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R A Butow

Publications and source records attributed to R A Butow.

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

In vivo double-strand breaks occur at recombinogenic G + C-rich sequences in the yeast mitochondrial genome.

An optional 46-base-pair G + C-rich element (GC cluster) in the coding region of the yeast mitochondrial var1 gene inserts preferentially in crosses into recipient alleles that lack the sequence. Unlike a similar gene conversion event involving the insertion of an optional 1143-base-pair intron, the mitochondrial 21S rRNA gene, which requires the action of a protein encoded by a gene within that intron, conversion of the var1 GC cluster does not require any protein product of the mitochondrial genome. We have detected double-strand breaks in the var1 gene in mitochondrial DNA isolated from unmated haploid rho+ and rho- strains at or near the boundaries of the optional GC cluster, as well as at a conserved copy of that sequence 160 base pairs upstream. No double-strand breaks were detected in the recipient var1 DNA molecules in the vicinity of the optional GC cluster target sequence. This contrasts with 21S rRNA-encoding DNA (rDNA) intron conversion where the recipient, but not the donor DNA, is cleaved at the element insertion site. These results suggest that although the 21S rDNA intron and the var1 GC cluster are preferentially inserted into their respective short alleles, these conversions probably occur by different mechanisms.

Base Composition↗
PubMed1987

The mitochondrial genotype can influence nuclear gene expression in yeast.

Isochromosomal, respiratory-deficient yeast strains, such as a mit-, a hypersuppressive petite, and a petite lacking mitochondrial DNA, are phenotypically identical in spite of differences in their mitochondrial genomes. Subtractive hybridizations of complementary DNA's to polyadenylated RNA isolated from derepressed cultures of these strains reveal the presence of nuclear-encoded transcripts whose abundance varies not only between them and their respiratory-competent parent, but among the respiratory-deficient strains themselves. Transcripts of some nuclear-encoded mitochondrial proteins, like cytochrome c and the alpha and beta subunits of the mitochondrial adenosine triphosphatase, whose abundance is affected by glucose or heme, do not vary. In the absence of major metabolic variables, yeast cells seem to respond to the quality and quantity of mitochondrial DNA and modulate the levels of nuclear-encoded RNA's, perhaps as a means of intergenomic regulation.

Base Sequence↗
PubMed1987

Kinetic and segregational analysis of mitochondrial DNA recombination in yeast.

A pair of yeast strains of opposite mating type was constructed to contain polymorphisms at three loci on the mitochondrial genome--the 21 S rRNA gene, var1, and cob--such that parental and recombinant forms of these genes could be easily detected by Southern blot analysis. These polymorphisms were used to measure in a single cross gene conversions at the 21 S rRNA and var1 loci and a reciprocal recombination at cob. For all three loci, recombination initiates at about the same time, 4 to 6 h after mixing cells, and increases with similar kinetics over a 24-h period. The segregation of parental and recombinant forms of these genes was then followed by pedigree analysis. The results, which show a high variance in the distribution of parental and recombinant forms of all three genes in cells derived from both the first bud and the mother zygote, are consistent with the segregation of a small number of mitochondrial DNA molecules from the zygote to diploid buds. Based on these results and previous experiments of this type, a limited "zone of mixing" of parental mitochondrial DNA molecules probably exists in the zygote. The extent of sampling from this zone, together with the intrinsic properties of the recombination events themselves, is likely to determine the observed pattern of recombination of mitochondrial DNA sequences at the population level.

Alleles↗
PubMed1987

RNA processing and expression of an intron-encoded protein in yeast mitochondria: role of a conserved dodecamer sequence.

The 3' ends of most Saccharomyces cerevisiae mitochondrial mRNAs terminate at a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3', of unknown function. We have studied the consequences of mutations within a dodecamer found in an 1,143-base-pair optional intron of the mitochondrial large (21S) rRNA gene on RNA processing. The dodecamer is situated at the 3' end of an expressed open reading frame (ORF) within that intron, and the mutations are two adjacent transversions that extend the intron ORF by 51 nucleotides. The strain harboring these mutations, L5-10-1, is defective in biased intron transmission in crosses to strains that lack the intron, as are other mutants which contain nucleotide changes within the ORF (I. G. Macreadie, R. M. Scott, A. R. Zinn, and R. A. Butow, Cell 41:395-402, 1985). However, unlike these other mutants, wild-type strains, or petites which retain the intron allele, L5-10-1 is defective in processing at the intron dodecamer. In addition, L5-10-1 lacks a prominent 2.7-kilobase RNA containing both intron and exon sequences and at least two of four RNAs that correspond to various forms of the excised intron. We propose that these RNAs, missing in L5-10-1 but present in all other strains examined, arise in part by processing at the intron dodecamer. In addition, in all strains examined, we have detected a novel processing activity in which precursor 21S rRNA transcripts are cleaved in the upstream exon, about 1,500 nucleotides from the 5' end of the RNA. This activity, together with 3' intron dodecamer cleavage, probably accounts for the 2.7-kilobase RNA species, a candidate for the mRNA for the intron-encoded protein.

Base Sequence↗
PubMed1985

var1 Gene on the mitochondrial genome of Torulopsis glabrata.

We have cloned and sequenced a region of the Torulopsis glabrata mitochondrial genome homologous to the Saccharomyces cerevisiae var1 gene (var1Sc). An open reading frame that could encode a protein of 339 amino acids was found with 72.7% amino acid and 85.3% nucleotide sequence homology to the S. cerevisiae var1 gene. The T. glabrata gene (var1Tg) is transcribed yielding two stable RNAs, a more abundant 13.5 S RNA and a less abundant 18 S species. We have also identified a candidate for a T. glabrata var1 protein among mitochondrial translation products labeled in isolated mitochondria. The var1Tg gene is even more A + T-rich (93%) than var1Sc (89.6%) and has conserved the strong codon bias of var1Sc. Major differences between the two sequences were found. Significant among these are that no GC clusters are found in var1Tg and the sequences surrounding each of the sites where known polymorphisms exist in var1Sc have deletions at the corresponding sites in var1Tg. These data are discussed with respect to possible origins of these var1 genes and translocation of GC clusters in S. cerevisiae mitochondrial DNA.

Amino Acid Sequence↗
PubMed1985

The unusual varl gene of yeast mitochondrial DNA.

The var1 gene specifies the only mitochondrial ribosomal protein known to be encoded by yeast mitochondrial DNA. The gene is unusual in that its base composition is nearly 90 percent adenine plus thymine. It and its expression product show a strain-dependent variation in size of up to 7 percent; this variation does not detectably interfere with function. Furthermore, var1 is an expandable gene that participates in a novel recombinational event resembling gene conversion whereby shorter alleles are preferentially converted to longer ones. The remarkable features of var1 indicate that it may have evolved by a mechanism analogous to exon shuffling, although no introns are actually present.

Alleles↗
PubMed1985

Nonreciprocal exchange between alleles of the yeast mitochondrial 21S rRNA gene: kinetics and the involvement of a double-strand break.

A 1.1 kb intron containing an open reading frame (ORF) in one allele (omega+) of the yeast mitochondrial 21S rRNA gene is nearly quantitatively inserted in crosses into a 21S rRNA allele lacking that intron (omega-). We have determined that this nonreciprocal exchange initiates soon after cells fuse to form zygotes and is complete by 10-16 hr after mating. We have discovered a unique in vivo double-strand cut in omega- mitochondrial DNA (mtDNA) at or near the site of intron insertion that is implicated in the process. Markers flanking the intron insertion site are coconverted with frequencies inversely proportional to their distance from that site. There is no net conversion of omega- to omega+ in crosses between petites retaining these alleles, nor do we observe the unique double-strand cut in the mtDNA from zygotes of such crosses. The data suggest that a translation product of the intron ORF is required for the double-strand cut and nonreciprocal recombination at omega.

Alleles↗
PubMed1985

Transposition of an intron in yeast mitochondria requires a protein encoded by that intron.

The optional 1143 bp intron in the yeast mitochondrial 21S rRNA gene (omega +) is nearly quantitatively inserted in genetic crosses into 21S rRNA alleles that lack it (omega -). The intron contains an open reading frame that can encode a protein of 235 amino acids, but no function has been ascribed to this sequence. We previously found an in vivo double-strand break in omega - DNA at or close to the intron insertion site only in zygotes of omega + X omega - crosses that appears with the same kinetics as intron insertion. We now show that mutations in the intron open reading frame that would alter the translation product simultaneously inhibit nonreciprocal omega recombination and the in vivo double-strand break in omega - DNA. These results provide evidence that the open reading frame encodes a protein required for intron transposition and support the role of the double-strand break in the process.

Base Sequence↗
PubMed1984

Expression of GC clusters in the yeast mitochondrial var 1 gene. Transcription into stable RNAs.

We have determined whether two 46-base pair (bp) GC-rich clusters present in the coding region of the yeast mitochondrial var 1 gene are transcribed and retained in the putative mRNA for the var 1 protein. One GC cluster is found in all var 1 alleles, while the other is optional; both are identical in sequence and, when present together in var 1, are arranged in opposite orientation 158 bp apart. Because of their dyad symmetry, these GC clusters would form stem and loop structures if present in RNA. We have exploited this potential for secondary structure to show that these GC clusters are transcribed. Using a 46-bp RsaI-MboI var 1 restriction fragment as primer and 16 S RNA (the putative var 1 mRNA (Farrelly, F., Zassenhaus, H. P., and Butow, R. A. (1982) J. Biol. Chem. 257, 6581-6587) purified from strains whose var 1 allele contains one or both of these GC clusters as template for AMV reverse transcriptase, we find the accumulation of a major class of cDNAs whose size is consistent with an impediment of reverse transcription at the predicted positions of the GC clusters in the RNA template. Moreover, depending upon the RNA template, we find that the small fraction of full length cDNA molecules made in the reaction contain one or two SstII sites. Because of fold-back, these sites are predicted to be present in the otherwise single-stranded cDNAs if the GC clusters are transcribed.

Base Sequence↗
PubMed1984

Expression of GC clusters in the yeast mitochondrial var 1 gene. Translation and secondary structure implications.

Alleles of the yeast mitochondrial var 1 gene, which encode a protein (var 1) associated with the small mitochondrial ribosomal subunit, contain one or two identical GC clusters within the coding region that are transcribed and retained in the putative var 1 mRNA (Zassenhaus, H.P., and Butow, R. A. (1984) J. Biol. Chem. 259, 8417-8421). By comparing peptide fragments generated by defined chemical and enzymatic cleavages of the products of these alleles, we show that these GC clusters encode amino acids in the var 1 protein. First, there is a strict correlation between the presence of an optional GC cluster in the var 1 gene and a corresponding increase in size of the peptide that would contain the "extra" amino acids encoded by that GC cluster. Second, we find proline residues in specific peptides of var 1 that, from DNA sequence, would only be present if the GC clusters were translated. Thus, although the yeast mitochondrial genome contains 70-100 GC clusters similar to those in var 1, the var 1 protein is the only mitochondrial translation product now known to contain amino acids encoded by these elements. We have also examined predictions of var 1 secondary structure and find little resemblance to the secondary structures predicted for most other ribosomal proteins. Finally, our analysis suggests a significant conformational difference between the var 1 protein containing amino acids encoded by the optional GC cluster and the form of the protein lacking those amino acids.

Amino Acid 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

Rearranged mitochondrial genes in the yeast nuclear genome.

We have found a contiguous DNA sequence in the yeast nuclear genome with extensive homology to non-contiguous yeast mitochondrial DNA sequences. Closely linked to this nuclear sequence in some, but not all, yeast strains is a tandem pair of transposable (Ty) elements. Certain features of the content and organization of this nuclear DNA sequence suggest that it may have originated from petite mitochondrial DNA which integrated into the nuclear genome.

Base Sequence↗
PubMed1983

Transcriptional analysis of the Saccharomyces cerevisiae mitochondrial var1 gene: anomalous hybridization of RNA from AT-rich regions.

A family of mitochondrial RNAs hybridizes specifically to the var1 region on Saccharomyces cerevisiae mitochondrial DNA (Farrelly et al., J. Biol. Chem. 257:6581-6587, 1982). We constructed a fine-structure transcription map of this region by hybridizing DNA probes containing different portions of the var1 region and some flanking sequences to mitochondrial RNAs isolated from var1-containing petites. We also report the nucleotide sequence of more than 1.2 kilobases of DNA flanking the var1 gene. Our primary findings are: (i) The family of RNAs we detect with homology to var1 DNA is colinear with the var1 gene. Their direction of transcription is olil to cap, as it is for most other mitochondrial genes. (ii) Extensive hybridization anomalies are present, most likely due to the high A-T (A-U) content of the hybridizing species and to the asymmetric distribution of their G-C residues. An important conclusion is that failure to detect transcripts from A-T-rich regions of the yeast mitochondrial genome by standard blot transfer hybridizations cannot be interpreted to mean that such sequences, which are commonly supposed to be spacer DNA, are noncoding or lack direct function in the expression of mitochondrial genes.

Base Composition↗
PubMed1982

Characterization of transcripts from the Var1 region on mitochondrial DNA of Saccharomyces cerevisiae.

We have identified transcripts with sequence homology to the var1 region on yeast mtDNA. In wild type, and in cytoplasmic petite strains retaining the var1 region, we detect four RNA species, 19 S, 16 S, 14 S, and 13 S, which hybridize to var1 DNA probes. The 16 S RNA is by far the most abundant of these RNAs in wild type cells. We also observe hybridization of the 15 S rRNA and a 10 S RNA species to var1 DNA probes. This hybridization is most likely due to the presence in these RNAs of a GC-rich cluster which has near perfect sequence homology to a GC-rich cluster in the var1 region. We find that the 16 S RNA, estimated to be 2000 to 2100 nucleotides long, varies in size in direct correspondence with the size of var1 polypeptide; it is about 100 nucleotides longer in strains with the var1 (44,000) allele than in strains with var1 (40,000). The amount of the 16 S RNA also varies in correspondence with the amount of var1 polypeptide made; it is increased in an oxi-3 mit- strain (CAD245) which makes about 10 times more var1 protein than wild type, and is barely detected in PZ200, a strain with very low levels of mitochondrial protein synthesis harboring two mutations within var1. We have purified the 16 S RNA following chromatography over oligo(dT) cellulose columns. When the RNA is end-labeled and hybridized to a HincII digest of wild type mtDNA, we observe hybridization only to the fragment containing the entire var1 region, HincII fragment 10.

DNA Restriction Enzymes↗