The THI5 gene family of Saccharomyces cerevisiae: distribution of homologues among the hemiascomycetes and functional redundancy in the aerobic biosynthesis of thiamin from pyridoxine

Microbiology ◽  
2003 ◽  
Vol 149 (6) ◽  
pp. 1447-1460 ◽  
Author(s):  
Raymond Wightman ◽  
Peter A. Meacock
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Family ◽  
Biosynthetic Pathway ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Gene Copy ◽  
Phylogenetic Study ◽  
Vitamin B ◽  
Mrna Levels ◽  
Quadruple Mutant

The THI5 gene family of Saccharomyces cerevisiae comprises four highly conserved members named THI5 (YFL058w), THI11 (YJR156c), THI12 (YNL332w) and THI13 (YDL244w). Each gene copy is located within the subtelomeric region of a different chromosome and all are homologues of the Schizosaccharomyces pombe nmt1 gene which is thought to function in the biosynthesis of hydroxymethylpyrimidine (HMP), a precursor of vitamin B1, thiamin. A comprehensive phylogenetic study has shown that the existence of THI5 as a gene family is exclusive to those yeasts of the Saccharomyces sensu stricto subgroup. To determine the function and redundancy of each of the S. cerevisiae homologues, all combinations of the single, double, triple and quadruple deletion mutants were constructed using a PCR-mediated gene-disruption strategy. Phenotypic analyses of these mutant strains have shown the four genes to be functionally redundant in terms of HMP formation for thiamin biosynthesis; each promotes synthesis of HMP from the pyridoxine (vitamin B6) biosynthetic pathway. Furthermore, growth studies with the quadruple mutant strain support a previous proposal of an alternative HMP biosynthetic pathway that operates in yeast under anaerobic growth conditions. Comparative analysis of mRNA levels has revealed subtle differences in the regulation of the four genes, suggesting that they respond differently to nutrient limitation.

scholarly journals Identification and Characterization ofMAE1, the Saccharomyces cerevisiae Structural Gene Encoding Mitochondrial Malic Enzyme

1998 ◽  
Vol 180 (11) ◽  
pp. 2875-2882 ◽  
Author(s):  
Eckhard Boles ◽  
Patricia de Jong-Gubbels ◽  
Jack T. Pronk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzyme Activity ◽  
Pyruvate Kinase ◽  
Malic Enzyme ◽  
Fold Increase ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Oxidative Decarboxylation ◽  
Mrna Levels ◽  
Malic Enzyme Activity

ABSTRACT Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression ofYKL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of MAE1 alone did not result in a clear phenotype. Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring β-galactosidase activities in a strain carrying a MAE1::lacZ fusion. Both in shake flask cultures and in carbon-limited chemostat cultures,MAE1 was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in S. cerevisiae is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.

scholarly journals Structure of the transcriptionally repressed phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (1) ◽  
pp. 38-46 ◽  
Author(s):  
L W Bergman ◽  
M C Stranathan ◽  
L H Preis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Copy Number ◽  
Regulatory Control ◽  
Gene Copy ◽  
Growth Media ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Acid Phosphatase Gene ◽  
Negative Supercoils

We developed a high-copy-number plasmid system containing the entire structural and regulatory sequences of the phosphate-repressible acid phosphatase (PHO5) gene and the TRP1/ARS1 replicator sequences of the yeast Saccharomyces cerevisiae to investigate the mechanism of repression-derepression of transcription. The resulting plasmid was used to transform either wild-type cells or a number of strains which contain mutations in various trans-acting regulatory loci for the production of acid phosphatase. Results of analysis of mRNA levels isolated from the transformed strains grown under repressed or derepressed conditions suggested that normal transcriptional regulation of the gene persisted, although gene copy number was significantly increased. Analysis of changes in linking number (i.e., the number of negative supercoils) of the plasmid isolated under repressed and derepressed growth conditions revealed that the transcriptionally inactive plasmid contained approximately three more negative supercoils than the transcriptionally active plasmid. This difference in topological state was similarly seen in a plasmid containing a sequence-related acid phosphatase gene (PHO11) under the same regulatory control system, but it was not seen in plasmids isolated from some strains containing mutations which caused either fully constitutive or nonderepressible production of acid phosphatase. Finally, analysis of the nucleosome positioning along the inactive gene sequence revealed that an abnormally broad internucleosomal spacer is present in a region presumed to function in the regulation of transcription by the level of Pi in the growth media.

Structure of the transcriptionally repressed phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae

1986 ◽  
Vol 6 (1) ◽  
pp. 38-46
Author(s):  
L W Bergman ◽  
M C Stranathan ◽  
L H Preis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Copy Number ◽  
Regulatory Control ◽  
Gene Copy ◽  
Growth Media ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Acid Phosphatase Gene ◽  
Negative Supercoils

We developed a high-copy-number plasmid system containing the entire structural and regulatory sequences of the phosphate-repressible acid phosphatase (PHO5) gene and the TRP1/ARS1 replicator sequences of the yeast Saccharomyces cerevisiae to investigate the mechanism of repression-derepression of transcription. The resulting plasmid was used to transform either wild-type cells or a number of strains which contain mutations in various trans-acting regulatory loci for the production of acid phosphatase. Results of analysis of mRNA levels isolated from the transformed strains grown under repressed or derepressed conditions suggested that normal transcriptional regulation of the gene persisted, although gene copy number was significantly increased. Analysis of changes in linking number (i.e., the number of negative supercoils) of the plasmid isolated under repressed and derepressed growth conditions revealed that the transcriptionally inactive plasmid contained approximately three more negative supercoils than the transcriptionally active plasmid. This difference in topological state was similarly seen in a plasmid containing a sequence-related acid phosphatase gene (PHO11) under the same regulatory control system, but it was not seen in plasmids isolated from some strains containing mutations which caused either fully constitutive or nonderepressible production of acid phosphatase. Finally, analysis of the nucleosome positioning along the inactive gene sequence revealed that an abnormally broad internucleosomal spacer is present in a region presumed to function in the regulation of transcription by the level of Pi in the growth media.

scholarly journals Transcriptional Regulation of the Two Sterol Esterification Genes in the Yeast Saccharomyces cerevisiae

2001 ◽  
Vol 183 (17) ◽  
pp. 4950-4957 ◽  
Author(s):  
Kristen Jensen-Pergakes ◽  
Zhongmin Guo ◽  
Mara Giattina ◽  
Stephen L. Sturley ◽  
Martin Bard
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Initiation ◽  
Physiological Role ◽  
Growth Conditions ◽  
Product Inhibition ◽  
Anaerobic Growth ◽  
Primary Role ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sterol Esterification

ABSTRACT Saccharomyces cerevisiae transcribes two genes,ARE1 and ARE2, that contribute disproportionately to the esterification of sterols. Are2p is the major enzyme isoform in a wild-type cell growing aerobically. This likely results from a combination of differential transcription initiation and transcript stability. By using ARE1 andARE2 promoter fusions to lacZ reporters, we demonstrated that transcriptional initiation from theARE1 promoter is significantly reduced compared to that from the ARE2 promoter. Furthermore, the half-life of the ARE2 mRNA is approximately 12 times as long as that of the ARE1 transcript. We present evidence that the primary role of the minor sterol esterification isoform encoded byARE1 is to esterify sterol intermediates, whereas the role of the ARE2 enzyme is to esterify ergosterol, the end product of the pathway. Accordingly, the ARE1promoter is upregulated in strains that accumulate ergosterol precursors. Furthermore, ARE1 and ARE2are oppositely regulated by heme. Under heme-deficient growth conditions, ARE1 was upregulated fivefold whileARE2 was down-regulated. ARE2 requires the HAP1 transcription factor for optimal expression, and both ARE genes are derepressed in arox1 (repressor of oxygen) mutant genetic background. We further report that the ARE genes are not subject to end product inhibition; neither ARE1 nor ARE2transcription is altered in an are mutant background, nor does overexpression of either ARE gene alter the response of the ARE-lacZ reporter constructs. Our observations are consistent with an important physiological role for Are1p during anaerobic growth when heme is limiting and sterol precursors may accumulate. Conversely, Are2p is optimally required during aerobiosis when ergosterol is plentiful.

scholarly journals Vitamin B12-Mediated Restoration of Defective Anaerobic Growth Leads to Reduced Biofilm Formation in Pseudomonas aeruginosa

2012 ◽  
Vol 80 (5) ◽  
pp. 1639-1649 ◽  
Author(s):  
Kang-Mu Lee ◽  
Junhyeok Go ◽  
Mi Young Yoon ◽  
Yongjin Park ◽  
Sang Cheol Kim ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Dna Replication ◽  
Cell Elongation ◽  
Biofilm Formation ◽  
Shape Change ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Vitamin B ◽  
Content Type ◽  
Cell Shape Change

ABSTRACTPseudomonas aeruginosaundergoes cell elongation and forms robust biofilms during anaerobic respiratory growth using nitrate (NO3−) as an alternative electron acceptor. Understanding the mechanism of cell shape change induced upon anaerobiosis is crucial to the development of effective treatments againstP. aeruginosabiofilm infection. Here, we uncovered the molecular basis of anaerobiosis-triggered cell elongation and identified vitamin B12to be a molecule that can reinstate defective anaerobic growth ofP. aeruginosa. The ratio of total cellular DNA content to protein content was significantly decreased in the PAO1 strain grown under anaerobic conditions, indicating that DNA replication is impaired during anaerobic growth. Anaerobic growth of PAO1 reached a higher cell density in the presence of vitamin B12, an essential coenzyme of class II ribonucleotide reductase. In addition, cell morphology returned to a normal rod shape and transcription of stress-response genes was downregulated under the same anaerobic growth conditions. These results suggest that vitamin B12, the production of which was suppressed during anaerobic growth, can restore cellular machineries for DNA replication and therefore facilitate better anaerobic growth ofP. aeruginosawith normal cell division. Importantly, biofilm formation was substantially decreased when grown with vitamin B12, further demonstrating that anaerobiosis-induced cell elongation is responsible for robust biofilm formation. Taken together, our data reveal mechanistic details of a morphological change that naturally occurs during anaerobic growth ofP. aeruginosaand illustrates the ability of vitamin B12to modulate the biofilm-forming capacity ofP. aeruginosaunder such condition.

Identification of REO1, a gene involved in negative regulation of COX5b and ANB1 in aerobically grown Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 120 (3) ◽  
pp. 671-680 ◽  
Author(s):  
C E Trueblood ◽  
R O Poyton
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Gene Family ◽  
Cytochrome C Oxidase ◽  
Carbon Sources ◽  
Phenotypic Expression ◽  
Growth Conditions ◽  
Complementation Group ◽  
Negative Regulation ◽  
Yeast Cells

Abstract In Saccharomyces cerevisiae, the COX5a and COX5b genes constitute a small gene family that encodes two forms of cytochrome c oxidase subunit V, Va and Vb, either of which can provide a function essential for cytochrome c oxidase activity and respiration. In aerobically grown wild-type yeast cells, Va is the predominant form of subunit V. The COX5b gene alone does not produce enough Vb to support a respiration rate sufficient to allow growth on nonfermentable carbon sources. By selecting for mutations that increase the respiratory capacity of a strain deleted for COX5a, we have identified a gene that is involved in negative regulation of COX5b expression under aerobic growth conditions. Each of four independently isolated reo1 mutations are shown to be recessive, unlinked to COX5b, but dependent on COX5b for phenotypic expression. The mutations define a single complementation and linkage group: designated as REO1 for regulator of expression of oxidase. reo1 mutations increase expression of COX5b in aerobically grown cells, but not in anaerobically grown cells, where expression is already elevated. These mutations have no effect on COX5a, the other member of this small gene family which is positively regulated by heme and oxygen. The REO1 gene does play a role in repression of ANB1, a gene that is normally repressed under aerobic but not anaerobic conditions. Neither rox1 or rox3 mutations, which have previously been shown to increase ANB1 expression, are in the same complementation group as reo1 mutations.

scholarly journals Expansion and accelerated evolution of 9-exon odorant receptors in Polistes paper wasps

2021 ◽  
Author(s):  
Andrew W Legan ◽  
Christopher M Jernigan ◽  
Sara E Miller ◽  
Matthieu F Fuchs ◽  
Michael J Sheehan
Keyword(s):  
Gene Family ◽  
Odorant Receptor ◽  
Receptor Gene ◽  
Gene Tree ◽  
Gene Family Evolution ◽  
Paper Wasp ◽  
Gene Copy ◽  
Evolutionary Divergence ◽  
Wasp Species ◽  
Or Gene

Abstract Independent origins of sociality in bees and ants are associated with independent expansions of particular odorant receptor (OR) gene subfamilies. In ants, one clade within the OR gene family, the 9-exon subfamily, has dramatically expanded. These receptors detect cuticular hydrocarbons (CHCs), key social signaling molecules in insects. It is unclear to what extent 9-exon OR subfamily expansion is associated with the independent evolution of sociality across Hymenoptera, warranting studies of taxa with independently derived social behavior. Here we describe odorant receptor gene family evolution in the northern paper wasp, Polistes fuscatus, and compare it to four additional paper wasp species spanning ∼40 million years of evolutionary divergence. We find 200 putatively functional OR genes in P. fuscatus, matching predictions from neuroanatomy, and more than half of these are in the 9-exon subfamily. Most OR gene expansions are tandemly arrayed at orthologous loci in Polistes genomes, and microsynteny analysis shows species-specific gain and loss of 9-exon ORs within tandem arrays. There is evidence of episodic positive diversifying selection shaping ORs in expanded subfamilies. Values of omega (d  N/dS) are higher among 9-exon ORs compared to other OR subfamilies. Within the Polistes OR gene tree, branches in the 9-exon OR clade experience relaxed negative (purifying) selection relative to other branches in the tree. Patterns of OR evolution within Polistes are consistent with 9-exon OR function in CHC perception by combinatorial coding, with both natural selection and neutral drift contributing to interspecies differences in gene copy number and sequence.

scholarly journals Regulatory Mechanisms Controlling Expression of the DAN/TIR Mannoprotein Genes During Anaerobic Remodeling of the Cell Wall in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1169-1177
Author(s):  
Natalia E Abramova ◽  
Brian D Cohen ◽  
Odeniel Sertil ◽  
Rachna Kapoor ◽  
Kelvin J A Davies ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Consensus Sequence ◽  
Ectopic Expression ◽  
Constitutive Expression ◽  
Anaerobic Growth ◽  
Zinc Cluster ◽  
Sterol Transport ◽  
Genes Encoding ◽  
Altered Regulation ◽  
Repression Domain

Abstract The DAN/TIR genes of Saccharomyces cerevisiae encode homologous mannoproteins, some of which are essential for anaerobic growth. Expression of these genes is induced during anaerobiosis and in some cases during cold shock. We show that several heme-responsive mechanisms combine to regulate DAN/TIR gene expression. The first mechanism employs two repression factors, Mox1 and Mox2, and an activation factor, Mox4 (for mannoprotein regulation by oxygen). The genes encoding these proteins were identified by selecting for recessive mutants with altered regulation of a dan1::ura3 fusion. MOX4 is identical to UPC2, encoding a binucleate zinc cluster protein controlling expression of an anaerobic sterol transport system. Mox4/Upc2 is required for expression of all the DAN/TIR genes. It appears to act through a consensus sequence termed the AR1 site, as does Mox2. The noninducible mox4Δ allele was epistatic to the constitutive mox1 and mox2 mutations, suggesting that Mox1 and Mox2 modulate activation by Mox4 in a heme-dependent fashion. Mutations in a putative repression domain in Mox4 caused constitutive expression of the DAN/TIR genes, indicating a role for this domain in heme repression. MOX4 expression is induced both in anaerobic and cold-shocked cells, so heme may also regulate DAN/TIR expression through inhibition of expression of MOX4. Indeed, ectopic expression of MOX4 in aerobic cells resulted in partially constitutive expression of DAN1. Heme also regulates expression of some of the DAN/TIR genes through the Rox7 repressor, which also controls expression of the hypoxic gene ANB1. In addition Rox1, another heme-responsive repressor, and the global repressors Tup1 and Ssn6 are also required for full aerobic repression of these genes.

scholarly journals Cloning and functional analysis of the FAD2 gene family from desert shrub Artemisia sphaerocephala

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Xiumei Miao ◽  
Lijing Zhang ◽  
Xiaowei Hu ◽  
Shuzhen Nan ◽  
Xiaolong Chen ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Linoleic Acid ◽  
Gene Family ◽  
Fatty Acid Biosynthesis ◽  
Family Members ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Acid Biosynthesis ◽  
Desert Shrub ◽  
Artemisia Sphaerocephala

Abstract Background Linoleic acid is an important polyunsaturated fatty acid, required for all eukaryotes. Microsomal delta-12 (Δ12) oleate desaturase (FAD2) is a key enzyme for linoleic acid biosynthesis. Desert shrub Artemisia sphaerocephala is rich in linoleic acid, it has a large FAD2 gene family with twenty-six members. The aim of this work is to unveil the difference and potentially functionality of AsFAD2 family members. Results Full-length cDNAs of twenty-one AsFAD2 genes were obtained from A. sphaerocephala. The putative polypeptides encoded by AsFAD2 family genes showed a high level of sequence similarity and were relatively conserved during evolution. The motif composition was also relatively conservative. Quantitative real-time PCR analysis revealed that the AsFAD2–1 gene was strongly expressed in developing seeds, which may be closely associated with the high accumulating ability of linoleic acid in A. sphaerocephala seeds. Although different AsFAD2 family members showed diverse response to salt stress, the overall mRNA levels of the AsFAD2 family genes was stable. Transient expression of AsFAD2 genes in the Nicotiana benthamiana leaves revealed that the encoded proteins were all located in the endoplasmic reticulum. Heterologous expression in Saccharomyces cerevisiae suggested that only three AsFAD2 enzymes, AsFAD2–1, − 10, and − 23, were Δ12 oleate desaturases, which could convert oleic acid to linoleic acid, whereas AsFAD2–1 and AsFAD2–10 could also produce palmitolinoleic acid. Conclusions This research reported the cloning, expression studies, subcellular localization and functional identification of the large AsFAD2 gene family. These results should be helpful in understanding fatty acid biosynthesis in A. sphaerocephala, and has the potential to be applied in the study of plant fatty acids traits.

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