells. An open reading frame in this TRM1 gene is essential for complementation of the trm1 phenotype. Expression of this open reading frame in Escherichia coli converts the organism from one that neither makes N2,N2-dimethylguanosine nor has N2,N2-dimethylguanosine-specific tRNA methyltransferase activity into one that does. This result suggests that the TRM1 locus is the structural gene for the tRNA modification enzyme and that both nuclear/cytoplasmic and mitochondrial forms of the methyltransferase are produced from the same gene.

Base Sequence↗
PubMed1986

In vitro transcription and promoter strength analysis of five mitochondrial tRNA promoters in yeast.

The in vitro transcriptional initiation sites of four yeast mitochondrial tRNA genes have been investigated in a run-off transcription assay. Precise initiation originating within the 9-nucleotide mitochondrial promoter sequence was detected for the phenylalanine, initiator formyl methionine, cysteine, and one of the two threonine tRNA genes. The relative promoter strength of each of these tRNA promoters as well as that for the previously described glutamate tRNA promoter was determined in a competition assay. This assay measured the utilization of a particular tRNA promoter relative to the amount of transcription arising from a control 14 S rRNA promoter present in the same reaction. The competition strength of the tRNAPhe, tRNAMetf, and tRNAGlu promoters is 20-fold greater than that for the tRNAThrACN and tRNACys promoters. Comparison of the nucleotide sequences at the +2 and +3 positions in the transcripts reveals a homology among the strong promoters not duplicated in the weak promoters.

Base Sequence↗
PubMed1986

RNase P activity in the mitochondria of Saccharomyces cerevisiae depends on both mitochondrion and nucleus-encoded components.

A requisite step in the biosynthesis of tRNA is the removal of 5' leader sequences from tRNA precursors. We have detected an RNase P activity in yeast mitochondrial extracts that can carry out this reaction on a homologous precursor tRNA. This mitochondrial RNase P was sensitive to both micrococcal nuclease and protease, demonstrating that it requires both a nucleic acid and protein for activity. The presence of RNase P activity in vitro directly correlated with the presence of a locus on yeast mitochondrial DNA previously shown by genetic and biochemical studies to be required for tRNA maturation. The product of the locus, the 9S RNA, and this newly described mitochondrial RNase P activity cofractionated, providing further evidence that the 9S RNA is the RNA component of yeast mitochondrial RNase P.

Cell Nucleus↗
PubMed1985

Polymorphism in the structure of the yeast mitochondrial tRNA synthesis locus.

Yeast mitochondrial DNA contains a genetic locus, called the tRNA synthesis locus, which codes for information necessary for mitochondrial tRNA biosynthesis. A 9S RNA molecule coded by this locus is thought to be the trans-acting element required for the removal of 5' extensions from tRNA precursors. The DNA coding for this RNA maps to a region of mitochondrial DNA known to contain strain specific restriction site polymorphisms. Comparison of the tRNA synthesis locus in two such strains by sequence analysis demonstrates that the restriction enzyme polymorphisms are due to the deletion/insertion of a 50 base pair GC-rich element in the 5' flanking sequence of the 9S RNA coding region. There are also several differences between the 9S RNA coding region of these two strains which do not interfere with the tRNA synthesis function.

Base Sequence↗
PubMed1985

Structure of a precursor to the yeast mitochondrial tRNAMetf. Implications for the function of the tRNA synthesis locus.

A transcript from the yeast mitochondrial tRNAMetf gene has been isolated from a petite deletion mutant, ND40. RNA sequence analysis demonstrates that it has a 5' unprocessed extension of 28 nucleotides, and capping experiments with guanylyltransferase reveal that the first nucleotide has a 5' di- or triphosphate. A comparison of the RNA sequence with the tRNAMetf gene sequence reported here shows that transcription initiation occurs at a sequence homologous to a nonanucleotide segment implicated as a promoter element of yeast mitochondrial DNA. The 3' end of this transcript is identical to the mature tRNAMetf and carries a CCA sequence. The transcript can be processed in vitro to yield a mature tRNAMetf and thus appears to be a bona fide tRNA precursor. This tRNA precursor accumulates in the absence of the mitochondrial tRNA synthesis locus whereas mature tRNAMetf can be made from the same gene in the presence of the locus. This data provides clear and convincing evidence that the synthesis locus codes for a 5' tRNA processing function.

Base Sequence↗
PubMed1984

Origins of transcripts of the yeast mitochondrial var 1 gene.

Transcription of the yeast mitochondrial var 1 gene encoding the mitochondrial small ribosomal subunit protein, var 1, has been examined in wild type and petite strains by Northern hybridization, S 1 nuclease protection experiments, and analysis of primary transcripts by in vitro labeling of mitochondrial RNAs with [alpha-32P]GTP and guanylyl transferase. We have identified a transcription unit in which a polycistronic precursor gives rise to the mRNAs for ATPase subunit 9 (the product of the oli 1 gene), var 1, and tRNASerUCN. Potential cleavage sites have been identified within this precursor that separates oli 1, tRNASerUCN, and var 1 transcripts. Transcriptional initiation of the polycistronic precursor occurs from one of two nearby copies of the sequence, 5'-ATATAAGTA-3', located approximately 550 and 630 base pairs 5' to the oli 1 gene. A closely related sequence, 5'-TTATAAGTA-3', is found within the var 1 gene and may serve as a transcriptional initiation site for one of a pair of primary transcripts detected in a petite retaining the var 1[40.0] allele. The other primary transcript in this strain initiates upstream in a region of the var 1 gene containing no recognizable initiation sites.

Base Sequence↗
PubMed1983

Identification and consequences of a guanosine-15 to adenosine-15 change in the yeast mitochondrial tRNASerUCX gene.

We have characterized a mutation affecting the yeast mitochondrial tRNASerUCX. The mutation is a single nucleotide substitution located within the structural portion of the tRNASerUCX gene which causes the strain to be respiratory deficient. The substitution is a G leads to A transition located in the dihydrouridine arm. The tRNASerUCX transcripts from the mutant gene are present in the same amount and are the same size as transcripts from the wild-type gene. The mutant tRNASerUCX can be charged in vitro with mitochondrial aminoacyl-tRNA synthetase. Mitochondrial protein synthesis does occur in the mutant, but the amount of cytochrome oxidase subunit I is significantly decreased relative to other mitochondrial translation products.

Adenosine↗
PubMed1983

Characterization of a yeast mitochondrial locus necessary for tRNA biosynthesis. Deletion mapping and restriction mapping studies.

Yeast mitochondrial DNA codes for a complete set of tRNAs. Although most components necessary for the biosynthesis of mitochondrial tRNA are coded by nuclear genes, there is one genetic locus on mitochondrial DNA necessary for the synthesis of mitochondrial tRNAs other than the mitochondrial tRNA genes themselves. Characterization of mutants by deletion mapping and restriction enzyme mapping studies has provided a precise location of this yeast mitochondrial tRNA synthesis locus. Deletion mutants retaining various segments of mitochondrial DNA were examined for their ability to synthesize tRNAs from the genes they retain. A subset of these strains was also tested for the ability to provide the tRNA synthesis function in complementation tests with deletion mutants unable to synthesize mature mitochondrial tRNAs. By correlating the tRNA synthetic ability with the presence or absence of certain wild-type restriction fragments, we have confined the locus to within 780 base pairs of DNA located between the tRNAMetf gene and tRNAPro gene, at 29 units on the wild-type map. Heretofore, no genetic function or gene product had been localized in this area of the yeast mitochondrial genome.

Chromosome Deletion↗
PubMed1983

Characterization of the yeast mitochondrial locus necessary for tRNA biosynthesis: DNA sequence analysis and identification of a new transcript.

Most components necessary for the biosynthesis of mitochondrial tRNAs are coded by nuclear genes, but one mitochondrial locus other than the tRNA genes themselves is required to make functional tRNAs in the yeast Saccharomyces cerevisiae. DNA sequence analysis of this yeast mitochondrial tRNA synthesis locus is reported here. This region of mitochondrial DNA is almost exclusively A+T-rich DNA with one G+C-rich element. Despite the unusual structure of the DNA in this region, we have demonstrated that it codes for a heretofore unidentified mitochondrial transcript about 450 bases in length. Since this RNA is the only RNA encoded by the tRNA synthesis locus, it must be the active agent of the locus. This RNA could either act autonomously through RNA-RNA interactions or as part of an RNA-protein complex to effect tRNA biosynthesis.

Base Sequence↗
PubMed1982

Isopentenylation of both cytoplasmic and mitochondrial tRNA is affected by a single nuclear mutation.

Cytoplasmic rRNA from the yeast mutant, mod5-1 is deficient in the modified base isopentenyladenosine and consequently migrates differently from isopentenylated wild type tRNAs on certain chromatographic systems. To determine if the mod5-1 mutation affects mitochondrial tRNA structure, the migration of mitochondrial tRNA from mod5-1 and wild type cells has been compared by reverse-phase chromatography. We conclude from this analysis that the single nuclear mutation that affects the isopentenylation of cytoplasmic tRNA also affects the isopentenylation of mitochondrial tRNA.

Alkenes↗
PubMed1982

Defects in modification of cytoplasmic and mitochondrial transfer RNAs are caused by single nuclear mutations.

Many nucleus-encoded mitochondrial enzymes differ in physical and chemical properties from analogous cytoplasmic enzymes, and it is therefore generally assumed that different genes encode analogous mitochondrial and cytoplasmic enzymes. However, our genetic studies show that for at least two different tRNA modifications, mutations in nuclear genes affect cytoplasmic as well as mitochondrial tRNAs. These studies utilize two yeast genes: TRM1 and TRM2. trm1 cells do not have the enzyme activity necessary to methylate guanosine to N2,N2-dimethylguanosine. trm2 is a new mutation that we describe here. trm2 cells are deficient in tRNA-(uridine-5)methyltransferase, and hence contain tRNA lacking 5-methyluridine or ribothymidine. Other than lacking 5-methyluridine trm2 cells have no obvious physiological defect. These studies also show that the N2,N2-dimethylguanosine and 5-methyluridine modifications are not added to tRNA in an obligatory order, and that 5-methyluridine is not required for removal of intervening sequences from precursor tRNA.

Cell Nucleus↗
PubMed1981

A mutation in the tRNAAsp gene from yeast mitochondria. Effects on RNA and protein synthesis.

We have characterized a mutation that affects the tRNAAsp coded by yeast mitochondrial DNA. Comparison of the DNA sequences of the tRNAAsp gene from a wild type strain and the mutant demonstrates that the mutant differs by a C to U base change in position 72 of the tRNA. This mutation abolishes mitochondrial protein synthesis, presumably because the tRNAAsp made from this gene cannot be charged with aspartic acid (FAye, G., Bolotin-Fukuhara, M., and Fukuhara, H. (1976) in The Genetics and Biogenesis of Chloroplasts and Mitochondria (Bucher, C. T., Neupert, W., Sebalt, W., and Werner, S., eds) pp. 547-555, North Holland Publishing Co., Amsterdam). It also reduces the amount of tRNAAsp transcripts in the mutant as compared to the wild type.

Base Sequence↗
PubMed1981

A mitochondrial locus is necessary for the synthesis of mitochondrial tRNA in the yeast Saccharomyces cerevisiae.

We have used a cloned yeast mitochondrial tRNAUCNSer gene as a probe to detect RNA species that are transcripts from this gene in wild-type Saccharomyces cerevisiae and in petite deletion mutants. In RNA from wild-type cells, the tRNA is the most prominent transcript of the gene. In RNA from deletion mutants that retain this gene but have lost other regions of mtDNA, high molecular weight transcripts containing the tRNAUCNSer sequences accumulate but tRNAUCNSer is not made. tRNAUCNSer synthesis can be restored in these mutants when they are mated to other deletion mutants that retain a different portion of the mitochondrial genome. Protein synthesis is not necessary for the restoration, and the restoration is not due to a nuclear effect or to an effect of mating alone, because strains without mtDNA are not able to restore tRNA synthesis. These results definitively demonstrate the existence of a yeast mitochondrial locus that is necessary for tRNA synthesis and, because the restoration of tRNAUCNSer synthesis appears to result from intergenic complementation, not recombination, indicate that this locus acts in trans.

DNA, Mitochondrial↗