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T D Fox

Publications and source records attributed to T D Fox.

At least 73 records · Page 4Linked to original sources
PubMed1988

Specific translational activation by nuclear gene products occurs in the 5' untranslated leader of a yeast mitochondrial mRNA.

Translation of the yeast mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII) is specifically activated by the products of at least three nuclear genes, PET494, PET54, and PET122. To investigate whether the target site for translational activation is within the 5' untranslated leader of the coxIII mRNA, we asked whether translation of another mitochondrial protein, apo-cytochrome b, from a chimeric mRNA bearing the coxIII mRNA leader required PET494, PET54, or PET122. Mutations in any of these three genes abolished translation of cytochrome b from an mRNA bearing the 5' two-thirds of the coxIII mRNA 5' untranslated leader, showing that all three gene products are required for translation of the chimeric mRNA and must act within the 5' two-thirds of the coxIII mRNA leader. Our data suggest that in wild-type cells, the specific activation of coxIII translation by PET494, PET54, and PET122 occurs by the action of these three gene products at a site or sites in a region of the 5' untranslated leader at least 172 nucleotides upstream of the initiation codon.

Cytochrome b Group↗
PubMed1988

Transformation of yeast by agitation with glass beads.

We have found that agitation of Saccharomyces cerevisiae with glass beads and plasmid DNA using a vortex mixer results in genetic transformation of the yeast cells. This method is less efficient, but considerably more convenient, than other yeast transformation procedures. The fact that the minimal requirements for transformation are simply physical damage and the presence of DNA in an osmotically supportive environment suggests that this process may occur in nature.

Cell Cycle↗
PubMed1987

The yeast nuclear gene CBS1 is required for translation of mitochondrial mRNAs bearing the cob 5' untranslated leader.

Mitochondrial translation of the cob mRNA to yield apocytochrome b is specifically dependent on the nuclear gene CBS1, while mitochondrial translation of the oxi2 mRNA to yield cytochrome oxidase subunit III (cox III) is specifically dependent on the nuclear gene PET494. Chimeric oxi2 mRNAs bearing the 5' leaders of other mitochondrial mRNAs, transcribed from rho- mitochondrial DNAs termed MSU494, are translated in pet494 mutants. In this study, we examined translation of coxIII from MSU494-encoded chimeric mRNAs in zygotes of defined nuclear and mitochondrial genotype. CoxIII was translated from a chimeric mRNA bearing the cob leader only when the zygotes contained a wild-type CBS1 gene. CoxIII translation from an mRNA bearing the 5' leader of the mitochondrial gene aap1 was not dependent on CBS1 activity. We conclude that the product of the nuclear gene CBS1, or something under its control, acts in the mitochondrion on the cob mRNA 5' leader to activate translation of down-stream coding sequences.

Cell Nucleus↗
PubMed1987

PET111, a Saccharomyces cerevisiae nuclear gene required for translation of the mitochondrial mRNA encoding cytochrome c oxidase subunit II.

Mutations in the nuclear gene PET111 are recessive and specifically block accumulation of cytochrome c oxidase subunit II (coxII), the product of a mitochondrial gene. However, the coxII mRNA is present in pet111 mutants at a level approximately one-third that of wild type. The simplest explanation for this phenotype is that PET111 is required for translation of the coxII mRNA. The reduced steady-state level of this mRNA is probably a secondary effect, caused by increased degradation of the untranslated transcript. Mitochondrial suppressors of pet111, carried on rho-mtDNAs, bypass the requirement for PET111 in coxII translation. Three suppressors are fusions between the coxII structural gene and other mitochondrial genes, that encode chimeric proteins consisting of the N-terminal portions of other mitochondrially coded proteins fused to the coxII precursor protein. When present together with rho+ mtDNA in a heteroplasmic state, these suppressors allow coxII synthesis in pet111 mutants. Thus in wild type, the PET111 product, or something under its control, probably acts at a site coded in the proximal portion of the gene for coxII to promote translation of the mRNA. PET111 was isolated by molecular cloning and genetically mapped to a position approximately midway between rna1 and SUP8 on chromosome XIII.

Base Sequence↗
PubMed1987

Saccharomyces cerevisiae positive regulatory gene PET111 encodes a mitochondrial protein that is translated from an mRNA with a long 5' leader.

The yeast nuclear gene PET111 is required specifically for translation of the mitochondrion-coded mRNA for cytochrome c oxidase subunit II. We have determined the nucleotide sequence of a 3-kilobase segment of DNA that carries PET111. The sequence contains a single long open reading frame that predicts a basic protein of 718 amino acids. The PET111 gene product is a mitochondrial protein, since a hybrid protein which includes the amino-terminal 154 amino acids of PET111 fused to beta-galactosidase is specifically associated with mitochondria. PET111 is translated from a 2.9-kilobase mRNA which, interestingly, has an extended 5'-leader sequence containing four short open reading frames upstream of the long open reading frame. These open reading frames exhibit an interesting pattern of overlap with each other and with the PET111 reading frame.

Amino Acid Sequence↗
PubMed1986

At least two nuclear gene products are specifically required for translation of a single yeast mitochondrial mRNA.

Mitochondrial translation of the oxi2 mRNA, encoding yeast cytochrome c oxidase subunit III (coxIII), has previously been shown to specifically require the mitochondrially located protein product of the nuclear gene PET494. We show here that this specific translational activation involves at least one other newly identified gene termed PET54. Mutations in PET54 cause an absence of the coxIII protein despite the presence of normal levels of its mRNA. pet494 mutations are known to be suppressible by mitochondrial gene rearrangements that replace the normal 5'-untranslated leader of the oxi2 mRNA with the leaders of other mitochondrial mRNAs. In this study we show that pet54, pet494 double mutants are suppressed by the same mitochondrial gene rearrangements, showing that the PET54 product is specifically required, in addition to the PET494 protein, for translation of the oxi2 mRNA. Since, as we show here, PET54 is not an activator of PET494 gene expression, our results suggest that the products of both of these genes may act together to stimulate coxIII translation.

Cell Nucleus↗
PubMed1986

Mitochondrial genome rearrangement leads to extension and relocation of the cytochrome c oxidase subunit I gene in sorghum.

The mitochondrial gene (COXI) encoding cytochrome c oxidase subunit I (COI) was isolated from two cytoplasmic genotypes of sorghum that synthesize different COI polypeptides. The Milo COI (Mr approximately 38,000) is encoded by a 530 codon structural gene sharing 98% homology with the corresponding maize gene. A variant COI observed in 9E cytoplasm (Mr approximately 42,000) is encoded by a 631 codon structural gene that diverges completely from the Milo COXI gene both 100 bp 5' to the presumed initiator methionine and within the 3' coding sequence. The 3' divergence results in a 101 C-terminal extension of the 9E COI that is not homologous to any known mitochondrial polypeptide. The novel 9E COXI, apparently arising from at least two rearrangements, affects transcription and gene product.

Base Sequence↗
PubMed1986

Biosynthesis of the Torpedo californica acetylcholine receptor alpha subunit in yeast.

Yeast cells were transformed with a plasmid containing complementary DNA encoding the alpha subunit of the Torpedo californica acetylcholine receptor. These cells synthesized a protein that had the expected molecular weight, antigenic specificity, and ligand-binding properties of the alpha subunit. The subunit was inserted into the yeast plasma membrane, demonstrating that yeast has the apparatus to express a membrane-bound receptor protein and to insert such a foreign protein into its plasma membrane. The alpha subunit constituted approximately 1 percent of the total yeast membrane. The alpha subunit constituted approximately 1 percent of the total yeast membrane proteins, and its density was about the same in the plasma membrane of yeast and in the receptor-rich electric organ of Electrophorus electricus. In view of the available technology for obtaining large quantities of yeast proteins, it may now be possible to obtain amplified amounts of interesting membrane-bound proteins for physical and biochemical studies.

Animals↗
PubMed1986

Primary structure of wild-type and mutant alleles of the PET494 gene of Saccharomyces cerevisiae.

The product of the yeast nuclear gene PET494 is required specifically for the translation of the mitochondrially encoded subunit III of cytochrome c oxidase. We have determined the DNA sequence of a 1.9 kb fragment carrying PET494. The sequence contains a single long open reading frame of 489 codons. This open reading frame encodes the PET494 protein since the DNA sequence of the corresponding fragment derived from a strain with a known pet494 amber mutation contained an in frame UAG codon. The results of S1 nuclease protection experiments demonstrated that this region is transcribed and that the 5' ends of the major transcripts lie 30 to 40 base-pairs upstream of the first AUG codon in the PET494 reading frame. The predicted PET494 protein has a highly basic amino-terminal domain of 66 amino acids followed by a stretch of 32 uncharged residues, half of which are hydrophobic. The remainder of the protein is not unusual in amino acid composition or distribution except that the carboxyterminal region is notably basic. The phenotype of mutations generated in vitro around codon 119 by exonuclease digestion and linker insertion indicated that this region is dispensable for function. A mutation caused by deletion of 101 bp of coding sequence behaved like a simple frameshift when inserted into the chromosome: it was partially suppressed by the recessive non-group specific frameshift suppressor suf13 and reverted to Pet+ phenotype by mutations linked to PET494.

Alleles↗
PubMed1986

Product of Saccharomyces cerevisiae nuclear gene PET494 activates translation of a specific mitochondrial mRNA.

The product of Saccharomyces cerevisiae nuclear gene PET494 is known to be required for a posttranscriptional step in the accumulation of one mitochondrial gene product, subunit III of cytochrome c oxidase (coxIII). Here we show that the PET494 protein probably acts in mitochondria by demonstrating that both a PET494-beta-galactosidase fusion protein and unmodified PET494 are specifically associated with mitochondria. To define the PET494 site of action, we isolated mutations that suppress a pet494 deletion. These mutations were rearrangements of the mitochondrial gene oxi2 that encodes coxIII. The suppressor oxi2 genes had acquired the 5'-flanking sequences of other mitochondrial genes and gave rise to oxi2 transcripts carrying the 5'-untranslated leaders of their mRNAs. These results demonstrate that in wild-type cells PET494 specifically promotes coxIII translation, probably by interacting with the 5'-untranslated leader of the oxi2 mRNA.

Base Sequence↗
PubMed1986

Expression of cDNAs for acetylcholine receptor subunits in the yeast cell plasma membrane.

Yeast cells transformed with a plasmid containing cDNA encoding the alpha or delta subunit of the Torpedo californica acetylcholine receptor synthesize a protein. The expected molecular mass, antigenic specificity, and ligand-binding properties (in the case of the alpha subunit) of the subunits in yeast are similar to those of the subunits in T. californica membranes. The subunits are inserted into the yeast plasma membrane, demonstrating for the first time that yeast has the apparatus to express and insert foreign proteins into its plasma membrane. The alpha subunit constitutes approximately 1% of the yeast membrane proteins, and its density is about the same in the plasma membrane of yeast as in the receptor-rich electric organ of Electrophorus electricus. In view of the widely available technology for obtaining large quantities of yeast proteins, yeast cells may prove ideal for amplifying the amounts of interesting membrane-bound proteins available so that physical and biochemical studies can be made easily.

Animals↗
PubMed1984

A nuclear mutation that post-transcriptionally blocks accumulation of a yeast mitochondrial gene product can be suppressed by a mitochondrial gene rearrangement.

The nuclear amber mutation, pet494-1, specifically blocks the accumulation of the product of the mitochondrial gene oxi2, cytochrome oxidase subunit III. The pet494-1 mutation does not prevent transcription of the mitochondrial gene since RNA--gel blot hybridizations showed that mutant cells contain normal amounts of an oxi2 transcript, indistinguishable in size from wild-type. A mitochondrial mutation that partially suppresses the nuclear mutation was isolated. The "mitochondrial revertant" behaved as though it contained two different mitochondrial DNAs: one rho+, the other rho-. The suppressor mutation is carried on the rho- mitochondrial DNA and is apparently the result of a gene fusion between oxi2 and another mitochondrial gene, oxi3. This gene rearrangement replaced the normal 5'-non-translated sequence of oxi2 with a portion of the open reading frame of the second intron of oxi3. Novel transcripts of the rearranged gene, containing oxi3 sequences upstream from oxi2 were detected in the mitochondrial revertant. The strain accumulated an electrophoretically variant form of cytochrome oxidase subunit III, probably translated from a new initiation codon. The data are consistent with models in which the PET494 protein acts within the mitochondria to specifically promote the translation of the oxi2 messenger RNA.

DNA, Mitochondrial↗
PubMed1984

Molecular cloning and genetic mapping of the PET494 gene of Saccharomyces cerevisiae.

The activity of the nuclear gene PET494 is required to allow expression of the yeast mitochondrial gene oxi2. To aid the study of the mechanism of action of PET494 we have isolated this gene from yeast DNA. A clone bank of yeast DNA fragments in a yeast-E. coli shuttle vector was screened by transformation for a plasmid able to complement the pet494-1 amber mutation. A complementing plasmid was obtained that contained a unique 4.4 kb yeast sequence. This 4.4 kb sequence contains the PET494 gene. Integration of a plasmid containing it into chromosomal DNA by homologous recombination, and subsequent genetic analysis, demonstrated that the 4.4 kb fragment was tightly linked to the pet494-1 mutation. In addition, the corresponding 4.4 kb sequence isolated from a pet494-1 mutant failed to complement the mutation. A 2 kb fragment, subcloned from the original plasmid retained the ability to complement the mutation. The pet494-1 mutation maps to chromosome XIV between rna2 and lys9, approximately 2.4 cm from lys9.

Base Sequence↗
PubMed1982

The maxicircle of Trypanosoma brucei kinetoplast DNA hybridizes with a mitochondrial gene encoding cytochrome oxidase subunit II.

A restriction endonuclease fragment of the maxicircle of Trypanosoma brucei brucei kinetoplast DNA hybridizes with a cloned mitochondrial DNA sequence which encodes cytochrome oxidase subunit II of Zea mays. A cloned mitochondrial DNA sequence encoding cytochrome oxidase subunit II of Saccharomyces cerevisiae also hybridized with kDNA, but exhibits less homology with the maxicircle than does the maize gene. The hybridizing maxicircle DNA was localized to a 2.8 kbp segment which is bounded by TaqI restriction endonuclease sites and nearby HindIII and EcoRI restriction sites. The TaqI restriction fragment is conserved between T. brucei brucei, T. brucei rhodesiense and T. brucei gambiense and hybridizes with the Zea mays probe in each case.

Animals↗
PubMed1982

Suppressor of yeast mitochondrial ochre mutations that maps in or near the 15S ribosomal RNA gene of mtDNA.

A polypeptide chain-terminating mutation in the yeast mitochondrial oxi 1 gene has been shown to be an ochre (TAA) mutation by DNA sequence analysis. Mitochondrially inherited revertants of this mutation include two types: In the first, the ochre codon has been changed to a sense codon by further mutation in the oxi 1 gene while, in the second, the ochre codon is still present, indicating the occurrence of an extrageneic ochre suppressor mutation. This mitochondrial ochre suppressor, termed MSU1, has been "cloned" in rho- strains of yeast and tested against other oxi 1 mutations. Several additional mutations are also suppressible, and those examined so far are also ochre mutations. MSU1 does not suppress known frameshift or missense mutations at oxi 1. Isoelectric focusing of the gene product (cytochrome oxidase subunit II) from a suppressed-mutant strain indicates that suppression does not involve insertion of charged amino acids. Physical mapping of the mtDNA retained in the MSU1-carrying rho- clones localizes the suppressor mutation to the gene coding the 15S rRNA or a site not more than 300 base pairs from it. No known tRNA genes occur this close to the 15S rRNA gene, and mtDNA from a suppressor-carrying rho- does not hybridize detectably to mitochondrial tRNAs. These results suggest that MSU1 may be an alteration in the 15S rRNA.

Base Sequence↗
PubMed1981

Synthesis and processing of ribosomal RNA in isolated yeast mitochondria.

The synthesis and processing of the 15S and 21S rRNAs have been studied in isolated yeast mitochondria. When mitochondrial transcripts were labeled with [alpha-32p]UTP in an incubation mixture containing 50 microM ATP, the transcripts from the genes for the large and small ribosomal RNAs accumulated in the form of putative precursor molecules. The labeled pre-21S rRNA was converted to mature 21S rRNA during a chase period in the presence of 1 mM ATP. Thus, the maturation of 21S rRNA, a process which includes trimming at the 3' end and, in omega+ strains, the excision of a 1.1 kb intervening sequence, can occur in isolated mitochondria and appears to be dependent on ATP. In contrast, the maturation of 15S rRNA by the removal of approximately 80 nucleotides from the 5' end of a 15.5S transcript is severely restricted in isolated mitochondria, even in the presence of 2.5 mM ATP.

DNA, Mitochondrial↗