scholarly journals Aspergillus nidulans Catalase-Peroxidase Gene (cpeA) Is Transcriptionally Induced during Sexual Development through the Transcription Factor StuA

2002 ◽  
Vol 1 (5) ◽  
pp. 725-735 ◽  
Author(s):  
Mario Scherer ◽  
Huijun Wei ◽  
Ralf Liese ◽  
Reinhard Fischer
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Aspergillus Nidulans ◽  
Sexual Development ◽  
Transcriptional Activation ◽  
Fluorescent Protein ◽  
Septoria Tritici ◽  
Protein Fusion ◽  
Homologous Proteins ◽  
Catalase Peroxidase

ABSTRACT Catalases, peroxidases, and catalase-peroxidases are important enzymes to cope with reactive oxygen species in pro- and eukaryotic cells. In the filamentous fungus Aspergillus nidulans three monofunctional catalases have been described, and a fourth catalase activity was observed in native polyacrylamide gels. The latter activity is probably due to the bifunctional enzyme catalase-peroxidase, which we characterized here. The gene, named cpeA, encodes an 81-kDa polypeptide with a conserved motif for heme coordination. The enzyme comprises of two similar domains, suggesting gene duplication and fusion during evolution. The first 439 amino acids share 22% identical residues with the C terminus. Homologous proteins are found in several prokaryotes, such as Escherichia coli and Mycobacterium tuberculosis (both with 61% identity). In fungi the enzyme has been noted in Penicillium simplicissimum, Septoria tritici, and Neurospora crassa (69% identical amino acids) but is absent from Saccharomyces cerevisiae. Expression analysis in A. nidulans revealed that the gene is transcriptionally induced upon carbon starvation and during sexual development, but starvation is not sufficient to reach high levels of the transcript during development. Besides transcriptional activation, we present evidence for posttranscriptional regulation. A green fluorescent protein fusion protein localized to the cytoplasm of Hülle cells. The Hülle cell-specific expression was dependent on the developmental regulator StuA, suggesting an activating function of this helix-loop-helix transcription factor.

scholarly journals The Predicted G-Protein-Coupled Receptor GPR-1 Is Required for Female Sexual Development in the Multicellular Fungus Neurospora crassa

2006 ◽  
Vol 5 (9) ◽  
pp. 1503-1516 ◽  
Author(s):  
Svetlana Krystofova ◽  
Katherine A. Borkovich
Keyword(s):  
Neurospora Crassa ◽  
G Protein ◽  
Sexual Development ◽  
Fluorescent Protein ◽  
Protein Fusion ◽  
Phenotypic Analysis ◽  
Reproductive Structures ◽  
Epistasis Analysis ◽  
Green Fluorescent ◽  
G Protein Coupled

ABSTRACTG-protein-coupled receptors (GPCRs) control important aspects of asexual and sexual development in eukaryotic organisms. We have identified a predicted GPCR in the filamentous fungusNeurospora crassawith similarity to cyclic AMP-receptor like GPCRs fromDictyostelium discoideumand GCR1 fromArabidopsis thaliana. Expression ofgpr-1is highest in female reproductive structures, and deletion ofgpr-1leads to defects during sexual development. Unfertilized female structures (protoperithecia) fromΔgpr-1strains are weakly pigmented, small, and submerged in the agar. The perithecia produced after fertilization have deformed beaks that lack ostioles, the openings through which ascospores are discharged. Localization studies using a GPR-1-green fluorescent protein fusion protein showed that GPR-1 is targeted to female reproductive structures. Genetic epistasis experiments with the three Gα genes were inconclusive due to the early block in mating exhibited by Δgna-1strains. Phenotypic analysis of mutants from a high-throughputN. crassaknockout project allowed identification of BEK-1, a homeodomain transcription factor that is a potential target of GPR-1. The perithecial defects ofΔbek-1strains are similar to those of theΔgpr-1strain, and epistasis analysis indicates thatbek-1could function downstream ofgpr-1during postfertilization events. The effect must be posttranscriptional, asbek-1transcript levels are not affected inΔgpr-1strains. The lack of ostioles inΔgpr-1and Δbek-1mutants has an undesirable effect on the ability to spread progeny (ascospores) by the normal ejection mechanism and would severely compromise the fitness of these strains in nature.

Cloning and characterization of the mouse polyamine-modulatedfactor-1 (mPMF-1) gene: an alternatively spliced homologue of the human transcription factor

2001 ◽  
Vol 359 (2) ◽  
pp. 387-392 ◽  
Author(s):  
Yanlin WANG ◽  
Wendy DEVEREUX ◽  
Patrick M. WOSTER ◽  
Robert A. CASERO
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Transcriptional Activation ◽  
Splice Variants ◽  
Coiled Coil ◽  
Human Protein ◽  
Related Factor ◽  
Polyamine Analogues ◽  
Coiled Coil Region ◽  
Alternatively Spliced

The natural polyamines and their analogues have been implicated in transcriptional regulation of specific genes. Human polyamine-modulated factor-1 (hPMF-1) was the first polyamine-responsive transcription factor identified. Here the mouse homologue of the hPMF-1 gene is described. Interestingly, the mouse gene (mPMF-1) codes for two alternatively spliced mRNAs. Both of the mouse splice variants, mPMF-1S and mPMF-1L, possess C-terminal coiled-coil domains nearly identical to that found in hPMF-1 and are highly homologous with the human protein. The C-terminal coiled-coil structure is necessary for transcriptional activation. However, the shorter protein, mPMF-1S, does not contain an N-terminal coiled-coil region as do both hPMF-1 and the longer mPMF-1L. mPMF-1L mRNA codes for a protein of 202 amino acids, 37 amino acids longer than the human protein. By contrast, mPMF-1S codes for only 133 amino acids, as a result of two exons being omitted compared with mPMF-1L. Both mouse transcription factors can interact with Nrf-2 (nuclear factor-E2-related factor 2), the normal partner of hPMF-1, substantiating the importance of the C-terminal coiled-coil region responsible for this interaction. Finally, the expression of mPMF-1 is induced when mouse M1 myeloid leukaemia cells are exposed to polyamine analogues, suggesting control similar to that observed for the hPMF-1.

scholarly journals A Subassembly of R27-Encoded Transfer Proteins Is Dependent on TrhC Nucleoside Triphosphate-Binding Motifs for Function but Not Formation

2004 ◽  
Vol 186 (6) ◽  
pp. 1606-1613 ◽  
Author(s):  
Matthew W. Gilmour ◽  
Diane E. Taylor
Keyword(s):  
Fluorescent Protein ◽  
Experimental System ◽  
Nucleoside Triphosphate ◽  
Conjugative Transfer ◽  
Bacterial Cells ◽  
Protein Fusion ◽  
Homologous Proteins ◽  
Focus Formation ◽  
Binding Motifs ◽  
Green Fluorescent

ABSTRACT The transfer of plasmid DNA molecules between bacterial cells is achieved by a large array of conjugative transfer proteins which assemble into both cytoplasmic and membrane-associated complexes. TrhC is a membrane-associated protein that is required for the transfer of the IncHI1 resistance plasmid R27. Homologous proteins are encoded in all known conjugative systems, and each contains characteristic nucleoside triphosphate (NTP)-binding domains. An assembly of R27-encoded proteins was previously visualized by use of a TrhC-green fluorescent protein fusion, which appeared as discrete membrane-associated fluorescent foci. We have utilized this experimental system to determine the requirements for assembly of this TrhC-associated protein complex, and we found that 12 of the other 18 R27 transfer proteins are required for focus formation. An individual focus possibly represents a subassembly comprised of some or all of these transfer proteins. These data support the notion that the transfer apparatus is a multicomponent structure. In contrast, substitutions and deletions within TrhC NTP-binding motifs had minor effects on focus formation, but these mutations did affect plasmid transfer and bacteriophage susceptibility. These results indicate that TrhC requires intact NTP-binding motifs to function during conjugative transfer but that these motifs are not essential for the assembly of TrhC into a complex with other transfer proteins.

scholarly journals FigA, a Putative Homolog of Low-Affinity Calcium System Member Fig1 in Saccharomyces cerevisiae, Is Involved in Growth and Asexual and Sexual Development in Aspergillus nidulans

2013 ◽  
Vol 13 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Shizhu Zhang ◽  
Hailin Zheng ◽  
Nanbiao Long ◽  
Natalia Carbó ◽  
Peiying Chen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Aspergillus Nidulans ◽  
Sexual Development ◽  
Calcium Uptake ◽  
Fluorescent Protein ◽  
Calcium Influx ◽  
Hyphal Growth ◽  
Uptake System ◽  
Content Type ◽  
Green Fluorescent

ABSTRACTCalcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. InSaccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 fromS. cerevisiae, was functionally characterized in the filamentous fungusAspergillus nidulans. Loss offigAresulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACSmidA. Interestingly, in afigAdeletion mutant, adding extracellular Ca2+rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed thatfigAdeletion sharply decreased the expression ofbrlAandnsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system inA. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development.

scholarly journals Saccharomyces cerevisiae Aqr1 Is an Internal-Membrane Transporter Involved in Excretion of Amino Acids

2004 ◽  
Vol 3 (6) ◽  
pp. 1492-1503 ◽  
Author(s):  
Isabel Velasco ◽  
Sandra Tenreiro ◽  
Isabel L. Calderon ◽  
Bruno André
Keyword(s):  
Amino Acids ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Negative Control ◽  
Yeast Cells ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Membrane Structures ◽  
Internal Membrane ◽  
Green Fluorescent

ABSTRACT Excretion of amino acids by yeast cells was reported long ago but has not been characterized in molecular terms. It is typically favored by overproduction of the amino acid and/or impairment of its uptake. Here we describe the construction of a yeast strain excreting threonine and homoserine. Using this excretor strain, we then applied a reverse-genetics approach and found that the transporter encoded by the YNL065w/AQR1 gene, a protein thought to mediate H+ antiport, is involved in homoserine and threonine excretion. Furthermore, overexpression of AQR1 led to increased excretion of several amino acids (alanine, aspartate, and glutamate) known to be relatively abundant in the cytosol. Transcription of the AQR1 gene is induced severalfold by a number of amino acids and appears to be under the negative control of Gcn4. An Aqr1-green fluorescent protein fusion protein is located in multiple internal membrane structures and appears to cycle continuously between these compartments and the plasma membrane. The Aqr1 sequence is significantly similar to the vesicular amine transporters of secretory vesicles of neuronal cells. We propose that Aqr1 catalyzes transport of excess amino acids into vesicles, which then release them in the extracellular space by exocytosis.

Two zinc finger transcription factors, CrzA and SltA, are involved in cation homoeostasis and detoxification in Aspergillus nidulans

2008 ◽  
Vol 414 (3) ◽  
pp. 419-429 ◽  
Author(s):  
Anja Spielvogel ◽  
Helen Findon ◽  
Herbert N. Arst ◽  
Lidia Araújo-Bazán ◽  
Patricia Hernández-Ortíz ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Transcription Factor ◽  
Aspergillus Nidulans ◽  
Fluorescent Protein ◽  
Alkaline Ph ◽  
Gene Encoding ◽  
Extreme Sensitivity ◽  
Green Fluorescent ◽  
P Type ◽  
Encoding Genes

To investigate cation adaptation and homoeostasis in Aspergillus nidulans, two transcription-factor-encoding genes have been characterized. The A. nidulans orthologue crzA of the Saccharomyces cerevisiae CRZ1 gene, encoding a transcription factor mediating gene regulation by Ca2+, has been identified and deleted. The crzA deletion phenotype includes extreme sensitivity to alkaline pH, Ca2+ toxicity and aberrant morphology connected with alterations of cell-wall-related phenotypes such as reduced expression of a chitin synthase gene, chsB. A fully functional C-terminally GFP (green fluorescent protein)-tagged form of the CrzA protein is apparently excluded from nuclei in the absence of added Ca2+, but rapidly accumulates in nuclei upon exposure to Ca2+. In addition, the previously identified sltA gene, which has no identifiable homologues in yeasts, was deleted, and the resulting phenotype includes considerably enhanced toxicity by a number of cations other than Ca2+ and also by alkaline pH. Reduced expression of a homologue of the S. cerevisiae P-type ATPase Na+ pump gene ENA1 might partly explain the cation sensitivity of sltA-null strains. Up-regulation of the homologue of the S. cerevisiae vacuolar Ca2+/H+ exchanger gene VCX1 might explain the lack of Ca2+ toxicity to null-sltA mutants, whereas down-regulation of this gene might be responsible for Ca2+ toxicity to crzA-null mutants. Both crzA and sltA encode DNA-binding proteins, and the latter exerts both positive and negative gene regulation.

scholarly journals Amino Acids Control Mammalian Gene Transcription: Activating Transcription Factor 2 Is Essential for the Amino Acid Responsiveness of the CHOP Promoter

2000 ◽  
Vol 20 (19) ◽  
pp. 7192-7204 ◽  
Author(s):  
Alain Bruhat ◽  
Céline Jousse ◽  
Valérie Carraro ◽  
Andreas M. Reimold ◽  
Marc Ferrara ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Transcription Factor ◽  
Amino Acid ◽  
Transcriptional Activation ◽  
Dominant Negative Mutant ◽  
Transient Expression Assay ◽  
Control Of Gene Expression ◽  
Mammalian Gene ◽  
Activating Transcription Factor

ABSTRACT In mammals, plasma concentration of amino acids is affected by nutritional or pathological conditions. It has been well established that nutrients, and particularly amino acids, are involved in the control of gene expression. Here we examined the molecular mechanisms involved in the regulation ofCHOP (a CCAAT/enhancer-binding protein [C/EBP]-related gene) expression upon amino acid limitation. We have previously shown that regulation of CHOP mRNA expression by amino acid concentration has both transcriptional and posttranscriptional components. We report the analysis ofcis- and trans-acting elements involved in the transcriptional activation of the human CHOPgene by leucine starvation. Using a transient expression assay, we show that a cis-positive element is essential for amino acid regulation of the CHOP promoter. This sequence is the first described that can regulate a basal promoter in response to starvation for several individual amino acids and therefore can be called an amino acid response element (AARE). In addition, we show that the CHOP AARE is related to C/EBP and ATF/CRE binding sites and binds in vitro the activating transcription factor 2 (ATF-2) in starved and unstarved conditions. Using ATF-2-deficient mouse embryonic fibroblasts and an ATF-2-dominant negative mutant, we demonstrate that expression of this transcription factor is essential for the transcriptional activation of CHOP by leucine starvation. Altogether, these results suggest that ATF-2 may be a member of a cascade of molecular events by which the cellular concentration of amino acids can regulate mammalian gene expression.

The “Pro” Sequence of the Sporulation-Specific ς Transcription Factor ςE Directs It to the Mother Cell Side of the Sporulation Septum

1999 ◽  
Vol 181 (19) ◽  
pp. 6171-6175 ◽  
Author(s):  
Jingliang Ju ◽  
W. G. Haldenwang
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Mother Cell ◽  
Fluorescent Protein ◽  
Amino Terminus ◽  
Cell Compartment ◽  
Intracellular Location ◽  
Specific Protease ◽  
Green Fluorescent

ABSTRACT ςE, a mother cell-specific transcription factor of sporulating Bacillus subtilis, is derived from an inactive precursor protein (pro-ςE). Activation of ςE occurs when a sporulation-specific protease (SpoIIGA) cleaves 27 amino acids from the pro-ςE amino terminus. This reaction is believed to take place at the mother cell-forespore septum. Using a chimera of pro-ςE and green fluorescent protein (GFP) to visualize the intracellular location of pro-ςE by fluorescence microscopy, and lysozyme treatment to separate the mother cell and forespore compartments, we determined that the pro-ςE::GFP signal, localized to the forespore septum prior to lysozyme treatment, is restricted to the mother cell compartment after treatment. Thus, pro-ςE::GFP had been sequestered to the mother cell side of the septum. This segregation of pro-ςE::GFP, and presumably pro-ςE, to the mother cell is likely to be the reason why ςE activity is restricted to that compartment.

Differentiation-Dependent Interactions Between Runx-1 and Fli-1 during Megakaryocyte Development

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 279-279
Author(s):  
Hui Huang ◽  
Ming Yu ◽  
Tyler B Moran ◽  
Nathan Tu ◽  
Thomas E Akie ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Gel Filtration ◽  
Cell Types ◽  
Myelogenous Leukemia ◽  
Activation Domains ◽  
Platelet Disorder ◽  
Familial Platelet Disorder

Abstract The transcription factor Runx-1 is required for the ontogeny of all definitive hematopoiesis, and plays a specific role in megakaryopoiesis during later stages of development. Germline mutations in Runx-1 cause Familial Platelet Disorder with Propensity to Develop AML (FPD/AML), and acquired mutations occur in a subset of patients with myelodysplastic syndrome (MDS) and acute myelogenous leukemia. Although many of the reported Runx-1 mutations affect DNA binding and/or its interaction with the cofactor CBF-beta, other mutations occur outside of these binding regions and have unknown mechanistic effects. In this study, we purified Runx-1 containing multiprotein complexes from murine megakaryocytic cells in order to identify potential novel Runx-1 associated factors whose interaction may be altered by Runx-1 mutations. Here we report the identification of the key megakaryocyte ets transcription factor Fli- 1 as a direct Runx-1 binding partner. This interaction involves the negative regulatory DNA binding and activation domains of Runx-1 (amino acids 179–370), and a region around the Ets DNA binding region of Fli-1 (amino acids 281–361). The interaction is lost in the MDS- associated Y254X Runx-1 mutation. We also show that Runx-1 and Fli-1 co-occupy the c-mpl promoter in primary megakaryocytes and act synergistically in transcriptional reporter assays. Interestingly, the interaction between Runx-1 and Fli- 1 occurs in murine L8057 megakaryoblastic cells only after they have been induced to differentiate, even though both proteins are expressed abundantly in uninduced cells. The interaction correlates with assembly of a large multiprotein complex that also includes the key megakaryocyte transcription factor GATA-1 and its cofactor Friend of GATA-1 (FOG- 1) based on gel filtration chromatography experiments. Furthermore, we show that Fli-1 from this large complex lacks phosphorylation of a specific residue that is phosphorylated on non-complexed Fli-1. Mutation of this site to aspartic acid, which mimics constitutive phosphorylation, disrupts the interaction between Fli-1 and Runx-1 and abrogates their transcriptional synergy. We propose that dephosphorylation of Fli-1 is a key event in the transcriptional activation of megakaryocyte terminal maturation by facilitating the assembly of a RUNX-1/FLI-1/GATA-1/FOG-1 enhancesome complex. These findings have implications for the differentiation of other cell types where interactions between Runx and ets family proteins occur.

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