scholarly journals GENETIC EVIDENCE FOR INTERACTION BETWEEN NONHOMOLOGOUS PROTEINS IN YEAST AND A CASE OF SUPPRESSION AT THE HIS1 LOCUS

Genetics ◽  
1980 ◽  
Vol 94 (2) ◽  
pp. 327-339 ◽  
Author(s):  
Richard Snow
Keyword(s):  
Enzyme Activity ◽  
Low Temperature ◽  
Enzymatic Activity ◽  
Temperature Effect ◽  
Conformational Change ◽  
Minimal Medium ◽  
Genetic Evidence ◽  
Structural Genes ◽  
Wild Type ◽  
Is Strain

ABSTRACT The HIS1 and THR4 loci are the structural genes for phosphoribosyl-ATP pyrophosphorylase and threonine synthetase, respectively. The allele his1-IS has no enzyme activity at 30", but does have activity at 15" provided the cell contains the wild-type THR4 allele or a suppressing allele at another locus, designated SUP(his1-1S). Under these conditions, cells with the hisl-IS mutation are capable of growth on minimal medium at 15". Three kinds of reversions of a hisl-IS thr4 sup(his1-IS) strain to histidine prototrophy have been obtained: (1) his1-IS locus reversions to HIS1 that restore growth without added histidine at 30", (2)  thr4 reversions to THR4 that simultaneously eliminate the requirement for threonine and restore the low-temperature effect on the his1-IS allele, and (3)mutations from sup to SUP. The SUP allele is not an ochre suppressor, and it is not linked to either HISI, THR4 or a centromere. It may represent a missense suppressor. I t is proposed that the effect ofTHR4 is caused by aggregation of the wild-type threonine synthetase with defective his1-IS monomers, causing a favorable conformational change in the histidine protein that restores limited enzymatic activity. This can be regarded as a case of complementation between nonhomologous proteins.

scholarly journals Catabolite repression of the lac operon. Separate repression of two enzymes

1969 ◽  
Vol 114 (2) ◽  
pp. 313-319 ◽  
Author(s):  
M D Yudkin
Keyword(s):  
Catabolite Repression ◽  
Minimal Medium ◽  
Wild Type Strain ◽  
Translational Repression ◽  
Genotypic Differences ◽  
Lac Operon ◽  
Structural Genes ◽  
Wild Type ◽  
Mutant Strains ◽  
K 12

1. Catabolite repression of β-galactosidase and of thiogalactoside transacetylase was studied in several strains of Escherichia coli K 12, in an attempt to show whether a single site within the structural genes of the lac operon co-ordinately controls translational repression for the two enzymes. In all experiments the rate of synthesis of the enzymes was compared in glycerol–minimal medium and in glucose–minimal medium. 2. In a wild-type strain, glucose repressed the synthesis of the two enzymes equally. 3. The possibility that repression was co-ordinate was investigated by studies of mutant strains that carry deletions in the genes for β-galactosidase or galactoside permease or both. In all of the strains with deletions, the repression of thiogalactoside transacetylase persisted, and it is concluded that there is no part of the structural gene for β-galactosidase that is essential for catabolite repression of thiogalactoside transacetylase. 4. Subculture of one strain through several transfers in rich medium greatly increased its susceptibility to catabolite repression by glucose. It is concluded that unknown features of the genotype can markedly affect sensitivity to catabolite repression. 5. These results make it clear that one cannot draw valid conclusions about the effect of known genotypic differences on catabolite repression from a comparison of two separate strains; to study the effect of a particular genetic change in a lac operon it is necessary to construct a partially diploid strain so that catabolite repression suffered by one lac operon can be compared with that suffered by another. 6. Four such partial diploids were constructed. In all of them catabolite repression of β-galactosidase synthesized by one operon was equal in extent to catabolite repression of thiogalactoside transacetylase synthesized by the other. 7. Taken together, these results suggest that catabolite repression of β-galactosidase and thiogalactoside transacetylase is separate but equal.

scholarly journals The Lipid Lysyl-Phosphatidylglycerol Is Present in Membranes of Rhizobium tropici CIAT899 and Confers Increased Resistance to Polymyxin B Under Acidic Growth Conditions

2007 ◽  
Vol 20 (11) ◽  
pp. 1421-1430 ◽  
Author(s):  
Christian Sohlenkamp ◽  
Kanaan A. Galindo-Lagunas ◽  
Ziqiang Guan ◽  
Pablo Vinuesa ◽  
Sally Robinson ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Minimal Medium ◽  
Polymyxin B ◽  
Growth Conditions ◽  
Membrane Lipid ◽  
Low Ph ◽  
Wild Type ◽  
Rhizobium Tropici ◽  
Gram Negative ◽  
Inducible Genes

Lysyl-phosphatidylglycerol (LPG) is a well-known membrane lipid in several gram-positive bacteria but is almost unheard of in gram-negative bacteria. In Staphylococcus aureus, the gene product of mprF is responsible for LPG formation. Low pH-inducible genes, termed lpiA, have been identified in the gram-negative α-proteobacteria Rhizobium tropici and Sinorhizobium medicae in screens for acid-sensitive mutants and they encode homologs of MprF. An analysis of the sequenced bacterial genomes reveals that genes coding for homologs of MprF from S. aureus are present in several classes of organisms throughout the bacterial kingdom. In this study, we show that the expression of lpiA from R. tropici in the heterologous hosts Escherichia coli and Sinorhizobium meliloti causes formation of LPG. A wild-type strain of R. tropici forms LPG (about 1% of the total lipids) when the cells are grown in minimal medium at pH 4.5 but not when grown in minimal medium at neutral pH or in complex tryptone yeast (TY) medium at either pH. LPG biosynthesis does not occur when lpiA is deleted and is restored upon complementation of lpiA-deficient mutants with a functional copy of the lpiA gene. When grown in the low-pH medium, lpiA-deficient rhizobial mutants are over four times more susceptible to the cationic peptide polymyxin B than the wild type.

scholarly journals Functional Assessment of 12 Rare Allelic CYP2C9 Variants Identified in a Population of 4773 Japanese Individuals

2021 ◽  
Vol 11 (2) ◽  
pp. 94
Author(s):  
Masaki Kumondai ◽  
Akio Ito ◽  
Evelyn Marie Gutiérrez Rico ◽  
Eiji Hishinuma ◽  
Akiko Ueda ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Three Dimensional ◽  
Warfarin Dose ◽  
Catalytic Efficiency ◽  
Therapeutic Window ◽  
Wild Type ◽  
Drug Metabolizing Enzyme ◽  
Novel Variants ◽  
Metabolizing Enzyme ◽  
Almost All

Cytochrome P450 2C9 (CYP2C9) is an important drug-metabolizing enzyme that contributes to the metabolism of approximately 15% of clinically used drugs, including warfarin, which is known for its narrow therapeutic window. Interindividual differences in CYP2C9 enzymatic activity caused by CYP2C9 genetic polymorphisms lead to inconsistent treatment responses in patients. Thus, in this study, we characterized the functional differences in CYP2C9 wild-type (CYP2C9.1), CYP2C9.2, CYP2C9.3, and 12 rare novel variants identified in 4773 Japanese individuals. These CYP2C9 variants were heterologously expressed in 293FT cells, and the kinetic parameters (Km, kcat, Vmax, catalytic efficiency, and CLint) of (S)-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation were estimated. From this analysis, almost all novel CYP2C9 variants showed significantly reduced or null enzymatic activity compared with that of the CYP2C9 wild-type. A strong correlation was found in catalytic efficiencies between (S)-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation among all studied CYP2C9 variants. The causes of the observed perturbation in enzyme activity were evaluated by three-dimensional structural modeling. Our findings could clarify a part of discrepancies among genotype–phenotype associations based on the novel CYP2C9 rare allelic variants and could, therefore, improve personalized medicine, including the selection of the appropriate warfarin dose.

scholarly journals Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

scholarly journals Loss of Ubp3 increases silencing, decreases unequal recombination in rDNA, and shortens the replicative life span in Saccharomyces cerevisiae

2014 ◽  
Vol 25 (12) ◽  
pp. 1916-1924 ◽  
Author(s):  
David Öling ◽  
Rehan Masoom ◽  
Kristian Kvint
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Life Span ◽  
Rna Polymerase Ii ◽  
Ribosomal Dna ◽  
Genetic Evidence ◽  
Wild Type ◽  
Replicative Life Span ◽  
Unequal Recombination

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes.

scholarly journals New Insights into the Spring-Loaded Conformational Change of Influenza Virus Hemagglutinin

2002 ◽  
Vol 76 (9) ◽  
pp. 4456-4466 ◽  
Author(s):  
Jennifer A. Gruenke ◽  
R. Todd Armstrong ◽  
William W. Newcomb ◽  
Jay C. Brown ◽  
Judith M. White
Keyword(s):  
Influenza Virus ◽  
Conformational Change ◽  
Conformational Changes ◽  
Limited Proteolysis ◽  
Coiled Coil ◽  
Fusion Peptide ◽  
Wild Type ◽  
Influenza Virus Hemagglutinin ◽  
Target Membrane ◽  
Mutant Forms

ABSTRACT Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix “spring-loaded” conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63P/F70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited proteolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only ∼50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.

Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone

1991 ◽  
Vol 11 (7) ◽  
pp. 3603-3612
Author(s):  
S Marcus ◽  
G A Caldwell ◽  
D Miller ◽  
C B Xue ◽  
F Naider ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biological Activity ◽  
Methyl Ester ◽  
Total Synthesis ◽  
Biologically Active ◽  
Structural Genes ◽  
Wild Type ◽  
Mating Pheromone ◽  
Carboxy Terminal

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[S-farnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.

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