scholarly journals Suppressor Analysis of Mutations in the 5′-Untranslated Region of COB mRNA Identifies Components of General Pathways for Mitochondrial mRNA Processing and Decay in Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1315-1325
Author(s):  
Wei Chen ◽  
Maria A Islas-Osuna ◽  
Carol L Dieckmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Untranslated Region ◽  
Mitochondrial Rna ◽  
Rna Turnover ◽  
Single Nucleotide ◽  
Mrna Stabilization ◽  
Recessive Mutations ◽  
A Subunit ◽  
Suppressor Analysis ◽  
Dominant Suppressor

Abstract The cytochrome b gene in Saccharomyces cerevisiae, COB, is encoded by the mitochondrial genome. Nuclear-encoded Cbp1 protein is required specifically for COB mRNA stabilization. Cbp1 interacts with a CCG element in a 64-nucleotide sequence in the 5′-untranslated region of COB mRNA. Mutation of any nucleotide in the CCG causes the same phenotype as cbp1 mutations, i.e., destabilization of both COB precursor and mature message. In this study, eleven nuclear suppressors of single-nucleotide mutations in CCG were isolated and characterized. One dominant suppressor is in CBP1, while the other 10 semidominant suppressors define five distinct linkage groups. One group of four mutations is in PET127, which is required for 5′ end processing of several mitochondrial mRNAs. Another mutation is linked to DSS1, which is a subunit of mitochondrial 3′ → 5′ exoribonuclease. A mutation linked to the SOC1 gene, previously defined by recessive mutations that suppress cbp1 ts alleles and stabilize many mitochondrial mRNAs, was also isolated. We hypothesize that the products of the two uncharacterized genes also affect mitochondrial RNA turnover.

scholarly journals Suppressor Analysis of the Saccharomyces cerevisiae Gene REC104 Reveals a Genetic Interaction With REC102

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1261-1272 ◽  
Author(s):  
Laura Salem ◽  
Natalie Walter ◽  
Robert Malone
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Null Allele ◽  
Genetic Interaction ◽  
In Vitro Mutagenesis ◽  
Conditional Mutations ◽  
Suppressor Analysis

Abstract REC104 is a gene required for the initiation of meiotic recombination in Saccharomyces cerevisiae. To better understand the role of REC104 in meiosis, we used an in vitro mutagenesis technique to create a set of temperature-conditional mutations in REC104 and used one ts allele (rec104-8) in a screen for highcopy suppressors. An increased dosage of the early exchange gene REC102 was found to suppress the conditional recombinational reduction in rec104-8 as well as in several other conditional rec104 alleles. However, no suppression was observed for a null allele of REC104, indicating that the suppression by REC102 is not “bypass” suppression. Overexpression of the early meiotic genes REC114, RAD50, HOP1, and RED1 fails to suppress any of the rec104 conditional alleles, indicating that the suppression might be specific to REC102.

Translational bypassing: a new reading alternative of the genetic code

1995 ◽  
Vol 73 (11-12) ◽  
pp. 1055-1059 ◽  
Author(s):  
Irina Groisman ◽  
Hanna Engelberg-Kulka
Keyword(s):  
16S Rrna ◽  
Genetic Code ◽  
Untranslated Region ◽  
Mrna Sequence ◽  
Cellular Gene ◽  
Single Nucleotide ◽  
Frameshift Event ◽  
Lac Z ◽  
Bypass Mechanism ◽  
Alternative Reading

The translation of the genetic code, once thought to be rigid, has been found to be quite flexible, and several alternatives in its reading have been described. An unusual alternative is translational bypassing, a frameshift event where the transition from frame 0 to another frame occurs by translational bypassing of an extended region of the mRNA sequence rather than by slippage past a single nucleotide, as has been described for most examples of frameshifting. Translational bypassing has been characterized in two cases, T4 gene 60 coding for a topoisomerase subunit and in a trpR–lac′Z fusion. The latter was discovered in our laboratory, and the unique bypass mechanism is investigated further in this study. Using a trpR+1–lac′Z fusion system, we show that the Gln codon at the beginning of lacZ end at the 3′ side of the gap is required for bypassing to occur. The Gln codon is part of an mRNA segment that can (potentially) base pair with a segment at the 5′ and of Escherichia coli 16S rRNA. A model of trpR+1–lac′Z bypassing is suggested in which the untranslated region of the mRNA is looped out through base pairing between a segment in the 5′ end of the 16S rRNA and two sites in the mRNA. Translational bypassing is a newly discovered mechanism of gene expression, and trpR is the first cellular gene identified in which such a mechanism could operate. The understanding of this mechanism and its associated signals may be considered a paradigm for the expression of other genes by this alternative reading of the genetic code.Key words: genetic code, translation, frameshifting, trpR.

scholarly journals Saccharomyces cerevisiae contains an RNase MRP that cleaves at a conserved mitochondrial RNA sequence implicated in replication priming.

1992 ◽  
Vol 12 (6) ◽  
pp. 2561-2569 ◽  
Author(s):  
L L Stohl ◽  
D A Clayton
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Dna Replication ◽  
Rna Processing ◽  
Mitochondrial Rna ◽  
Rna Sequence ◽  
Rnase Mrp ◽  
Processing Activity ◽  
Yeast Mitochondrial Dna ◽  
Human And Mouse

Yeast mitochondrial DNA contains multiple promoters that sponsor different levels of transcription. Several promoters are individually located immediately adjacent to presumed origins of replication and have been suggested to play a role in priming of DNA replication. Although yeast mitochondrial DNA replication origins have not been extensively characterized at the primary sequence level, a common feature of these putative origins is the occurrence of a short guanosine-rich region in the priming strand downstream of the transcriptional start site. This situation is reminiscent of vertebrate mitochondrial DNA origins and raises the possibility of common features of origin function. In the case of human and mouse cells, there exists an RNA processing activity with the capacity to cleave at a guanosine-rich mitochondrial RNA sequence at an origin; we therefore sought the existence of a yeast endoribonuclease that had such a specificity. Whole cell and mitochondrial extracts of Saccharomyces cerevisiae contain an RNase that cleaves yeast mitochondrial RNA in a site-specific manner similar to that of the human and mouse RNA processing activity RNase MRP. The exact location of cleavage within yeast mitochondrial RNA corresponds to a mapped site of transition from RNA to DNA synthesis. The yeast activity also cleaved mammalian mitochondrial RNA in a fashion similar to that of the mammalian RNase MRPs. The yeast endonuclease is a ribonucleoprotein, as judged by its sensitivity to nucleases and proteinase, and it was present in yeast strains lacking mitochondrial DNA, which demonstrated that all components required for in vitro cleavage are encoded by nuclear genes. We conclude that this RNase is the yeast RNase MRP.

scholarly journals Identification and characterization of a 44 kDa protein that binds specifically to the 3′-untranslated region of CYP2a5 mRNA: inducibility, subcellular distribution and possible role in mRNA stabilization

1996 ◽  
Vol 313 (3) ◽  
pp. 1029-1037 ◽  
Author(s):  
Olivier GENESTE ◽  
Françoise RAFFALLI ◽  
Matti A. LANG
Keyword(s):  
Half Life ◽  
Untranslated Region ◽  
Translational Regulation ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Blocking Agent ◽  
Subcellular Fractionation ◽  
Mrna Stabilization ◽  
Nuclear Extracts

Stabilization of mRNA is important in the regulation of CYP2a5 expression but the factors involved in the process are not known [Aida and Negishi (1991) Biochemistry 30, 8041–8045]. In this paper, we describe, for the first time, a protein that binds specifically to the 3′-untranslated region of CYP2a5 mRNA and which is inducible by pyrazole, a compound known to increase the half-life of CYP2a5 mRNA. We also demonstrate that pyrazole treatment causes an elongation of the CYP2a5 mRNA poly(A) tail, and that phenobarbital, which is transcriptional activator of the CYP2a5 gene that does not affect the mRNA half-life, neither induces the RNA-binding protein nor affects the poly(A) tail size. SDS/PAGE of the UV-cross-linked RNA–protein complex demonstrated that the RNA-binding protein has an apparent molecular mass of 44 kDa. The protein-binding site was localized to a 70-nucleotide region between bases 1585 and 1655. Treatment of cytoplasmic extracts with an SH-oxidizing agent, diamide, an SH-blocking agent, N-ethylmaleimide or potato acid phosphatase abolished complex-formation, suggesting that the CYP2a5 mRNA-binding protein is subject to post-translational regulation. Subcellular fractionation showed that the 44 kDa protein is present in polyribosomes and nuclei, and that its apparent induction is much stronger in polyribosomes than in nuclear extracts. We propose that this 44 kDA RNA-binding protein is involved in the stabilization of CYP2a5 mRNA by controlling the length of the poly(A) tail.

The Mitochondrial Nucleoid Protein, Mgm101p, of Saccharomyces cerevisiae Is Involved in the Maintenance of ρ+ and ori/rep-Devoid Petite Genomes but Is Not Required for Hypersuppressive ρ− mtDNA

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1389-1400
Author(s):  
Xiao Ming Zuo ◽  
G Desmond Clark-Walker ◽  
Xin Jie Chen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mitochondrial Rna ◽  
Mtdna Copy Number ◽  
Mitochondrial Rna Polymerase ◽  
Mitochondrial Nucleoids ◽  
Mtdna Replication ◽  
Sensitive Allele ◽  
Discriminatory Effect

Abstract The Saccharomyces cerevisiae MGM101 gene encodes a DNA-binding protein targeted to mitochondrial nucleoids. MGM101 is essential for maintenance of a functional ρ+ genome because meiotic segregants, with a disrupted mgm101 allele, cannot undergo more than 10 divisions on glycerol medium. Quantitative analysis of mtDNA copy number in a ρ+ strain carrying a temperature-sensitive allele, mgm101-1, revealed that the amount of mtDNA is halved each cell division upon a shift to the restrictive temperature. These data suggest that mtDNA replication is rapidly blocked in cells lacking MGM101. However, a small proportion of meiotic segregants, disrupted in MGM101, have ρ− genomes that are stably maintained. Interestingly, all surviving ρ− mtDNAs contain an ori/rep sequence. Disruption of MGM101 in hypersuppressive (HS) strains does not have a significant effect on the propagation of HS ρ− mtDNA. However, in petites lacking an ori/rep, disruption of MGM101 leads to either a complete loss or a dramatically decreased stability of mtDNA. This discriminatory effect of MGM101 suggests that replication of ρ+ and ori/rep-devoid ρ− mtDNAs is carried out by the same process. By contrast, the persistence of ori/rep-containing mtDNA in HS petites lacking MGM101 identifies a distinct replication pathway. The alternative mtDNA replication mechanism provided by ori/rep is independent of mitochondrial RNA polymerase encoded by RPO41 as a HS ρ− genome is stably maintained in a mgm101, rpo41 double mutant.

RPD3 encodes a second factor required to achieve maximum positive and negative transcriptional states in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (12) ◽  
pp. 6317-6327 ◽  
Author(s):  
M Vidal ◽  
R F Gaber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Target Genes ◽  
Current Data ◽  
Regulatory Function ◽  
Fourfold Increase ◽  
Recessive Mutations ◽  
Activation Of Transcription ◽  
Transcriptional Regulatory ◽  
Low Levels ◽  
Full Activation

In Saccharomyces cerevisiae, TRK1 and TRK2 encode the high- and low-affinity K+ transporters, respectively. In cells containing a deletion of TRK1, transcription levels of TRK2 are extremely low and are limiting for growth in media containing low levels of K+ (Trk- phenotype). Recessive mutations in RPD1 and RPD3 suppress the TRK2, conferring an approximately fourfold increase in transcription. rpd3 mutations confer pleiotropic phenotypes, including (i) mating defects, (ii) hypersensitivity to cycloheximide, (iii) inability to sporulate as homozygous diploids, and (iv) constitutive derepression of acid phosphatase. RPD3 was cloned and is predicted to encode a 48-kDa protein with no extensive similarity to proteins contained in current data bases. Deletion of RPD3 is not lethal but confers phenotypes identical to those caused by spontaneous mutations. RPD3 is required for both full repression and full activation of transcription of target genes including PHO5, STE6, and TY2. RPD3 is the second gene required for this function, since RPD1 is also required. The effects of mutations in RPD1 and RPD3 are not additive, suggesting that these genes are involved in the same transcriptional regulatory function or pathway.

scholarly journals Translational Regulation of Nuclear Gene COX4 Expression by Mitochondrial Content of Phosphatidylglycerol and Cardiolipin in Saccharomyces cerevisiae

2006 ◽  
Vol 26 (3) ◽  
pp. 743-753 ◽  
Author(s):  
Xuefeng Su ◽  
William Dowhan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translational Regulation ◽  
Nuclear Gene ◽  
Green Fluorescence Protein ◽  
Untranslated Regions ◽  
Loss Of Function ◽  
Green Fluorescence ◽  
Recessive Mutations ◽  
Protein Factor ◽  
Fluorescence Protein

ABSTRACT Previous results indicated that translation of four mitochondrion-encoded genes and one nucleus-encoded gene (COX4) is repressed in mutants (pgs1Δ) of Saccharomyces cerevisiae lacking phosphatidylglycerol and cardiolipin. COX4 translation was studied here using a mitochondrially targeted green fluorescence protein (mtGFP) fused to the COX4 promoter and its 5′ and 3′ untranslated regions (UTRs). Lack of mtGFP expression independent of carbon source and strain background was established to be at the translational level. The translational defect was not due to deficiency of mitochondrial respiratory function but was rather caused directly by the lack of phosphatidylglycerol and cardiolipin in mitochondrial membranes. Reintroduction of a functional PGS1 gene under control of the ADH1 promoter restored phosphatidylglycerol synthesis and expression of mtGFP. Deletion analysis of the 5′ UTR COX4 revealed the presence of a 50-nucleotide fragment with two stem-loops as a cis-element inhibiting COX4 translation. Binding of a protein factor(s) specifically to this sequence was observed with cytoplasm from pgs1Δ but not PGS1 cells. Using HIS3 and lacZ as reporters, extragenic spontaneous recessive mutations that allowed expression of His3p and β-galactosidase were isolated, which appeared to be loss-of-function mutations, suggesting that the genes mutated may encode the trans factors that bind to the cis element in pgs1Δ cells.

scholarly journals Loss of Heterozygosity and Base Mutation Rates Vary Among Saccharomyces cerevisiae Hybrid Strains

2020 ◽  
Vol 10 (9) ◽  
pp. 3309-3319 ◽  
Author(s):  
Ajith V Pankajam ◽  
Suman Dash ◽  
Asma Saifudeen ◽  
Abhishek Dutta ◽  
Koodali T Nishant
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Loss Of Heterozygosity ◽  
Mutation Rates ◽  
Single Nucleotide ◽  
Single Nucleotide Mutation ◽  
Genome Wide ◽  
Nucleotide Mutation ◽  
Specific Variation ◽  
Chromosomal Changes ◽  
Species Specific

Abstract A growing body of evidence suggests that mutation rates exhibit intra-species specific variation. We estimated genome-wide loss of heterozygosity (LOH), gross chromosomal changes, and single nucleotide mutation rates to determine intra-species specific differences in hybrid and homozygous strains of Saccharomyces cerevisiae. The mutation accumulation lines of the S. cerevisiae hybrid backgrounds - S288c/YJM789 (S/Y) and S288c/RM11-1a (S/R) were analyzed along with the homozygous diploids RM11, S288c, and YJM145. LOH was extensive in both S/Y and S/R hybrid backgrounds. The S/Y background also showed longer LOH tracts, gross chromosomal changes, and aneuploidy. Short copy number aberrations were observed in the S/R background. LOH data from the S/Y and S/R hybrids were used to construct a LOH map for S288c to identify hotspots. Further, we observe up to a sixfold difference in single nucleotide mutation rates among the S. cerevisiae S/Y and S/R genetic backgrounds. Our results demonstrate LOH is common during mitotic divisions in S. cerevisiae hybrids and also highlight genome-wide differences in LOH patterns and rates of single nucleotide mutations between commonly used S. cerevisiae hybrid genetic backgrounds.

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