SSN20 is an essential gene with mutant alleles that suppress defects in SUC2 transcription in Saccharomyces cerevisiae.

Dominant and recessive mutations at the SSN20 locus were previously isolated as extragenic suppressors of mutations in three genes (SNF2, SNF5, and SNF6) that are required in trans to derepress invertase expression. All ssn20 alleles cause recessive, temperature-sensitive lethality. In this study we cloned the SSN20 gene, identified a 4.6-kilobase poly(A)-containing RNA, and showed that disruption of the gene is lethal in a haploid cell. Genetic mapping of SSN20 to a locus on chromosome VII 10 centimorgans distal to cly8 led to the finding that SSN20 is the same gene as SPT6, which affects expression of delta insertions in the 5' noncoding region of HIS4 (F. Winston, D. T. Chaleff, B. Valent, and G. R. Fink, Genetics 107:179-197, 1984). We also showed that an ssn20 mutation restored expression of secreted invertase from deletions of the SUC2 upstream regulatory region; ssn20 restored derepression of SUC2 mRNA in strains with a SUC2 upstream region deletion or a snf2 mutation. Increased or decreased gene dosage of SSN20 also suppressed defects that are suppressed by ssn20 missense mutations. These findings suggest that SSN20 plays a role in general transcriptional processes.

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Genetic interactions between CDC31 and KAR1, two genes required for duplication of the microtubule organizing center in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/137.2.407 ◽

1994 ◽

Vol 137 (2) ◽

pp. 407-422 ◽

Cited By ~ 8

Author(s):

E A Vallen ◽

W Ho ◽

M Winey ◽

M D Rose

Keyword(s):

Copy Number ◽

Gene Dosage ◽

Spindle Pole Body ◽

Direct Interaction ◽

Spindle Pole ◽

High Copy Number ◽

Temperature Sensitive ◽

Microtubule Organizing Center ◽

Recessive Mutations ◽

Organizing Center

Abstract KAR1 encodes an essential component of the yeast spindle pole body (SPB) that is required for karyogamy and SPB duplication. A temperature-sensitive mutation, kar1-delta 17, mapped to a region required for SPB duplication and for localization to the SPB. To identify interacting SPB proteins, we isolated 13 dominant mutations and 3 high copy number plasmids that suppressed the temperature sensitivity of kar1-delta 17. Eleven extragenic suppressor mutations mapped to two linkage groups, DSK1 and DSK2. The extragenic suppressors were specific for SPB duplication and did not suppress karyogamy-defective alleles. The major class, DSK1, consisted of mutations in CDC31. CDC31 is required for SPB duplication and encodes a calmodulin-like protein that is most closely related to caltractin/centrin, a protein associated with the Chlamydomonas basal body. The high copy number suppressor plasmids contained the wild-type CDC31 gene. One CDC31 suppressor allele conferred a temperature-sensitive defect in SPB duplication, which was counter-suppressed by recessive mutations in KAR1. In spite of the evidence for a direct interaction, the strongest CDC31 alleles, as well as both DSK2 alleles, suppressed a complete deletion of KAR1. However, the CDC31 alleles also made the cell supersensitive to KAR1 gene dosage, arguing against a simple bypass mechanism of suppression. We propose a model in which Kar1p helps localize Cdc31p to the SPB and that Cdc31p then initiates SPB duplication via interaction with a downstream effector.

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The SPT6 gene is essential for growth and is required for delta-mediated transcription in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.679 ◽

1987 ◽

Vol 7 (2) ◽

pp. 679-686 ◽

Cited By ~ 53

Author(s):

C D Clark-Adams ◽

F Winston

Keyword(s):

Saccharomyces Cerevisiae ◽

Null Allele ◽

Gene Dosage ◽

Essential Gene ◽

Gene Dosage Effects ◽

Dosage Effects ◽

High Copy Number Plasmid ◽

The Right ◽

Extragenic Suppressors ◽

Insertion Mutations

Mutations in the Saccharomyces cerevisiae SPT6 gene were originally identified as one class of extragenic suppressors of Ty and delta insertion mutations in the 5' noncoding regions of HIS4 and LYS2. We cloned SPT6 and constructed a null allele by gene disruption. Haploid spores carrying the spt6 null allele were inviable, indicating that the SPT6 gene is essential for mitotic growth. SPT6 was mapped to the right arm of chromosome VII, 44 centimorgans (cM) from ADE6 and 9 cM from CLY8. We showed that spt6 mutations suppress delta insertion mutations at the level of transcription but have no qualitative or quantitative effect on Ty transcription. In addition, we observed interesting SPT6 gene dosage effects. An SPT6 strain containing a high-copy-number plasmid clone of SPT6 showed suppression of delta insertion mutations, and a diploid strain with half its normal dose of SPT6 (SPT6/spt6 null) also exhibited suppression of delta insertion mutations. Therefore, having either too many or too few copies of SPT6 causes a mutant phenotype. Finally, this study and that in the accompanying paper (L. Neigeborn, J. L. Celenza, and M. Carlson, Mol. Cell. Biol. 7:679-686, 1986) showed that spt6 and ssn20 mutations (isolated as suppressors of snf2 and snf5 [sucrose nonfermenting] mutations) identify the same gene. SPT6 and SSN20 have the same genetic map position and share an identical restriction map. Furthermore, spt6 and ssn20 mutations fail to complement each other, and ssn20 mutations suppress solo delta insertion mutations at HIS4 and LYS2. These results, taken in conjunction with the SPT6 dosage effects and the fact that SPT6 is an essential gene, suggest that SPT6 plays a fundamental role in cellular transcription, perhaps by interaction with other transcription factors.

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Antibiotic-Sensitive TolC Mutants and Their Suppressors

Journal of Bacteriology ◽

10.1128/jb.186.6.1851-1860.2004 ◽

2004 ◽

Vol 186 (6) ◽

pp. 1851-1860 ◽

Cited By ~ 48

Author(s):

Anne Marie Augustus ◽

Teresa Celaya ◽

Fasahath Husain ◽

Matthew Humbard ◽

Rajeev Misra

Keyword(s):

Efflux Pump ◽

Regulatory Region ◽

Single Amino Acid ◽

Missense Mutations ◽

Antibiotic Resistant ◽

Isolation And Characterization ◽

Tolc Protein ◽

Acrab Efflux Pump ◽

Extragenic Suppressors ◽

Intragenic Suppressor

ABSTRACT The TolC protein of Escherichia coli, through its interaction with AcrA and AcrB, is thought to form a continuous protein channel that expels inhibitors from the cell. Consequently, tolC null mutations display a hypersensitive phenotype. Here we report the isolation and characterization of tolC missense mutations that direct the synthesis of mutant TolC proteins partially disabled in their efflux role. All alterations, consisting of single amino acid substitutions, were localized within the periplasmic α-helical domain. In two mutants carrying an I106N or S350F substitution, the hypersensitivity phenotype may be in part due to aberrant TolC assembly. However, two other alterations, R367H and R390C, disrupted efflux function by affecting interactions among the helices surrounding TolC's periplasmic tunnel. Curiously, these two TolC mutants were sensitive to a large antibiotic, vancomycin, and exhibited a Dex+ phenotype. These novel phenotypes of TolCR367H and TolCR390C were likely the result of a general influx of molecules through a constitutively open tunnel aperture, which normally widens only when TolC interacts with other proteins during substrate translocation. An intragenic suppressor alteration (T140A) was isolated from antibiotic-resistant revertants of the hypersensitive TolCR367H mutant. T140A also reversed, either fully (R390C) or partially (I106N and S350F), the hypersensitivity phenotype of other TolC mutants. Our data suggest that this global suppressor phenotype of T140A is the result of impeded antibiotic influx caused by tapering of the tunnel passage rather than by correcting individual mutational defects. Two extragenic suppressors of TolCR367H, mapping in the regulatory region of acrAB, uncoupled the AcrR-mediated repression of the acrAB genes. The resulting overexpression of AcrAB reduced the hypersensitivity phenotype of all the TolC mutants. Similar results were obtained when the chromosomal acrR gene was deleted or the acrAB genes were expressed from a plasmid. Unlike the case for the intragenic suppressor T140A, the overexpression of AcrAB diminished hypersensitivity towards only erythromycin and novobiocin, which are substrates of the TolC-AcrAB efflux pump, but not towards vancomycin, which is not a substrate of this pump. This showed that the two types of suppressors produced their effects by fundamentally different means, as the intragenic suppressor decreased the general influx while extragenic suppressors increased the efflux of TolC-AcrAB pump-specific antibiotics.

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Upstream regulatory region for inducible expression of the chicken skeletal myosin alkali light-chain gene.

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2581 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2581-2588 ◽

Cited By ~ 9

Author(s):

M Shirakata ◽

Y Nabeshima ◽

K Konishi ◽

Y Fujii-Kuriyama

Keyword(s):

Light Chain ◽

Transcription Initiation ◽

Fusion Gene ◽

Regulatory Region ◽

Direct Repeat ◽

Upstream Region ◽

Chain Gene ◽

Temperature Sensitive ◽

Flanking Sequence ◽

Light Chain Gene

The expression of the fast type of myosin alkali light chain 1 is induced during the differentiation of muscle cells. To study the mechanism of its gene regulation, we joined the sequence of the 5'-flanking and upstream region of the chicken myosin alkali light-chain gene to the structural gene for chloramphenicol acetyltransferase (CAT). The fusion gene was introduced either into quail myoblasts transformed by a temperature-sensitive mutant of Rous sarcoma virus (tsNY68) or into chicken myoblasts, and the transiently expressed CAT activity was assayed after the differentiation of the myoblasts. From the experiments with the external and internal deletion mutants of the fusion gene, the cis-acting regulatory region responsible for the enhanced expression of the CAT activity in response to the cell differentiation was found to be localized at 2 kilobases upstream of the transcription initiation site. This region of 160 nucleotides contained two pairs of short sequences worthy of note, a direct repeat of 12 nucleotides, and an inverted repeat of 8 nucleotides. The nucleotide sequences of the 5'-flanking sequence up to nucleotide -3381 were determined and compared with those of the upstream activating elements of actin genes.

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Upstream regulatory region for inducible expression of the chicken skeletal myosin alkali light-chain gene

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2581-2588.1988 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2581-2588

Author(s):

M Shirakata ◽

Y Nabeshima ◽

K Konishi ◽

Y Fujii-Kuriyama

Keyword(s):

Light Chain ◽

Transcription Initiation ◽

Fusion Gene ◽

Regulatory Region ◽

Direct Repeat ◽

Upstream Region ◽

Chain Gene ◽

Temperature Sensitive ◽

Flanking Sequence ◽

Light Chain Gene

The expression of the fast type of myosin alkali light chain 1 is induced during the differentiation of muscle cells. To study the mechanism of its gene regulation, we joined the sequence of the 5'-flanking and upstream region of the chicken myosin alkali light-chain gene to the structural gene for chloramphenicol acetyltransferase (CAT). The fusion gene was introduced either into quail myoblasts transformed by a temperature-sensitive mutant of Rous sarcoma virus (tsNY68) or into chicken myoblasts, and the transiently expressed CAT activity was assayed after the differentiation of the myoblasts. From the experiments with the external and internal deletion mutants of the fusion gene, the cis-acting regulatory region responsible for the enhanced expression of the CAT activity in response to the cell differentiation was found to be localized at 2 kilobases upstream of the transcription initiation site. This region of 160 nucleotides contained two pairs of short sequences worthy of note, a direct repeat of 12 nucleotides, and an inverted repeat of 8 nucleotides. The nucleotide sequences of the 5'-flanking sequence up to nucleotide -3381 were determined and compared with those of the upstream activating elements of actin genes.

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Cloning and characterization of ERG8, an essential gene of Saccharomyces cerevisiae that encodes phosphomevalonate kinase

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.620-631.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 620-631

Author(s):

Y H Tsay ◽

G W Robinson

Keyword(s):

Saccharomyces Cerevisiae ◽

Genomic Library ◽

Essential Gene ◽

Single Copy ◽

Yeast Cells ◽

Upstream Region ◽

Ergosterol Biosynthesis ◽

Temperature Sensitive ◽

Coding Region ◽

Phosphomevalonate Kinase

Saccharomyces cerevisiae strains that contain the ery8-1 mutation are temperature sensitive for growth due to a defect in phosphomevalonate kinase, an enzyme of isoprene and ergosterol biosynthesis. A plasmid bearing the yeast ERG8 gene was isolated from a YCp50 genomic library by functional complementation of the erg8-1 mutant strain. Genetic analysis demonstrated that integrated copies of an ERG8 plasmid mapped to the erg8 locus, confirming the identity of this clone. Southern analysis showed that ERG8 was a single-copy gene. Subcloning and DNA sequencing defined the functional ERG8 regulon as an 850-bp upstream region and an adjacent 1,272-bp open reading frame. The deduced 424-amino-acid ERG8 protein showed no homology to known proteins except within a putative ATP-binding domain present in many kinases. Disruption of the chromosomal ERG8 coding region by integration of URA3 or HIS3 marker fragments was lethal in haploid cells, indicating that this gene is essential. Expression of the ERG8 gene in S. cerevisiae from the galactose-inducible galactokinase (GAL1) promoter resulted in 1,000-fold-elevated levels of phosphomevalonate kinase enzyme activity. Overproduction of a soluble protein with the predicted 48-kDa size for phosphomevalonate kinase was also observed in the yeast cells.

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Mutant thermal proteome profiling for characterization of missense protein variants and their associated phenotypes within the proteome

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014576 ◽

2020 ◽

Vol 295 (48) ◽

pp. 16219-16238 ◽

Cited By ~ 3

Author(s):

Sarah A. Peck Justice ◽

Monica P. Barron ◽

Guihong D. Qi ◽

H. R. Sagara Wijeratne ◽

José F. Victorino ◽

...

Keyword(s):

Genetic Manipulation ◽

Essential Gene ◽

26S Proteasome ◽

Temperature Sensitive ◽

Missense Mutations ◽

Proteome Profiling ◽

Global Impacts ◽

Dynamics Simulations ◽

Experimental Findings

Temperature-sensitive (TS) missense mutants have been foundational for characterization of essential gene function. However, an unbiased approach for analysis of biochemical and biophysical changes in TS missense mutants within the context of their functional proteomes is lacking. We applied MS-based thermal proteome profiling (TPP) to investigate the proteome-wide effects of missense mutations in an application that we refer to as mutant thermal proteome profiling (mTPP). This study characterized global impacts of temperature sensitivity–inducing missense mutations in two different subunits of the 26S proteasome. The majority of alterations identified by RNA-Seq and global proteomics were similar between the mutants, which could suggest that a similar functional disruption is occurring in both missense variants. Results from mTPP, however, provide unique insights into the mechanisms that contribute to the TS phenotype in each mutant, revealing distinct changes that were not obtained using only steady-state transcriptome and proteome analyses. Computationally, multisite λ-dynamics simulations add clear support for mTPP experimental findings. This work shows that mTPP is a precise approach to measure changes in missense mutant–containing proteomes without the requirement for large amounts of starting material, specific antibodies against proteins of interest, and/or genetic manipulation of the biological system. Although experiments were performed under permissive conditions, mTPP provided insights into the underlying protein stability changes that cause dramatic cellular phenotypes observed at nonpermissive temperatures. Overall, mTPP provides unique mechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in a rapid, nonbiased fashion.

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Nuclear Proteins Nut1p and Nut2p Cooperate To Negatively Regulate a Swi4p-Dependent lacZ Reporter Gene inSaccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.18.8.4707 ◽

1998 ◽

Vol 18 (8) ◽

pp. 4707-4718 ◽

Cited By ~ 37

Author(s):

Ramon K. Tabtiang ◽

Ira Herskowitz

Keyword(s):

Binding Sites ◽

Regulatory Region ◽

Nuclear Proteins ◽

Upstream Region ◽

Temperature Sensitive ◽

Wild Type ◽

Lacz Reporter Gene ◽

Reading Frame ◽

Sensitive Allele ◽

Activation Sequence

ABSTRACT The URS2 region of the Saccharomyces cerevisiae HOupstream region contains 10 binding sites for the Swi4p/Swi6p transcription factor and confers Swi4p dependence for transcription. Using a hybrid promoter, UAS GAL (upstream activation sequence of GAL1)-URS2R, in which theGAL1-10 regulatory region is fused to the proximal 360 bp of URS2, we isolated mutants in which Swi4p is no longer required for transcription. Mutations of SIN4, ROX3,SRB8, SRB9, SRB10,SRB11, and two novel genes, NUT1 andNUT2, relieve the requirement of Swi4p for expression of this reporter. We found that NUT1 (open reading frame [ORF] YGL151w) is a nonessential gene, that NUT2 (ORF YPR168w) is essential, and that both Nut1p and Nut2p encode nuclear proteins. Deletion of NUT1 causes a constitutive, Swi4p-independent phenotype only in combination with thenut2-1 allele or an allele of CCR4. In contrast, inactivation of a temperature-sensitive allele ofNUT2, nut2-ts70, alone causes constitutivity.nut1Δ nut2-1 cells and sin4Δ cells exhibit Swi4p-independent expression of an ho-lacZ reporter but not of an intact ho gene. Likewise, a pPHO5-lacZconstruct is constitutively expressed in nut1 nut2 mutants relative to their wild-type counterparts. These results suggest that Nut1p, Nut2p, Sin4p, and Ccr4p define a group of proteins that negatively regulate transcription in a subtle manner which is revealed by artificial reporter genes.

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Yeast pheromone response pathway: characterization of a suppressor that restores mating to receptorless mutants.

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2682 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2682-2694 ◽

Cited By ~ 48

Author(s):

K L Clark ◽

G F Sprague

Keyword(s):

Essential Gene ◽

Receptor Gene ◽

Cell Types ◽

Recessive Mutation ◽

Temperature Sensitive ◽

Haploid Cell ◽

Nonpermissive Temperature ◽

Pheromone Response ◽

Pheromone Response Pathway ◽

Alpha Factor

Saccharomyces cerevisiae haploid cells, alpha and a, mate after being appropriately stimulated by the pheromone secreted by the opposite cell type (a-factor and alpha-factor, respectively). The binding of a pheromone to its receptor is a signal that initiates a series of intracellular changes that lead to the specific physiological alterations required for mating. To identify components of the signal transduction pathway, we sought pseudorevertants that restored mating competence to receptor mutants (MAT alpha ste3::LEU2). The suppressor srm1-1 was isolated as a recessive mutation that conferred temperature-sensitive growth to all strains and mating ability to MAT alpha ste3::LEU2 strains at the nonpermissive temperature. In addition, when srm1-1 mutants were shifted to the nonpermissive temperature, they exhibited two phenotypes characteristic of pheromone response, induction of FUS1 transcription and accumulation of cells in the G1 phase of the cell cycle. The srm1-1 mutation also suppressed a deletion of the alpha-factor-receptor gene in a cells. Together, these phenotypes suggest that the wild-type SRM1 product is a component of the pheromone response pathway. Deletion of STE4 or STE5, which are required in both haploid cell types for mating and response to pheromone, was not suppressed by srm1-1, suggesting that the SRM1 product may function before the STE4 and STE5 products. SRM1 is an essential gene and is expressed in both haploid cell types as well as in the product of their mating, a/alpha diploids. Homozygous srm1-1 a/alpha diploids were temperature sensitive although they did not arrest in G1. Thus, the SRM1 product may also have a role in the vegetative life cycle of cells.

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