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R A Butow

Publications and source records attributed to R A Butow.

At least 55 records · Page 3Linked to original sources

The yeast nuclear gene suv3 affecting mitochondrial post-transcriptional processes encodes a putative ATP-dependent RNA helicase.

Mitochondrial gene expression is controlled largely through the action of products of the nuclear genome. The yeast nuclear gene suv3 has been implicated in a variety of mitochondrial posttranscriptional processes and in translation and, thus, represents a key control element in nuclear-mitochondrial interactions. We have exploited a property of a mutant allele of suv3, SUV3-1, that causes, among other effects, a massive increase in the abundance of excised group I introns to clone the wild-type gene by a strategy of colony Northern hybridization. We have determined that the 84-kDa deduced protein product of the suv3 gene, which maps to chromosome XVI, has a typical mitochondrial targeting presequence and additional sequence motifs that suggest that it belongs to a family of ATP-dependent RNA helicases, enzymes whose importance in post-transcriptional and translational events has recently become apparent. We have identified the SUV3-1 mutation as a G----T transversion that creates a Val----Leu substitution in a 10-amino acid block that is highly conserved among ATP-dependent RNA helicases. We discuss some implications of this mutation on the effects of the SUV3-1 allele on mitochondrial RNA metabolism.

Adenosine Triphosphate↗

Complex recognition site for the group I intron-encoded endonuclease I-SceII.

We have characterized features of the site recognized by a double-stranded DNA endonuclease, I-SceII, encoded by intron 4 alpha of the yeast mitochondrial COX1 gene. We determined the effects of 36 point mutations on the cleavage efficiency of natural and synthetic substrates containing the Saccharomyces capensis I-SceII site. Most mutations of the 18-bp I-SceII recognition site are tolerated by the enzyme, and those mutant sites are cleaved between 42 and 100% as well as the wild-type substrate is. Nine mutants blocked cleavage to less than or equal to 33% of the wild-type, whereas only three point mutations, G-4----C, G-12----T, and G-15----C, block cleavage completely. Competition experiments indicate that these three substrates are not cleaved, at least in part because of a marked reduction in the affinity of the enzyme for those mutant DNAs. About 90% of the DNAs derived from randomization of the nucleotide sequence of the 4-bp staggered I-SceII cleavage site are not cleaved by the enzyme. I-SceII cleaves cloned DNA derived from human chromosome 3 about once every 110 kbp. The I-SceII recognition sites in four randomly chosen human DNA clones have 56 to 78% identity with the 18-bp site in yeast mitochondrial DNA; they are cleaved at least 50% as well as the wild-type mitochondrial substrate despite the presence of some substitutions that individually compromise cleavage of the mitochondrial substrate. Analysis of these data suggests that the effect of a given base substitution in I-SceII cleavage may depend on the sequence at other positions.

Base Sequence↗

Splicing-defective mutants of the yeast mitochondrial COXI gene can be corrected by transformation with a hybrid maturase gene.

We have developed a recombinant vector, termed pMIT, for transient expression of genes delivered to yeast mitochondria by biolistic transformation. Using that vector, we introduced a hybrid RNA maturase (splicing) gene into mitochondria of rho 0 petite cells and showed the gene to be functional in crosses. The hybrid maturase is an in-frame fusion between the N-terminal half of the maturase encoded by intron 1 of the COXI (cytochrome oxidase) gene and the C-terminal half of a similar maturase encoded by COXI intron 2. pMIT transformants can provide a functional maturase in crosses to restore respiration and COXI polypeptide synthesis to a respiratory-deficient strain defective in the synthesis of a maturase encoded by COXI intron 1; the transformant will also restore respiration to two splicing-defective cis mutants of COXI introns 1 and 3. We detect a 68-kDa polypeptide comparable in abundance to other major mitochondrial translation products as a likely product of the hybrid maturase gene. Transformants containing an internal 218-amino acid deletion mutation of the hybrid maturase gene no longer express a functional maturase in crosses and produce the expected shortened polypeptide of approximately 40 kDa; however, those transformants still restore respiration to the COXI cis mutants. These studies show the utility of the pMIT transformation system for the expression and reverse genetic analysis of yeast mitochondrial genes.

Cloning, Molecular↗

Introns in pieces.

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Journal Article↗

Intramitochondrial functions regulate nonmitochondrial citrate synthase (CIT2) expression in Saccharomyces cerevisiae.

We have examined the effects of perturbation of mitochondrial function on expression of two nuclear genes encoding the mitochondrial and peroxisomal forms of citrate synthase in Saccharomyces cerevisiae, CIT1 and CIT2. CIT2 expression was as much as 30-fold higher in [rho0] petites, than in isochromosomal [rho+] cells, whereas CIT1 expression was slightly down regulated in [rho0] cells. CIT2 expression was also increased in [rho+] cells by inhibition of respiration with antimycin A or in [rho+] cells containing a disruption of the CIT1 gene. These effects were additive, and together they approached the level of CIT2 expression seen in [rho0] cells. Experiments using heterologous gene fusions showed that all of the effects leading to increased expression of CIT2 were transcriptionally controlled through 5'-flanking CIT2 DNA sequences. Analysis of [rho+] and [rho0] cells containing disruptions of CIT1 and CIT2, singly and in combination, showed that the peroxisomal citrate synthase could partially spare the mitochondrial isoform for growth yield in [rho+] but not in [rho0] cells. These studies suggest a physiological role for increased expression of CIT2 in cells with altered mitochondrial function. They also provide additional evidence for a retrograde path of communication from mitochondria to the nucleus in yeast cells.

Amino Acid Sequence↗

Purification of a site-specific endonuclease, I-Sce II, encoded by intron 4 alpha of the mitochondrial coxI gene of Saccharomyces cerevisiae.

We have purified to near homogeneity a site-specific, double-stranded DNA endonuclease (I-Sce II) encoded by intron 4 alpha (aI4 alpha) of the yeast mitochondrial coxI gene. Our purification starts with a high salt extract of mitochondria isolated from a yeast strain that overproduces the enzyme because of a block in splicing of aI4 alpha. The final step of purification is an affinity column consisting of covalently bound double-stranded DNA multimers of a synthetic sequence, 5'-TTGGTCATCCAGAAGTAT-3', which contains the I-Sce II cleavage/recognition site. Typical yields of enzyme are 3-5% with a specific activity of approximately 500,000 units/mg, where 1 unit of activity cleaves 50 ng of DNA substrate/h at 30 degrees C. I-Sce II has a monomer molecular mass of 31 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Active enzyme purifies as a 55-kDa species, which we presume to be a homodimer. I-Sce II monomer comigrates with an in vivo synthesized mitochondrial translation product made in the strain that overproduces the enzyme. We conclude that I-Sce II is derived by proteolytic processing of a precursor polypeptide, p62, encoded by an in-frame fusion of coxI exons 1-4 with the downstream aI4 alpha reading frame. I-Sce II is most active at pH 7.5 and at 20-30 degrees C. Endonuclease activity is sensitive to salt and is dependent upon Mg2+ or Mn2+, but is unaffected by inclusion of ATP or GTP. I-Sce II is the first intron-encoded protein to be purified and characterized from yeast mitochondria.

Base Sequence↗

The nuclear SUV3-1 mutation affects a variety of post-transcriptional processes in yeast mitochondria.

The SUV3-1 mutation was isolated earlier as a suppressor of a deletion of a conserved RNA processing site (dodecamer) near the 3' end of the var1 gene. Previous studies indicate that the suppressor enhances translation of mutant var1 messages; unexpectedly, it also causes over-accumulation of excised intron RNA of the large rRNA gene intron and blocks cleavage at the dodecamer site within that intron. In this study most mitochondrial genes in SUV3-1 and suv3 nuclear contexts are surveyed for changes in levels of mRNA, for interference with dodecamer cleavage and splicing and for levels of excised intron RNAs. SUV3-1 has little or no effect on the size or abundance of unspliced RNAs tested. It results, however, in a marked increase in the abundance of seven of eight excised group I intron RNAs tested, most of which are not detectable in wild-type (suv3) strains. The suppressor lowers levels of the cob and coxl mRNAs about 2-5 and 20-fold, respectively. The effect on coxl mRNA results from a decrease in the splicing of its intron 5 beta. Despite the reduction in these mRNA levels, the amounts of coxl and cyt b polypeptides were close to wild-type levels in SUV3-1 cells. These data show that the suv3 gene plays a prominent role in post-transcriptional and translation events in yeast mitochondria.

Cell Nucleus↗

Organelle transformation: shoot first, ask questions later.

Transformation of mitochondria and chloroplasts is now possible by the application of microprojectile bombardment for the direct delivery of DNA to these organelles in living cells. Recent experiments have shown the feasibility of reverse genetic studies on mitochondrial and chloroplast genomes. Moreover, foreign or novel genes can now be stably introduced into mitochondria and chloroplasts, allowing the study of a much wider range of molecular genetic problems involving organelles than was previously possible.

Chlamydomonas↗

Mobile introns: definition of terms and recommended nomenclature.

A number of introns in mitochondrial, chloroplast, nuclear or prokaryotic genes have recently been shown to encode double-strand sequence-specific endonucleases. Such introns are mobile genetic elements that insert themselves at or near the cleaved sites. A uniform nomenclature to designate the molecular elements involved in the phenomenon of intron mobility is proposed.

Base Sequence↗

A latent intron-encoded maturase is also an endonuclease needed for intron mobility.

Some yeast mitochondrial introns encode proteins that promote either splicing (maturases) or intron propagation via gene conversion (the fit1 endonuclease). We surveyed introns in the coxl gene for their ability to engage in gene conversion and found that the group I intron, al4 alpha, was efficiently transmitted to genes lacking it. An endonucleolytic cleavage is detectable in recipient DNA molecules near the site of intron insertion in vivo and in vitro. Conversion is dependent on an intact al4 alpha open reading frame. This intron product is a latent maturase, but these data show that it is also a potent endonuclease involved in recombination. Dual function proteins that cleave DNA and facilitate RNA splicing may have played a pivotal role in the propagation and tolerance of introns.

Base Sequence↗

Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3' end of the gene.

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-I, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the var1 gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit1, a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. The fit1 dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-type fit1 dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit1 gene is essential for fit1 expression.

Base Sequence↗

Interaction between the yeast mitochondrial and nuclear genomes influences the abundance of novel transcripts derived from the spacer region of the nuclear ribosomal DNA repeat.

We have identified stable transcripts from the so-called nontranscribed spacer region (NTS) of the nuclear ribosomal DNA repeat in certain respiration-deficient strains of Saccharomyces cerevisiae. These RNAs, which are transcribed from the same strand as is the 37S rRNA precursor, are 500 to 800 nucleotides long and extend from the 5' end of the 5S rRNA gene to three major termination sites about 1,780, 1,830, and 1,870 nucleotides from the 3' end of the 26S rRNA gene. A survey of various wild-type and respiration-deficient strains showed that NTS transcript abundance depended on the mitochondrial genotype and a single codominant nuclear locus. In strains with that nuclear determinant, NTS transcripts were barely detected in [rho+] cells, were slightly more abundant in various mit- derivatives, and were most abundant in petites. However, in one petite that was hypersuppressive and contained a putative origin of replication (ori5) within its 757-base-pair mitochondrial genome, NTS transcripts were no more abundant than in [rho+] cells. The property of low NTS transcript abundance in the hypersuppressive petite was unstable, and spontaneous segregants that contained NTS transcripts as abundant as in the other petites examined could be obtained. Thus, respiration deficiency per se is not the major factor contributing to the accumulation of these unusual RNAs. Unlike RNA polymerase I transcripts, the abundant NTS RNAs were glucose repressible, fractionated as poly(A)+ RNAs, and were sensitive to inhibition by 10 micrograms of alpha-amanitin per ml, a concentration that had no effect on rRNA synthesis. Abundant NTS RNAs are therefore most likely derived by polymerase II transcription.

Base Sequence↗

The role of a conserved dodecamer sequence in yeast mitochondrial gene expression.

All mRNAs on the yeast mitochondrial genome terminate at a conserved dodecamer sequence 5'-AAUAAUAUUCUU-3'. We have characterized two mutants with altered dodecamers. One contains a deletion of the dodecamer at the end of the var1 gene, and the other contains two adjacent transversions in the dodecamer at the end of the reading frame of fit1, a gene within the omega+ allele of the 21S rRNA gene. In each mutant, expression of the respective gene is blocked completely. A dominant nuclear suppressor, SUV3-1, was isolated that suppresses the var1 deletion but is without effect on the fit1 dodecamer mutations. Unexpectedly, however, we found that SUV3-1 blocks expression of the wild-type fit1 allele by blocking processing at its dodecamer. SUV3-1 has pleiotropic effects on mitochondrial gene expression, affecting RNA processing, RNA stability, and translation. Our results suggest that RNA metabolism and translation may be part of a multicomponent complex within mitochondria.

Base Sequence↗

Mitochondrial transformation in yeast by bombardment with microprojectiles.

The genetic transformation of mitochondria and chloroplasts has been an intractable problem. The newly developed "biolistic" (biological ballistic) process was used to deliver DNA into yeast cells to stably transform their mitochondria. A nonreverting strain, which is respiratory deficient because of a deletion in the mitochondrial oxi3 gene, was bombarded with tungsten microprojectiles coated with DNA bearing sequences that could correct the oxi3 deletion. Respiratory-competent transformants were obtained in which the introduced oxi3 DNA is integrated at the homologous site in the mitochondrial genome. Organelle genomes can now be manipulated by molecular genetic techniques in the same way as nuclear genomes.

DNA, Mitochondrial↗

A path from mitochondria to the yeast nucleus.

We have identified a path in yeast, from mitochondria to the nucleus, which may have a regulatory function in mitochondrial biogenesis. This path is evident as an elevated expression of a number of nuclear DNA sequences in response to specific defects in the mitochondrial genome, including the absence of mitochondrial DNA in rho 0 petites. Among those nuclear sequences preferentially expressed in certain respiratory-deficient cells are stable poly(A)+ transcripts derived from the so-called non-transcribed spacer region of the nuclear ribosomal DNA repeat, where they are most abundant in the rho 0 petite. Although the function of these unusual RNAs is unclear, the observations may reflect the presence of a mitochondrial homeostatic control system in yeast, which we suggest could function to adjust the mass of mitochondria and mitochondrial DNA in the cell in response to inequities in organelle apportionment during cell budding.

Cell Nucleus↗