scholarly journals Unigenic evolution: a novel genetic method localizes a putative leucine zipper that mediates dimerization of the Saccharomyces cerevisiae regulator Gcr1p.

Genetics ◽  
1995 ◽  
Vol 141 (4) ◽  
pp. 1263-1274
Author(s):  
S J Deminoff ◽  
J Tornow ◽  
G M Santangelo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Leucine Zipper ◽  
Single Gene ◽  
Point Mutations ◽  
Random Mutagenesis ◽  
Genetic Method ◽  
Dna Binding Sites ◽  
Surface Domains

Abstract The GCR1 gene of Saccharomyces cerevisiae encodes a transcriptional activator that complexes with Rap1p and, through UASRPG elements (Rap1p DNA binding sites), stimulates efficient expression of glycolytic and translational component genes. To map the functionally important domains in Gcr1p, we combined multiple rounds of random mutagenesis in vitro with in vivo selection of functional genes to locate conserved, or hypomutable, regions. We name this method unigenic evolution, a statistical analysis of mutations in evolutionary variants of a single gene in an otherwise isogenic background. Examination of the distribution of 315 mutations in 24 variant alleles allowed the localization of four hypomutable regions in GCR1 (A, B, C, and D). Dispensable N-terminal (intronic) and C-terminal portions of the evolved region of GCR1 were included in the analysis as controls and were, as expected, not hypomutable. The analysis of several insertion, deletion, and point mutations, combined with a comparison of the hypomutability and hydrophobicity plots of Gcr1p, suggested that some of the hypomutable regions may individually or in combination correspond to functionally important surface domains. In particular, we determined that region D contains a putative leucine zipper and is necessary and sufficient for Gcr1p homodimerization.

scholarly journals Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875 ◽  
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme

1990 ◽  
Vol 10 (11) ◽  
pp. 5679-5687
Author(s):  
C K Barlowe ◽  
D R Appling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Point Mutations ◽  
Gene Replacement ◽  
Molecular Genetic Analysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purine Synthesis

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

scholarly journals A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions.

1997 ◽  
Vol 17 (11) ◽  
pp. 6683-6692 ◽  
Author(s):  
A J Peterson ◽  
M Kyba ◽  
D Bornemann ◽  
K Morgan ◽  
H W Brock ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Leucine Zipper ◽  
Point Mutations ◽  
Direct Interaction ◽  
Molecular Complexes ◽  
Polycomb Group ◽  
Ph Domain ◽  
Binding Studies

The Sex comb on midleg (Scm) and polyhomeotic (ph) proteins are members of the Polycomb group (PcG) of transcriptional repressors. PcG proteins maintain differential patterns of homeotic gene expression during development in Drosophila flies. The Scm and ph proteins share a homology domain with 38% identity over a length of 65 amino acids, termed the SPM domain, that is located at their respective C termini. Using the yeast two-hybrid system and in vitro protein-binding assays, we show that the SPM domain mediates direct interaction between Scm and ph. Binding studies with isolated SPM domains from Scm and ph show that the domain is sufficient for these protein interactions. These studies also show that the Scm-ph and Scm-Scm domain interactions are much stronger than the ph-ph domain interaction, indicating that the isolated domain has intrinsic binding specificity determinants. Analysis of site-directed point mutations identifies residues that are important for SPM domain function. These binding properties, predicted alpha-helical secondary structure, and conservation of hydrophobic residues prompt comparisons of the SPM domain to the helix-loop-helix and leucine zipper domains used for homotypic and heterotypic protein interactions in other transcriptional regulators. In addition to in vitro studies, we show colocalization of the Scm and ph proteins at polytene chromosome sites in vivo. We discuss the possible roles of the SPM domain in the assembly or function of molecular complexes of PcG proteins.

scholarly journals Opposite roles of trehalase activity in heat-shock recovery and heat-shock survival in Saccharomyces cerevisiae

1999 ◽  
Vol 343 (3) ◽  
pp. 621-626 ◽  
Author(s):  
Stefaan WERA ◽  
Ellen DE SCHRIJVER ◽  
Ilse GEYSKENS ◽  
Solomon NWAKA ◽  
Johan M. THEVELEIN
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Specific Activity ◽  
Point Mutations ◽  
Activity Levels ◽  
Protective Function ◽  
Trehalase Activity ◽  
Neutral Trehalase

A variety of results has been obtained consistent with activation of neutral trehalase in Saccharomyces cerevisiae through direct phosphorylation by cAMP-dependent protein kinase (PKA). A series of neutral trehalase mutant alleles, in which all evolutionarily conserved putative phosphorylation sites were changed into alanine, was tested for activation in vitro (by PKA) and in vivo (by glucose addition). None of the mutations alone affected the activation ratio, whereas all mutations combined resulted in an inactive enzyme. All mutant alleles were expressed to similar levels, as shown by Western blotting. Several of the point mutations significantly lowered the specific activity. Using this series of mutants with different activity levels we show an inverse relationship between trehalase activity and heat-shock survival during glucose-induced trehalose mobilization. This is consistent with a stress-protective function of trehalose. On the other hand, reduction of trehalase activity below a certain threshold level impaired recovery from a sublethal heat shock. This suggests that trehalose breakdown is required for efficient recovery from heat shock, and that the presence of trehalase protein alone is not sufficient for efficient heat-stress recovery.

scholarly journals Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

2016 ◽  
Vol 113 (13) ◽  
pp. 3669-3674 ◽  
Author(s):  
Min-Suk Song ◽  
Gyanendra Kumar ◽  
William R. Shadrick ◽  
Wei Zhou ◽  
Trushar Jeevan ◽  
...  
Keyword(s):  
Point Mutations ◽  
Random Mutagenesis ◽  
Resistant Mutant ◽  
Inhibitor Binding ◽  
New Class ◽  
Identification And Characterization ◽  
Modest Reduction

The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. These mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containing the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. Using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.

scholarly journals A primary study on genes with selected mutations by in vitro passage of Chlamydia muridarum strains

2019 ◽  
Vol 77 (3) ◽  
Author(s):  
Zhou Zhou ◽  
Na Liu ◽  
Yingzi Wang ◽  
Arthur Wirekoh Emmanuel ◽  
Xiaoxing You ◽  
...  
Keyword(s):  
Single Gene ◽  
Point Mutations ◽  
Primary Study ◽  
Chlamydia Muridarum ◽  
In Vivo Experiments ◽  
High Incidence ◽  
Identification And Characterization

ABSTRACTObjectiveThis study is to investigate the functions of newly discovered genes in Chlamydia muridarum (C. muridarum) strains with single gene differences.MethodsUsing whole genome sequencing and plaque formation assays, C. muridarum parental and passaging strains were established, and the isogenic clones expressing certain genotypes were isolated. Strains with single gene differences were obtained. Based on prediction, the valuable strains with single gene differences of tc0412, tc0668 or tc0237 were subjected to the in vitro and in vivo experiments for biological characterization and virulence analysis.ResultsInsertional -472840T mutation of the tc0412 gene (T28T/B3 type) matching with the nonmutant tc0668 gene and tc0237 gene with point mutations G797659T (Q117E) might slow the growth of Chlamydia due to the lack of a plasmid. The nonmutant tc0668 in the strain might induce a high incidence of hydrosalpinx in mice, while tc0668 with a G797659T point mutation was significantly attenuated. Compared with the nonmutant tc0237, the strains containing mutant tc0237 were characterized by reduced centrifugation dependence during infection.ConclusionThe identification and characterization of these genes might contribute to the comprehensive understanding of the pathogenic mechanism of Chlamydia.

scholarly journals Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme.

1990 ◽  
Vol 10 (11) ◽  
pp. 5679-5687 ◽  
Author(s):  
C K Barlowe ◽  
D R Appling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Point Mutations ◽  
Gene Replacement ◽  
Molecular Genetic Analysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purine Synthesis

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

scholarly journals Adjacent upstream activation sequence elements synergistically regulate transcription of ADH2 in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (1) ◽  
pp. 34-42 ◽  
Author(s):  
J Yu ◽  
M S Donoviel ◽  
E T Young
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Repression ◽  
Point Mutations ◽  
Single Copy ◽  
Upstream Activation Sequence ◽  
Sequence Elements ◽  
Adh2 Promoter ◽  
Activation Sequence

A 22-base-pair (bp) inverted repeat present in the ADH2 promoter is an upstream activation sequence (UAS1) which confers ADR1-dependent activation upon a heterologous Saccharomyces cerevisiae promoter. UAS1 was nonfunctional when placed within an intron 3' to the transcription start site. The 11-bp sequence which constitutes one-half of the UAS1 palindrome did not activate transcription in a single copy, as direct repeats, or in an inverted orientation opposite to that of ADH2 UAS1. Furthermore, two pairs of symmetrical point mutations within UAS1 significantly reduced activation. This result suggests that a specific orientation of sequences within UAS1 is necessary for ADR1-dependent activation. We determined that an ADR1-dependent complex was formed with UAS1 and, to a lesser extent, with the nonfunctional 11-bp half palindrome. However, the 11 bp did not confer UAS activity, suggesting that ADR1 binding is not sufficient for activation in vivo. ADR1 did not bind to mutant UAS1 sequences in vitro, indicating that their decreased activation is attributable to a reduced affinity of ADR1 for these sequences. We also identified an additional 20-bp ADH2 element (UAS2) that increased the expression of CYC1-lacZ 20-fold when combined with UAS1. UAS2 permitted ADR1-independent, glucose-regulated expression of the hybrid gene. Consistent with this observation, ADR1 did not form a detectable complex with UAS2. Deletion of UAS2 at the chromosomal ADH2 locus virtually abolished ADH2 derepression and had no effect on glucose repression.

scholarly journals Mutational analysis of the Saccharomyces cerevisiae ABD1 gene: cap methyltransferase activity is essential for cell growth.

1996 ◽  
Vol 16 (2) ◽  
pp. 475-480 ◽  
Author(s):  
X Mao ◽  
B Schwer ◽  
S Shuman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Yeast Cells ◽  
Terminal Deletion ◽  
Methyltransferase Activity

RNA (guanine-7-)-methyltransferase is the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA. The Saccharomyces cerevisiae enzyme is a 436-amino-acid protein encoded by the essential ABD1 gene. In this study, deletion and point mutations in ABD1 were tested for the ability to support growth of an abd1 null strain. Elimination of 109 amino acids from the N terminus had no effect on cell viability, whereas a more extensive N-terminal deletion of 155 residues was lethal, as was a C-terminal deletion of 55 amino acids. Alanine substitution mutations were introduced at eight conserved residues within a 206-amino-acid region of similarity between ABD1 and the methyltransferase domain of the vaccinia virus capping enzyme. ABD1 alleles H253A (encoding a substitution of alanine for histidine at position 253), T282A, E287A, E361A, and Y362A were viable, whereas G174A, D178A, and Y254A were either lethal or severely defective for growth. Alanine-substituted and amino-truncated ABD1 proteins were expressed in bacteria, purified, and tested for cap methyltransferase activity in vitro. Mutations that were viable in yeast cells had either no effect or only a moderate effect on the specific methyltransferase activity of the mutated ABD1 protein, whereas mutations that were deleterious in vivo yielded proteins that were catalytically defective in vitro. These findings substantiate for the first time the long-held presumption that cap methylation is an essential function in eukaryotic cells.

Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

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