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Fred Sherman - One of the best experts on this subject based on the ideXlab platform.
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The importance of mutation, then and now: studies with yeast cytochrome c
Mutation Research-reviews in Mutation Research, 2020Co-Authors: Fred ShermanAbstract:Abstract The development of a genetic system based on the CYC1 gene was initiated over 40 years ago, primarily because of the anticipated ease of sequencing of the corresponding encoded protein, iso-1-cytochrome c from Saccharomyces cerevisiae . The success of the iso-cytochrome c system was dependent on the early development of methods for detecting and selecting CYC1 defective mutants and CYC1 functional revertants, and of methods for fine-structure genetic mapping using deletions and single-site mutations. The nonsense codons TAA and TAG, and the initiation codon ATG, were determined from the amino acid alterations of iso-1-cytochromes c from intragenic revertants; this represented the first assignments of such codons in a eukaryotic organism. The types of desired sequences were expanded by selecting recombinants from CYC1 × CYC1 nonfunctional mutants or CYC1 × CYC1 functional mutants, permitting the early determination of the rules of translation, which differed from those of prokaryotes by use of the most 5′ AUG codon for initiation of translation. The sequence of 44 base pairs of CYC1 was determined with altered iso-1-cytochromes c from revertants of frameshift and initiation mutants, allowing the early cloning of the gene. A method was developed for transforming yeast directly with synthetic oligonucleotides, resulting in the convenient production of CYC1 mutants with defined sequences. At this point in time, Sherman and colleagues have published approximately 240 papers on or using the iso-cytochrome c system, dealing with such diverse topics as translation, informational suppressors, transcription and transcription termination, recombination, ectopic recombination, mutagen specificity, regulation by Ty1 elements, evolution of duplicated chromosomal segments, structure–function relationships of cytochrome c , protein stability and degradation, biosynthesis and mitochondrial import of cytochrome c , mitochondrial proteases, co- and post-translational modifications, and mRNA degradation. Current work on degradation of proteins in mitochondria, on degradation of mRNA in the nucleus, and on N-terminal acetylation stems from properties of CYC1 mutants isolated in early screens more than a decade ago.
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The Role of Nuclear Cap Binding Protein Cbc1p of Yeast in mRNA Termination and Degradation
Molecular and Cellular Biology, 2000Co-Authors: Patrick Russo, Patrick Chartrand, Fred ShermanAbstract:The CYC1-512 mutation in Saccharomyces cerevisiae causes a 90% reduction in the level of iso-1-cytochrome c because of the lack of a proper 3'-end-forming signal, resulting in low levels of eight aberrantly long CYC1-512 mRNAs which differ in length at their 3' termini. CYC1-512 can be suppressed by deletion of either of the nonessential genes CBC1 and CBC2, which encode the CBP80 and CBP20 subunits of the nuclear cap binding complex, respectively, or by deletion of the nonessential gene UPF1, which encodes a major component of the mRNA surveillance complex. The upf1-Delta deletion suppressed the CYC1-512 defect by diminishing degradation of the longer subset of CYC1-512 mRNAs, suggesting that downstream elements or structures occurred in the extended 3' region, similar to the downstream elements exposed by transcripts bearing premature nonsense mutations. On the other hand, suppression of CYC1-512 defects by cbc1-Delta occurred by two different mechanisms. The levels of the shorter CYC1-512 transcripts were enhanced in the cbc1-Delta mutants by promoting 3'-end formation at otherwise-weak sites, whereas the levels of the longer CYC1-512 transcripts, as well as of all mRNAs, were slightly enhanced by diminishing degradation. Furthermore, cbc1-Delta greatly suppressed the degradation of mRNAs and other phenotypes of a rat7-1 strain which is defective in mRNA export. We suggest that Cbc1p defines a novel degradation pathway that acts on mRNAs partially retained in nuclei.
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degradation of CYC1 mrna in the yeast saccharomyces cerevisiae does not require translation
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Fred ShermanAbstract:Several studies have indicated that degradation of certain mRNAs is tightly coupled to their translation, whereas, in contrast, other observations suggested that translation can be inhibited without changing the stability of the mRNA. We have addressed this question with the use of altered CYC1 alleles, which encode iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. The CYC1-1249 mRNA, which lacks all in-frame and out-of-frame AUG triplets, was as stable as the normal mRNA. This finding established that translation is not required for the degradation of CYC1 mRNAs. Furthermore, poly(G)18 tracks were introduced within the CYC1 mRNA translated regions to block exonuclease degradation. The recovery of 3' fragments revealed that the translatable and the AUG-deficient mRNAs are both degraded 5'-->3'. Also, the increased stability of CYC1 mRNAs in xrn1-delta strains lacking Xrn1p, the major 5'-->3' exonuclease, established that the normal and AUG-deficient mRNAs are degraded by the same pathway. In addition, deadenylylation, which activates the action of Xrn1p, occurred at equivalent rates in both normal and AUG-deficient mRNAs. We conclude that translation is not required for the normal degradation of CYC1 mRNAs, and that translatable and untranslated mRNAs are degraded by the same pathway.
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redundant 3 end forming signals for the yeast CYC1 mrna
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: P Russo, J S Butler, Fred ShermanAbstract:Abstract The CYC1-512 mutation is a 38-bp deletion in the 3' untranslated region of the CYC1 gene, which encodes iso-1-cytochrome c in Saccharomyces cerevisiae. This deletion caused a 90% reduction in the levels of the CYC1 mRNA and protein because of the absence of the normal 3' end-forming signal. Although the 3' end-forming signal was not defined by previous analyses, we report that concomitant alteration by base-pair substitution of three 3' end-forming signals within and adjacent to the 38-bp region produced the same phenotype as the CYC1-512 mutation. Furthermore, these signals appear to be related to the previously identified 3' end-forming signal TATATA. A computer analysis revealed that TATATA and related sequences were present in the majority of 3' untranslated regions of yeast genes. Although TATATA may be the strongest and most frequently used signal in yeast genes, the CYC1+ gene concomitantly employed the weaker signals TT-TATA, TATGTT, and TATTTA, resulting in a strong signal.
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initiation of translation can occur only in a restricted region of the CYC1 mrna of saccharomyces cerevisiae
Molecular and Cellular Biology, 1995Co-Authors: Fred ShermanAbstract:: The steady-state levels and half-lives of CYC1 mRNAs were estimated in a series of mutant strains of Saccharomyces cerevisiae containing (i) TAA nonsense codons, (ii) ATG initiator codons, or (iii) the sequence ATA ATG ACT TAA (denoted ATG-TAA) at various positions along the CYC1 gene, which encodes iso-1-cytochrome c. These mutational alterations were made in backgrounds lacking all internal in-frame and out-of-frame ATG triplets or containing only one ATG initiator codon at the normal position. The results revealed a "sensitive" region encompassing approximately the first half of the CYC1 mRNA, in which nonsense codons caused Upf1-dependent degradation. This result and the stability of CYC1 mRNAs lacking all ATG triplets, as well as other results, suggested that degradation occurs unless elements associated with this sensitive region are covered with 80S ribosomes, 40S ribosomal subunits, or ribonucleoprotein particle proteins. While elongation by 80S ribosomes could be prematurely terminated by TAA codons, the scanning of 40S ribosomal units could not be terminated solely by TAA codons but could be disrupted by the ATG-TAA sequence, which caused the formation and subsequent prompt release of 80S ribosomes. The ATG-TAA sequence caused degradation of the CYC1 mRNA only when it was in the region spanning nucleotide positions -27 to +37 but not in the remaining 3' distal region, suggesting that translation could initiate only in this restricted initiation region. CYC1 mRNA distribution on polyribosomes confirmed that only ATG codons within the initiation region were translated at high efficiency. This initiation region was not entirely dependent on the distance from the 5' cap site and was not obviously dependent on the short-range secondary structure but may simply reflect an open structural requirement for initiation of translation of the CYC1 mRNA.
P Russo - One of the best experts on this subject based on the ideXlab platform.
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redundant 3 end forming signals for the yeast CYC1 mrna
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: P Russo, J S Butler, Fred ShermanAbstract:Abstract The CYC1-512 mutation is a 38-bp deletion in the 3' untranslated region of the CYC1 gene, which encodes iso-1-cytochrome c in Saccharomyces cerevisiae. This deletion caused a 90% reduction in the levels of the CYC1 mRNA and protein because of the absence of the normal 3' end-forming signal. Although the 3' end-forming signal was not defined by previous analyses, we report that concomitant alteration by base-pair substitution of three 3' end-forming signals within and adjacent to the 38-bp region produced the same phenotype as the CYC1-512 mutation. Furthermore, these signals appear to be related to the previously identified 3' end-forming signal TATATA. A computer analysis revealed that TATATA and related sequences were present in the majority of 3' untranslated regions of yeast genes. Although TATATA may be the strongest and most frequently used signal in yeast genes, the CYC1+ gene concomitantly employed the weaker signals TT-TATA, TATGTT, and TATTTA, resulting in a strong signal.
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signals that produce 3 termini in CYC1 mrna of the yeast saccharomyces cerevisiae
Molecular and Cellular Biology, 1993Co-Authors: P Russo, Wenzhuo Li, Fred ShermanAbstract:The CYC1-512 mutant was previously shown to contain a 38-bp deletion, 8 nucleotides upstream from the major wild-type poly(A) site, in the CYC1 gene, which encodes iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. This 38-bp deletion caused a 90% reduction in the CYC1 transcripts, which were heterogeneous in size, aberrantly long, and presumably labile (K. S. Zaret and F. Sherman, Cell 28:563-573, 1982). Site-directed mutagenesis in and adjacent to the 38-bp region was used to identify signals involved in the formation and positioning of CYC1 mRNA 3' ends. In addition, combinations of various putative 3' end-forming signals were introduced by in vitro mutagenesis into the 3' region of the CYC1-512 mutant. The combined results from both studies suggest that 3' end formation in yeast cells involves signals having the following three distinct but integrated elements acting in concert: (i) the upstream element, including sequences TATATA, TAG ... TATGTA, and TTTTTATA, which function by enhancing the efficiency of downstream elements; (ii) downstream elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation, which often occurs after cytidine residues that are 3' to the so-called downstream element. While the upstream element is required for efficient 3' end formation, alterations of the downstream element and poly(A) sites generally do not affect the efficiency of 3' end formation but appear to alter the positions of poly(A) sites. In addition, we have better defined the upstream elements by examining various derivatives of TATATA and TAG ... TATGTA, and we have examined the spatial requirements of the three elements by systematically introducing or deleting upstream and downstream elements and cytidine poly(A) sites.
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distinct cis acting signals enhance 3 endpoint formation of CYC1 mrna in the yeast saccharomyces cerevisiae
The EMBO Journal, 1991Co-Authors: P Russo, Kenneth S. Zaret, Wenzhuo Li, D M Hampsey, Fred ShermanAbstract:Abstract The CYC1-512 mutant of the yeast Saccharomyces cerevisiae contains a 38 bp deletion in the 3' untranslated region of the CYC1 gene, resulting in CYC1 mRNAs that are elongated, presumably labile, and reduced to 10% of the normal level. Analysis with S1 nuclease and a novel PCR procedure revealed that the low amount of CYC1-512 mRNA contained many discrete 3' termini at certain sites, ranging from the wild-type position to over 2000 nucleotides (nt) downstream. The CYC1-512 mRNA deficiency was completely or almost completely restored in eight intragenic revertants that contained six different single and multiple base-pair changes within a 300 bp region downstream from the translation terminator codon. Two of the six different reversions formed the sequence TAG...TATGTA, whereas the other four reversions created the sequences TATATA or TACATA. The positions of these revertant sequences varied, even though they caused an increased use of specific major downstream mRNA 3' endpoints, apparently identical to those seen in the CYC1-512 mRNA. However, several revertants contained minor end points not corresponding to any of the CYC1-512 mRNAs. The capacity of these three signals to form 3' ends was confirmed with sequences constructed by site-directed mutagenesis. We therefore suggest that the production of 3' termini of yeast mRNA may involve at least two functionally distinct elements working in concert. One type of element determines the sites of preferred 3' mRNA termini, as represented by the CYC1-512 termini. The second type of element, which includes TAG...TATGTA and TATATA motifs, operates at a distance to enhance the use of the downstream 3' preferred sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Wei Miao - One of the best experts on this subject based on the ideXlab platform.
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sexual cell cycle initiation is regulated by cdk19 and cyc9 in tetrahymena thermophila
Journal of Cell Science, 2020Co-Authors: Jing Zhang, Jie Xiong, Wei MiaoAbstract:To investigate the mechanisms underlying initiation of the sexual cell cycle in eukaryotes, we have focused on cyclins and cyclin-dependent kinases (CDKs) in the well-studied model ciliate, Tetrahyhymena thermophila. We identified two genes, CDK19 and CYC9, which are highly co-expressed with the mating-associated factors, MTA, MTB, and HAP2. Both CDK19 and CYC9 were found to be essential for mating in T. thermophila. Subcellular localization experiments suggested these proteins are located at the oral area, including the conjugation junction area, and that CDK19 or CYC9 knockout prevents mating. We found that CDK19 and CYC9 form a complex and also identified several additional subunits, which may have regulatory or constitutive functions. RNA-Seq analyses and cytological experiments showed that mating is abnormal both in ΔCDK19 and ΔCYC9, mainly at the entry to the costimulation stage. These results indicate that the CDK19/CYC9 complex initiates the sexual cell cycle in T. thermophila.
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a dp like transcription factor protein interacts with e2fl1 to regulate meiosis in tetrahymena thermophila
Cell Cycle, 2018Co-Authors: Jing Zhang, Miao Tian, Jie Xiong, Wei MiaoAbstract:ABSTRACTEvolutionarily conserved E2F family transcription factors regulate the cell cycle via controlling gene expression in a wide range of eukaryotes. We previously demonstrated that the meiosis-specific transcription factor E2fl1 had an important role in meiosis in the model ciliate Tetrahymena thermophila. Here, we report that expression of another E2F family transcription factor gene DPL2 correlates highly with that of E2FL1. Similar to e2fl1Δ cells, dpl2Δ cells undergo meiotic arrest prior to anaphase I, with the five chromosomes adopting an abnormal tandem arrangement. Immunofluorescence staining and immunoprecipitation experiments demonstrate that Dpl2 and E2fl1 form a complex during meiosis. We previously identified several meiotic regulatory proteins in T. thermophila. Cyc2 and Tcdk3 may cooperate to initiate meiosis and CYC17 is essential for initiating meiotic anaphase. We investigate the relationship of these regulators with Dpl2 and E2fl1, and then construct a meiotic regulatory network by m...
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CYC17 a meiosis specific cyclin is essential for anaphase initiation and chromosome segregation in tetrahymena thermophila
Cell Cycle, 2016Co-Authors: Huai Dang, Jing Zhang, Miao Tian, Jie Xiong, Anura Shodhan, Yingzhi Ning, Wei MiaoAbstract:Although the role of cyclins in controlling nuclear division is well established, their function in ciliate meiosis remains unknown. In ciliates, the cyclin family has undergone massive expansion which suggests that diverse cell cycle systems exist, and this warrants further investigation. A screen for cyclins in the model ciliate Tetrahymena thermophila showed that there are 34 cyclins in this organism. Only 1 cyclin, CYC17, contains the complete cyclin core and is specifically expressed during meiosis. Deletion of CYC17 led to meiotic arrest at the diakinesis-like metaphase I stage. Expression of genes involved in DNA metabolism and chromosome organization (chromatin remodeling and basic chromosomal structure) was repressed in CYC17 knockout matings. Further investigation suggested that CYC17 is involved in regulating spindle pole attachment, and is thus essential for chromosome segregation at meiosis. These findings suggest a simple model in which chromosome segregation is influenced by CYC17.
Michael Hampsey - One of the best experts on this subject based on the ideXlab platform.
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cis and trans acting suppressors of a translation initiation defect at the CYC1 locus of saccharomyces cerevisiae
Genetics, 1992Co-Authors: Ines Pinto, J G Na, F Sherman, Michael HampseyAbstract:The CYC1-362 mutant of Saccharomyces cerevisiae is deficient in iso-1-cytochrome c as a consequence of an aberrant ATG codon that initiates a short open reading frame (uORF) in the CYC1 transcribed leader region. We have isolated and characterized functional revertants of CYC1-362 in an effort to define cis- and trans-acting factors that can suppress the effect of the uORF. Genetic and DNA sequence analyses have defined three classes of revertants: (i) those that acquired point mutations in the upstream ATG (uATG), restoring iso-1-cytochrome c to its normal level; (ii) substitution of the normal A residue at position -1 relative to the uATG by either C or T, enhancing iso-1-cytochrome c production from &2% to 6% (C) or 10% (T) of normal, indicating that the nucleotide immediately preceding the initiator codon can affect the efficiency of AUG start codon recognition and that purines are preferred over pyrimidines at this site; and (iii) extragenic suppressors that enhance iso-1-cytochrome c expression to 10-40% of normal while retaining the uATG. These suppressors are represented by five different genes, designated sua1-sua4 and sua6. In contrast to the previously described sua7 and sua8 suppressors, they do not compensate for the uATG by affecting CYC1 transcription start site selection. Potential suppressor mechanisms are discussed.
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The yeast SUA7 gene encodes a homolog of human transcription factor TFIIB and is required for normal start site selection in vivo.
Cell, 1992Co-Authors: Ines Pinto, Dan E. Ware, Michael HampseyAbstract:Abstract Mutations in the Saccharomyces cerevisiae SUA7 gene were isolated as suppressors of an aberrant ATG translation initiation codon in the leader region of the CYC1 gene. Molecular and genetic analysis of the cloned SUA7 gene demonstrated that SUA7 is a single copy, essential gene encoding a basic protein (calculated M r of 38, 142) that is homologous to human transcription factor TFIIB. Analysis of CYC1 transcripts from sua7 strains revealed that suppression is a consequence of diminished transcription initiation at the normal start sites in favor of initiation at downstream sites, including a major site between the aberrant and normal ATG start codons. A similar effect was found at the ADH1 locus, establishing that this effect is not CYC1 gene-specific. Thus, SUA7 encodes a yeast TFIIB homolog and functions in transcription start site selection.
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extragenic suppressors of a translation initiation defect in the CYC1 gene of saccharomyces cerevisiae
Biochimie, 1991Co-Authors: Michael Hampsey, J G Na, Ines Pinto, D E Ware, R W BerroteranAbstract:Abstract The cycl-362 allele contains a point mutation that generates an aberrant AUG codon upstream of the normal CYC1 translation initiation codon. Mutants containing this allele express only about 2% of normal iso-1-cytochrome c, presumably due to translation initiation at the upstream AUG, termination at a UAA sequence six codons downstream, and failure to reinitiate at the normal AUG codon two nucleotides later. Both intragenic and extragenic revertants of cycl-362, expressing elevated levels of iso-1-cytochrome c, have been isolated simply by selecting for growth on lactate medium. Here we describe an improved method for isolating and readily distinguishing cis- from trans-acting suppressors of the upstream AUG. Eight different genes, designated sua1 - sua8, are represented in our current collection of extragenic suppressors; all the recessive and enhance iso-1-cytochrome c levels to 10–60% of normal. None of the sua genes is allelic to SU12 or sui3, which encode eIF-2α and eIF-2s, respectively, or to SU11. Many of the suppressors exhibit pleiotropic phenotypes, including slow growth, cold (16°C) and heat (37°C) sensitivity. These phenotypes have been exploited to clone the SUA5, SUA7 and SUA8 genes, which are presently being characterized. The structure of cucl-362 and the number of sua genes already uncovered suggest that the SUA genes are likely to encode factors affecting several different cellular processes, including translation initiation, mRNA stability and possibly transcription start site selection.
Ines Pinto - One of the best experts on this subject based on the ideXlab platform.
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cis and trans acting suppressors of a translation initiation defect at the CYC1 locus of saccharomyces cerevisiae
Genetics, 1992Co-Authors: Ines Pinto, J G Na, F Sherman, Michael HampseyAbstract:The CYC1-362 mutant of Saccharomyces cerevisiae is deficient in iso-1-cytochrome c as a consequence of an aberrant ATG codon that initiates a short open reading frame (uORF) in the CYC1 transcribed leader region. We have isolated and characterized functional revertants of CYC1-362 in an effort to define cis- and trans-acting factors that can suppress the effect of the uORF. Genetic and DNA sequence analyses have defined three classes of revertants: (i) those that acquired point mutations in the upstream ATG (uATG), restoring iso-1-cytochrome c to its normal level; (ii) substitution of the normal A residue at position -1 relative to the uATG by either C or T, enhancing iso-1-cytochrome c production from &2% to 6% (C) or 10% (T) of normal, indicating that the nucleotide immediately preceding the initiator codon can affect the efficiency of AUG start codon recognition and that purines are preferred over pyrimidines at this site; and (iii) extragenic suppressors that enhance iso-1-cytochrome c expression to 10-40% of normal while retaining the uATG. These suppressors are represented by five different genes, designated sua1-sua4 and sua6. In contrast to the previously described sua7 and sua8 suppressors, they do not compensate for the uATG by affecting CYC1 transcription start site selection. Potential suppressor mechanisms are discussed.
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The yeast SUA7 gene encodes a homolog of human transcription factor TFIIB and is required for normal start site selection in vivo.
Cell, 1992Co-Authors: Ines Pinto, Dan E. Ware, Michael HampseyAbstract:Abstract Mutations in the Saccharomyces cerevisiae SUA7 gene were isolated as suppressors of an aberrant ATG translation initiation codon in the leader region of the CYC1 gene. Molecular and genetic analysis of the cloned SUA7 gene demonstrated that SUA7 is a single copy, essential gene encoding a basic protein (calculated M r of 38, 142) that is homologous to human transcription factor TFIIB. Analysis of CYC1 transcripts from sua7 strains revealed that suppression is a consequence of diminished transcription initiation at the normal start sites in favor of initiation at downstream sites, including a major site between the aberrant and normal ATG start codons. A similar effect was found at the ADH1 locus, establishing that this effect is not CYC1 gene-specific. Thus, SUA7 encodes a yeast TFIIB homolog and functions in transcription start site selection.
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extragenic suppressors of a translation initiation defect in the CYC1 gene of saccharomyces cerevisiae
Biochimie, 1991Co-Authors: Michael Hampsey, J G Na, Ines Pinto, D E Ware, R W BerroteranAbstract:Abstract The cycl-362 allele contains a point mutation that generates an aberrant AUG codon upstream of the normal CYC1 translation initiation codon. Mutants containing this allele express only about 2% of normal iso-1-cytochrome c, presumably due to translation initiation at the upstream AUG, termination at a UAA sequence six codons downstream, and failure to reinitiate at the normal AUG codon two nucleotides later. Both intragenic and extragenic revertants of cycl-362, expressing elevated levels of iso-1-cytochrome c, have been isolated simply by selecting for growth on lactate medium. Here we describe an improved method for isolating and readily distinguishing cis- from trans-acting suppressors of the upstream AUG. Eight different genes, designated sua1 - sua8, are represented in our current collection of extragenic suppressors; all the recessive and enhance iso-1-cytochrome c levels to 10–60% of normal. None of the sua genes is allelic to SU12 or sui3, which encode eIF-2α and eIF-2s, respectively, or to SU11. Many of the suppressors exhibit pleiotropic phenotypes, including slow growth, cold (16°C) and heat (37°C) sensitivity. These phenotypes have been exploited to clone the SUA5, SUA7 and SUA8 genes, which are presently being characterized. The structure of cucl-362 and the number of sua genes already uncovered suggest that the SUA genes are likely to encode factors affecting several different cellular processes, including translation initiation, mRNA stability and possibly transcription start site selection.
