ABSTRACT
A system for the regulation of gene expression by ambient (extracellular) pH was first identified in Aspergillus nidulans. This system consists of the products of the pacC and palA, B, C, F, H, and I genes. pacC encodes a zinc finger transcription factor and these pal genes encode components of an ambient pH signal transduction pathway. pH regulatory systems have also been identified in other fungi. Components of these regulatory systems are homologous to those in A. nidulans. This review describes the pH regulatory system in A. nidulans and the history of this research and how it relates to other systems. pH regulation in bacteria and animal cells is also briefly discussed.
Subject(s)
Aspergillus nidulans/genetics , Gene Expression Regulation, Fungal , Animals , Aspergillus nidulans/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Genes, Fungal/physiology , Hydrogen-Ion Concentration , Phosphoric Monoester Hydrolases/metabolism , Saccharomyces cerevisiae/geneticsABSTRACT
In response to alkaline ambient pH, the Aspergillus nidulans PacC transcription factor mediating pH regulation of gene expression is activated by proteolytic removal of a negative-acting C-terminal domain. We demonstrate interactions involving the approximately 150 C-terminal PacC residues and two regions located immediately downstream of the DNA binding domain. Our data indicate two full-length PacC conformations whose relative amounts depend upon ambient pH: one 'open' and accessible for processing, the other 'closed' and inaccessible. The location of essential determinants for proteolytic processing within the two more upstream interacting regions probably explains why the interactions prevent processing, whereas the direct involvement of the C-terminal region in processing-preventing interactions explains why C-terminal truncating mutations result in alkalinity mimicry and pH-independent processing. A mutant PacC deficient in pH signal response and consequent processing behaves as though locked in the 'closed' form. Single-residue substitutions, obtained as mutations bypassing the need for pH signal transduction, identify crucial residues in each of the three interactive regions and overcome the processing deficiency in the 'permanently closed' mutant.
Subject(s)
Fungal Proteins , Transcription Factors/metabolism , Amino Acid Sequence , Amino Acid Substitution , Aspergillus nidulans/genetics , Aspergillus nidulans/metabolism , Binding Sites/genetics , Endopeptidases/metabolism , Gene Expression Regulation , Genes, Fungal , Hydrogen-Ion Concentration , Models, Biological , Molecular Sequence Data , Point Mutation , Protein Conformation , Protein Processing, Post-Translational , Protein Structure, Tertiary , Sequence Deletion , Signal Transduction , Transcription Factors/chemistry , Transcription Factors/genetics , Two-Hybrid System Techniques , Zinc Fingers/geneticsABSTRACT
Completing the molecular analysis of the six pal genes of the ambient pH signal transduction pathway in Aspergillus nidulans, we report the characterization of palC and palH. The derived translation product of palH contains 760 amino acids with prediction of seven transmembrane domains in its N-terminal moiety. Remarkably, a palH frameshift mutant lacking just over half the PalH protein, including almost all of the long hydrophilic region C-terminal to the transmembrane domains, retains some PalH function. The palC-derived translation product contains 507 amino acids, and the null phenotype of a frameshift mutation indicates that at least one of the C-terminal 142 residues is essential for function. Uniquely among the A. nidulans pH-signalling pal genes, palC appears to have no Saccharomyces cerevisiae homologue, although it does have a Neurospora crassa expressed sequence tag homologue. In agreement with findings for the palA, palB and palI genes of this signalling pathway, levels of the palC and palH mRNAs do not appear to be pH regulated.
Subject(s)
Aspergillus nidulans/genetics , Aspergillus nidulans/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Signal Transduction , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Frameshift Mutation , Hydrogen-Ion Concentration , Molecular Sequence Data , Sequence Homology, Amino Acid , Transcription, GeneticABSTRACT
The zinc finger regions of the Aspergillus nidulans PacC transcription factor, mediating regulation of gene expression by ambient pH, and the Saccharomyces cerevisiae Rim1p transcription factor, mediating control of meiosis and invasiveness, are homologous and both transcription factors undergo proteolytic processing of the C-terminus for conversion to the functional form. In both cases, functioning of a signal transduction pathway involving several gene products is a necessary prerequisite for processing. We now show that the Aspergillus PalI pH signal transduction component is homologous to the Saccharomyces Rim9p meiotic signal transduction component throughout a region containing four hydrophobic, putative membrane-spanning segments. This suggests that PalI might be a membrane sensor for ambient pH. Deletion of the palI gene established that the less extreme phenotype of palI mutations compared with mutations in the other five genes of the pH signalling pathway is a general feature of palI mutations.
Subject(s)
Aspergillus nidulans/genetics , Fungal Proteins/genetics , Fungal Proteins/metabolism , Meiosis , Membrane Proteins , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Signal Transduction , Amino Acid Sequence , Aspergillus nidulans/metabolism , Base Sequence , Blotting, Northern , Cloning, Molecular , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Deoxyribonucleases, Type II Site-Specific/genetics , Deoxyribonucleases, Type II Site-Specific/metabolism , Hydrogen-Ion Concentration , Molecular Sequence Data , Mutation , Repressor Proteins , Saccharomyces cerevisiae/metabolism , Sequence Homology, Amino Acid , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
We have cloned the palA gene of Aspergillus nidulans, one of six genes participating in ambient pH signal transduction in a regulatory circuit mediating pH regulation of gene expression. The derived 798-residue PalA protein is 29.4% identical over its entire length to a hypothetical protein from the nematode Caenorhabditis elegans and also has possible yeast homologs.
Subject(s)
Aspergillus nidulans/genetics , Fungal Proteins/genetics , Signal Transduction/genetics , Amino Acid Sequence , Aspergillus nidulans/growth & development , Aspergillus nidulans/metabolism , Base Sequence , Cloning, Molecular , Fungal Proteins/chemistry , Fungal Proteins/physiology , Gene Expression Regulation, Fungal , Genes, Fungal , Hydrogen-Ion Concentration , Molecular Sequence Data , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence AlignmentABSTRACT
In Aspergillus nidulans, the regulation of gene expression in response to changes in ambient pH is mediated by the PacC zinc finger transcriptional regulator. At alkaline ambient pH, PacC is proteolytically processed to a functional form serving as an activator of alkaline-expressed genes and a repressor of acid-expressed genes. This activation of PacC occurs in response to a signal mediated by the products of the pal genes. Thus, the products of the palA, -B, -C, -F, -H, and -I genes constitute an alkaline ambient pH signal transduction pathway. How the pal signal transduction pathway senses ambient pH and transduces a signal to trigger PacC processing is a fascinating unresolved problem. We have cloned and sequenced the palB gene. The predicted palB gene product has similarity to the catalytic domain of the calpain family of calcium-activated cysteine proteases. We have shown, however, that the PalB protein does not catalyze the final step of proteolytic processing of PacC.
Subject(s)
Aspergillus nidulans/metabolism , Cysteine Endopeptidases/metabolism , Fungal Proteins , Signal Transduction , Amino Acid Sequence , Animals , Aspergillus nidulans/enzymology , Base Sequence , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , Humans , Hydrogen-Ion Concentration , Hydrolysis , Molecular Sequence Data , Mutation , Sequence Homology, Amino AcidABSTRACT
We have examined the consequences of treatment with DNA-damaging agents of uvs mutants and the bimD6 mutant of Aspergillus nidulans. We first established that wild-type Aspergillus undergoes a cell cycle delay following treatment with the DNA-damaging agents methyl methanesulfonate (MMS) or ultraviolet light (UV). We have also determined that strains carrying the bimD6, uvsB110, uvsH77, uvsF201 and the uvsC114 mutations, all of which cause an increased sensitivity to DNA-damaging agents, undergo a cell-cycle delay following DNA damage. These mutations therefore do not represent nonfunctional checkpoints in Aspergillus. However, all of the mutant strains accumulated nuclear defects after a period of delay following mutagen treatment. The nuclear defects in the uvsB110 and bimD6 strains following MMS treatment were shown to be dependent on passage through mitosis after DNA damage, as the defects were prevented with benomyl. Checkpoint controls responding to DNA damage thus only temporarily halt cell-cycle progression in response to DNA damage. The conditional bimD6 mutation also results in a defective mitosis at restrictive temperatures. This mitotic defect is similar to that seen with MMS treatment at temperatures permissive for the mitotic defect. Thus the bimD gene product may perform dual roles, one in DNA repair and the other during the mitotic cell cycle in the absence of damage.
Subject(s)
Aspergillus nidulans/genetics , Cell Cycle Proteins , DNA Damage , Fungal Proteins/physiology , Genes, Fungal/genetics , Mitosis/physiology , Aspergillus nidulans/drug effects , Aspergillus nidulans/radiation effects , Cell Cycle/physiology , Cell Death , DNA Repair/genetics , Drug Tolerance , Fungal Proteins/genetics , Genes, Fungal/physiology , Methyl Methanesulfonate/pharmacology , Mutation , Radiation Tolerance , Time Factors , Ultraviolet RaysABSTRACT
Mutation in the bimD gene of Aspergillus nidulans results in a mitotic block in anaphase characterized by a defective mitosis. Mutation in bimD also confers, at temperatures permissive for the mitotic arrest phenotype, an increased sensitivity to DNA damaging agents, including methyl methanesulfonate and ultraviolet light. In order to better understand the relationship between DNA damage and mitotic progression, we cloned the bimD gene from Aspergillus. A cosmid containing the bimD gene was identified among pools of cosmids by cotransformation with the nutritional selective pyrG gene of a strain carrying the recessive, temperature-sensitive lethal bimD6 mutation. The bimD gene encodes a predicted polypeptide of 166,000 daltons in mass and contains amino acid sequence motifs similar to those found in some DNA-binding transcription factors. These sequences include a basic domain followed by a leucine zipper, which together are called a bZIP motif, and a carboxyl-terminal domain enriched in acidic amino acids. Overexpression of the wild-type bimD protein resulted in an arrest of the nuclear division cycle that was reversible and determined to be in either the G1 or S phase of the cell cycle. Our data suggest that bimD may play an essential regulatory role relating to DNA metabolism which is required for a successful mitosis.
Subject(s)
Aspergillus nidulans/genetics , Cell Cycle Proteins , DNA Damage , Fungal Proteins/genetics , Genes, Fungal , Mutation , Amino Acid Sequence , Aspergillus nidulans/drug effects , Base Sequence , Cloning, Molecular , DNA, Fungal/drug effects , Fungal Proteins/biosynthesis , Methyl Methanesulfonate/pharmacology , Mitosis/genetics , Molecular Sequence Data , Mutagens/pharmacology , Restriction Mapping , Sequence Homology, Amino Acid , Ultraviolet RaysABSTRACT
A conditionally lethal mutation in the bimB gene of Aspergillus nidulans disrupts the normal regulatory patterns associated with mitotic events. This results in DNA replication in the absence of the completion of mitosis in the mutant at restrictive temperature. This defect yields large polyploid nuclei after several hours at restrictive temperature. The bimB gene has been cloned by genetic mapping and chromosome walking from the previously cloned amdS gene. The cloned DNA complements the temperature-sensitive recessive bimB3 mutation. Sequence analysis of overlapping complementary DNA clones for bimB predicts a polypeptide of 2,068 amino acids. The predicted polypeptide of 227,958 Da is shown to have a carboxyl-terminal region similar to those of the budding yeast ESP1 and fission yeast cut1+ genes. In contrast these genes exhibit no other regions of similarity to one another. The conserved domain in these three proteins and the similarity of the terminal mutant phenotypes for these genes are suggestive of a conserved function for this domain in each of the predicted polypeptides. We also present evidence for a second gene in the genome of A. nidulans which also has this conserved carboxyl-terminal region, suggesting that bimB, ESP1, and cut1+ may be members of a small gene family.
Subject(s)
Aspergillus nidulans/genetics , DNA Replication/genetics , Gene Expression Regulation, Bacterial , Genes, Bacterial , Mitosis , Mutation , Amino Acid Sequence , Aspergillus nidulans/cytology , Aspergillus nidulans/growth & development , Base Sequence , Benomyl/pharmacology , Cell Nucleus/drug effects , Cell Nucleus/physiology , Cell Nucleus/ultrastructure , Cloning, Molecular , Fluorescent Dyes , Indoles , Microscopy, Fluorescence , Molecular Sequence Data , Phenotype , Sequence Homology, Nucleic Acid , Temperature , Tubulin/analysisABSTRACT
The core histone gene family of Aspergillus nidulans was characterized. The H2A, H2B and H3 genes are unique in the A. nidulans genome. In contrast there are two H4 genes, H4.1 and H4.2. As previously reported for the H2A gene (May and Morris 1987) introns also interrupt the other core histone genes. The H2B gene, like the H2A gene, is interrupted by three introns, the H3 and H4.1 gene are each interrupted by two introns and the H4.2 gene contains one intron. The position of the single intron in H4.2 is the same as that the first intron of the H4.1 gene. The H2A and H2B genes are arranged as a gene pair separated by approximately 600 bp and are divergently transcribed. The H3 and H4.1 genes are similarly arranged and are separated by approximately 800 bp. The H4.2 gene is not closely linked to either the H2A-H2B or H3-H4.1 gene pairs. Using pulse field gel electrophoresis an electrophoretic karyotype was established for A. nidulans. This karyotype was used to assign the H3-H4.1 gene pair and the H4.2 gene to linkage group VIII and the H2A-H2B gene pair to either linkage group III or VI. The abundance of each of the histone messenger RNAs was determined to be cell cycle regulated but the abundance of the H4.2 mRNA appears to be regulated differently from the others.
Subject(s)
Aspergillus nidulans/genetics , Genes, Fungal , Histones/genetics , Amino Acid Sequence , Base Sequence , Blotting, Southern , DNA, Fungal/genetics , DNA, Fungal/isolation & purification , Fungal Proteins/biosynthesis , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Genetic Linkage , Histones/biosynthesis , Introns , Karyotyping , Molecular Sequence Data , Multigene Family , Restriction Mapping , Sequence Homology, Nucleic Acid , Transcription, GeneticABSTRACT
A glucocorticoid response element of the tyrosine aminotransferase gene was demonstrated to mediate effects of androgen when located upstream of a heterologous promoter linked to the chloramphenicol acetyl transferase gene (CAT).