Genetic regulation of nitrogen metabolism in the fungi.

In the fungi, nitrogen metabolism is controlled by a complex genetic regulatory circuit which ensures the preferential use of primary nitrogen sources and also confers the ability to use many different secondary nitrogen sources when appropriate. Most structural genes encoding nitrogen catabolic enzymes are subject to nitrogen catabolite repression, mediated by positive-acting transcription factors of the GATA family of proteins. However, certain GATA family members, such as the yeast DAL80 factor, act negatively to repress gene expression. Selective expression of the genes which encode enzymes for the metabolism of secondary nitrogen sources is often achieved by induction, mediated by pathway-specific factors, many of which have a GAL4-like C6/Zn2 DNA binding domain. Regulation within the nitrogen circuit also involves specific protein-protein interactions, as exemplified by the specific binding of the negative-acting NMR protein with the positive-acting NIT2 protein of Neurospora crassa. Nitrogen metabolic regulation appears to play a significant role in the pathogenicity of certain animal and plant fungal pathogens.

Molecular cloning and analysis of the regulation of cys-14+, a structural gene of the sulfur regulatory circuit of Neurospora crassa.

The cys-14+ gene encodes sulfate permease II, which is primarily expressed in mycelia. cys-14+ is one of a set of sulfur-related structural genes under the control of cys-3+ and scon+, the regulatory genes of the sulfur control circuit. We have cloned cys-14+ from a cosmid library of Neurospora crassa DNA. A restriction fragment length polymorphism analysis showed that this clone maps to the region of chromosome IV corresponding to the cys-14+ locus. Northern blot analyses were used to examine the regulated expression of the cys-14+ gene. In the wild type, a 3-kilobase cys-14+ transcript was highly expressed under sulfur-derepressing conditions but completely absent during sulfur repression. A cys-3 mutant, which cannot synthesize any of the sulfur-controlled enzymes, including sulfate permease II, did not possess any cys-14+ transcript under either condition. A cys-3 temperature-sensitive revertant completely lacked any cys-14+ mRNA at the conditional temperature but expressed the cys-14+ transcript upon derepression at the permissive temperature. Mutation of a second sulfur regulatory gene, scon(c), causes the expression of sulfur-related enzymes in a constitutive fashion; the scon(c) mutant showed a corresponding constitutive expression of cys-14+ mRNA, such that it was present even in cells subjected to sulfur repression conditions. These results show that the cys-14+ gene is regulated through the modulation of message content by the cys-3+ and scon(c) control genes in response to the sulfur levels of the cells.

nit-2, the major nitrogen regulatory gene of Neurospora crassa, encodes a protein with a putative zinc finger DNA-binding domain.

The nitrogen regulatory circuit of Neurospora crassa consists of a set of unlinked structural genes which specify various nitrogen catabolic enzymes plus control genes and metabolic effectors which regulate their expression. The positive-acting nit-2 regulatory gene is required to turn on the expression of the nitrogen catabolic enzymes during conditions of nitrogen limitation. The complete nucleotide sequence of the nit-2 gene was determined. The nit-2 mRNA is 4.3 kilobases long and has a long nontranslated sequence at both its 5' and 3' ends. The nit-2 gene nucleotide sequence can be translated to yield a protein containing 1,036 amino acid residues with a molecular weight of approximately 110,000. Deletion analyses demonstrated that approximately 21% of the NIT2 protein at its carboxy terminus can be removed without loss of function. The nit-2 protein contains a single putative Cys2/Cys2 zinc finger domain which appears to function in DNA binding and which has striking homology to a mammalian trans-acting factor, GF-1.

Characterization of nit-2, the major nitrogen regulatory gene of Neurospora crassa.

The nit-2 gene is the major nitrogen-regulatory gene of Neurospora crassa, and under conditions of nitrogen limitations, it turns on the expression of various unlinked structural genes which specify nitrogen-catabolic enzymes. The nit-2 gene was subcloned as a 6-kilobase (kb) DNA fragment from a cosmid that carried approximately a 40-kb N. crassa DNA insert. The nit-2 gene was localized in a DNA segment of approximately 3.5 kb and was shown to correspond to a unique DNA sequence located on linkage group 1. Several N. crassa nit-2 transformants were characterized and were found to possess significantly different levels of the regulated enzyme nitrate reductase. Northern blot analysis of RNA from various strains was carried out to determine whether the nit-2 gene was expressed constitutively or was itself subject to regulation. The results revealed that the nit-2 gene is transcribed to give a single large mRNA of approximately 3.5 kb. Expression of the nit-2 gene is regulated such that its transcript is present at a substantially higher level in cells which are limited for nitrogen than in cells growing under nitrogen-repressed conditions. However, the nit-2 gene is not controlled by autogenous regulation. The nit-2 gene was transcribed only at a low level in nmr-1 and in gln-1b, under both nitrogen-repressed and derepressed conditions, suggesting that these unlinked loci may exert a positive regulatory effect on nit-2.

Interaction between major nitrogen regulatory protein NIT2 and pathway-specific regulatory factor NIT4 is required for their synergistic activation of gene expression in Neurospora crassa.

In Neurospora crassa, the major nitrogen regulatory protein, NIT2, a member of the GATA family of transcription factors, controls positively the expression of numerous genes which specify nitrogen catabolic enzymes. Expression of the highly regulated structural gene nit-3, which encodes nitrate reductase, is dependent upon a synergistic interaction of NIT2 with a pathway-specific control protein, NIT4, a member of the GAL4 family of fungal regulatory factors. The NIT2 and NIT4 proteins both bind at specific recognition elements in the nit-3 promoter, but, in addition, we show that a direct protein-protein interaction between NIT2 and NIT4 is essential for optimal expression of the nit-3 structural gene. Neurospora possesses at least five different GATA factors which control different areas of cellular function, but which have a similar DNA binding specificity. Significantly, only NIT2, of the several Neurospora GATA factors examined, interacts with NIT4. We propose that protein-protein interactions of the individual GATA factors with additional pathway-specific regulatory factors determine each of their specific regulatory functions.

Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa.

cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation. The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain. The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity. The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene. A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity. The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.

Gene disruption by transformation in Neurospora crassa.

To establish conditions which might permit deliberate gene disruptions in Neurospora crassa, we studied transformation with linear DNA fragments. The transformation frequency observed was increased about twofold in comparison with that obtained with circular plasmid DNA. However, only a low proportion, approximately 10%, of the integration events occurred at the homologous site, whereas most integrations of transforming DNA took place in nonhomologous regions. It was also found that multiple integration events frequently occurred in individual transformants. A plasmid, designated pJP12, was constructed that contains the N. crassa am+ gene interrupted by insertion into its coding region of a DNA segment carrying a functional Neurospora qa-2+ gene. A fragment of Neurospora DNA that contains this am qa-2+ construction was obtained from plasmid pJP12 and used to transform an am+ qa-2 strain in an attempt to disrupt the resident am+ gene. After the initial qa-2+ transformants were converted to homokaryons by appropriate crosses, 10 independent transformants with an am mutant phenotype were found among 117 examined. Each of these qa-2+ am transformants showed the loss of a hybridization band in Southern blots of genomic DNA that corresponded to the normal am+ gene and the presence of a new hybridization band, consistent with an alteration in the am+ region.

nit-4, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-binding domain.

nit-4, a pathway-specific regulatory gene in the nitrogen circuit of Neurospora crassa, is required for the expression of nit-3 and nit-6, the structural genes which encode nitrate and nitrite reductase, respectively. The complete nucleotide sequence of the nit-4 gene has been determined. The predicted NIT4 protein contains 1,090 amino acids and appears to possess a single Zn(II)2Cys6 binuclear-type zinc finger, which may mediate DNA binding. Site-directed mutagenesis studies demonstrated that cysteine and other conserved amino acid residues in this possible DNA-binding domain are necessary for nit-4 function. A stretch of 27 glutamines, encoded by a CAGCAA repeating sequence, occurs in the C terminus of the NIT4 protein, and a second glutamine-rich domain occurs further upstream. A NIT4 protein deleted for the polyglutamine region was still functional in vivo. However, nit-4 function was abolished when both the polyglutamine region and the glutamine-rich domain were deleted, suggesting that the glutamine-rich domain might function in transcriptional activation. The homologous regulatory gene from Aspergillus nidulans, nirA, encodes a protein whose amino-terminal half has approximately 60% amino acid identity with NIT4 but whose carboxy terminus is completely different. A hybrid nit-4-nirA gene was constructed and found to function in N. crassa.

cys-3, the positive-acting sulfur regulatory gene of Neurospora crassa, encodes a protein with a putative leucine zipper DNA-binding element.

The sulfur-regulatory circuit of Neurospora crassa consists of a set of unlinked structural genes which encode sulfur-catabolic enzymes and two major regulatory genes which govern their expression. The positive-acting cys-3 regulatory gene is required to turn on the expression of the sulfur-related enzymes, whereas the other regulatory gene, scon, acts in a negative fashion to repress the synthesis of the same set of enzymes. Expression of the cys-3 regulatory gene was found to be controlled by scon and by sulfur availability. The nucleotide sequence of the cys-3 gene was determined and can be translated to yield a protein of molecular weight 25,892 which displays significant homology with the oncogene protein Fos, yeast GCN4 protein, and sea urchin histone H1. Moreover, the putative cys-3 protein has a well-defined leucine zipper element plus an adjacent charged region which together may make up a DNA-binding site. A cys-3 mutant and a cys-3 temperature-sensitive mutant lead to substitutions of glutamine for basic amino acids within the charged region and thus may alter DNA-binding properties of the cys-3 protein.

Molecular cloning and characterization of the cys-3 regulatory gene of Neurospora crassa.

The regulatory gene cys-3+ controls the synthesis of a number of enzymes involved in sulfur metabolism. cys-3 mutants show a multiple loss of enzymes in different pathways of sulfur metabolism. The cys-3+ gene was isolated by transformation of an aro-9 qa-2 cys-3 inl strain with a clone bank followed by screening with the "sib selection" method. The library used (pRAL1) contained inserts of Sau3a partial digest fragments of about 9 kilobases as well as the Neurospora qa-2+ gene. Double selection for qa-2+ and cys-3+ function was carried out. The transformants obtained with the isolated cys-3+ clone show recovery of the enzyme activities associated with the cys-3 mutation (e.g., arylsulfatase and sulfate permease). Restriction fragment length polymorphism experiments confirmed the identity of the clone, mRNA studies with Northern blots show that the expression of the cys-3+ gene is inducible. In contrast to cys-3+, the cys-3 (P22) mutant gene was not expressed at a higher level under sulfur-derepressed conditions.

Sequence complexity and abundance classes of nuclear and polysomal polyadenylated RNA in Neurospora crassa.

The sequence complexity of nuclear and polysomal polyadenylated RNA in Neurospora crassa has been investigated by an analysis of the kinetics of RNA : cDNA hybridizations. There are about 2000 different messenger RNAs organized into three abundance classes of low, medium and high complexity which contain approx. 10, 150 and 1800 sequences, respectively. Taking 1300 nucleotides as the average length of mRNA, the total sequence complexity of polyadenylated polysomal RNA was calculated to represent 2.4 . 10(6) nucleotide pairs, which is 9% of the genome. Hybridization of polysomal polyadenylated RNA with nuclear DNA yielded results in good agreement and revealed that about 12% of the genome was transcribed into mRNA. Analysis of RNA : cDNA hybridizations with nuclear polyadenylated RNA gave results similar to that observed with polysomal RNA, and indicated that nuclear RNAs were also present in discrete abundance classes. Cross-hybridization experiments showed that all mRNA sequences are present in nuclear RNA, and that the sequence complexity detected in polysomal and nuclear polyadenylated RNA is identical or very similar. In total, approx. 15 to 20% of the Neurospora genome is transcribed into various RNA species, including messenger and ribosomal RNA, in cells growing vegetatively on minimal medium.

Intracellular processing and toxicity of the truncated androgen receptor: nuclear congophilia-associated cell death.

The pathogenesis of the selective motor neuron death in spinal bulbar muscular atrophy (SBMA) is not fully understood. Similar to observations with other mutant polyglutamine (poly Q) expanded proteins, truncated androgen receptor (AR) with expanded poly Q tract cause intracellular aggregates; however, the precise relationship between aggregates and disease pathogenesis is unresolved. In order to have a better understanding of the cellular processing and toxicity of the mutant AR, we focused on a short N-terminal portion of AR containing normal or expanded poly Q repeats, and have carried out biochemical, immunocytochemical, cytochemical and ultrastructural studies of BHK cells at different intervals after transfection. In cells expressing mutant truncated AR, using an anti-AR N-terminal antibody, we observed no immune staining in the nucleus and identified immune negative aggregates surrounded by immunopositive material in the cytoplasm. Congo red staining identified a component of aggregates with a beta-pleated secondary structure in both cytosol and nucleus, while electron microscopy revealed a fibrillary-granular material as the ultrastructural correlate. In addition, acid phosphatase staining and ubiquitin immunocytochemistry demonstrated that in transfected cells, both lysosomal and nonlysosomal degradation systems are actively involved in handling the mutant truncated AR. The temporal relationship of nuclear congophilia to a subsequent massive cell death suggests that entry of proteolytic cleavage products into the nucleus, perhaps the expanded poly Q stretch itself, may play an important role in cell toxicity.

Heterologous expression and regulation of the Neurospora crassa nit-4 pathway-specific regulatory gene for nitrate assimilation in Aspergillus nidulans.

The nirA gene of Aspergillus nidulans and the nit-4 gene of Neurospora crassa appear to be equivalent pathway-specific regulatory genes which mediate nitrate induction of nitrate reductase and nitrite reductase (NR and NiR) activities. We have transformed the nit-4 wild-type (wt) gene into the A. nidulans loss-of-function (pleiotropic negative) nirA 1 mutant strain. The nit-4 gene was found to complement the nirA 1 mutation, thus permitting the nirA 1 mutant strain to grow on nitrate or nitrite as the sole source of nitrogen. Integration of the nit-4 gene in transformants appears to have occurred at a number of 'ectopic', i.e. non-nirA, sites. Nitrate is required for the induction of NR activity in nit-4-transformed strains whilst NR production remains markedly subject to nitrogen-metabolite repression. However, NR levels are modestly higher than wt under all growth conditions.

The leu-1 gene of Neurospora crassa: nucleotide and deduced amino acid sequence comparisons.

The Neurospora crassa leu-1 gene encodes beta-isopropylmalate dehydrogenase (IPMDH; EC 1.1.1.85), an enzyme in the leucine biosynthetic pathway. We determined the nucleotide sequence of the entire leu-1 gene and of four independent cDNA clones. By comparing the genomic and cDNA sequences, four introns were identified in the 5' portion of the gene and a single open reading frame was established. One of the introns is located within the 5'-noncoding region of the transcript. The deduced amino acid sequence encoded by leu-1 was aligned with that of the homologous yeast enzyme and extensive sequence identity was uncovered. The lesion present in a conventional leu-1 mutant was identified as the insertion of a single base pair.

Molecular genetics of sulfur assimilation in filamentous fungi and yeast.

The filamentous fungi Aspergillus nidulans and Neurospora crassa and the yeast Saccharomyces cerevisiae each possess a global regulatory circuit that controls the expression of permeases and enzymes that function both in the acquisition of sulfur from the environment and in its assimilation. Control of the structural genes that specify an array of enzymes that catalyze reactions of sulfur metabolism occurs at the transcriptional level and involves both positive-acting and negative-acting regulatory factors. Positive trans-acting regulatory proteins that contain a basic region, leucine zipper-DNA binding domain, are found in Neurospora and yeast. Each of these fungi contain a sulfur regulatory protein of the beta-transducin family that acts in a negative fashion to control gene expression. Sulfur regulation in yeast also involves the general DNA binding protein, centromere binding factor I. Sulfate uptake is a highly regulated step and appears to occur in fungi, plants, and mammals via a family of related transporter proteins. Recent developments have provided new insight into the nature and control of the enzymes ATP sulfurylase and APS kinase, which catalyze the early steps of sulfate assimilation, and of the Aspergillus enzyme, cysteine synthase, which produces cysteine from O-acetylserine.

Regulation of sulfur and nitrogen metabolism in filamentous fungi.

In the filamentous fungi, N. crassa and A. nidulans, complex regulatory circuits control nitrogen metabolism and sulfur metabolism. The expression of entire sets of unlinked structural genes that encode metabolic enzymes is repressed when favored sulfur or nitrogen sources are available. These structural genes are coregulated by global positive-acting regulatory proteins and often are also controlled by metabolic inducers and pathway-specific regulatory proteins. The recent isolation of regulatory genes and representative structural genes of these circuits has provided significant new insight into the operation of both the nitrogen and the sulfur regulatory circuits, which involve sequence-specific DNA binding proteins, promoter control elements, metabolic inducers and repressors, and autogenous regulation.

Genetic and metabolic controls for sulfate metabolism in Neurospora crassa: isolation and study of chromate-resistant and sulfate transport-negative mutants.

Mutants of Neurospora resistant to chromate were selected and all were found to map at a single genetic locus designated as cys-13. The chromate-resistant mutants grow at a wild-type rate on minimal media but are partially deficient in the transport of inorganic sulfate, especially during the conidial stage. An unlinked mutant, cys-14, is sensitive to chromate but transports sulfate during the mycelial stage at only 25% of the wild-type rate; cys-14 also grows at a fully wild-type rate on minimal media. The double-mutant strain, cys-13;cys-14, cannot utilize inorganic sulfate for growth and completely lacks the capacity to transport this anion. The only biochemical lesion that has been detected for the double-mutant strain is its loss in capacity for sulfate transport. Neurospora appears to possess two distinct sulfate permease species encoded by separate genetic loci. The transport system (permease I) encoded by cys-13 predominates in the conidial stage and is replaced by sulfate permease II, encoded by the cys-14 locus, during outgrowth into the mycelial phase. The relationship of these new mutants to cys-3, a regulatory gene that appears to control their expression, is discussed.

Regeneration of invertase in Neurospora crassa.

In Neurospora, invertase is predominately an extracellular enzyme, and acid phosphatase is partially external in location. Both extracellular invertase and acid phosphatase were rapidly and quantitatively inactivated by acid treatment (pH 1.3). When such acid-treated cells were incubated with a suitable carbon source, a substantial regeneration of invertase activity occurred, but no restoration of acid phosphatase could be detected. The regeneration of invertase does not occur by renaturation of the inactivated enzyme, nor by secretion of a preexisting intracellular pool of invertase, but instead requires de novo enzyme synthesis. Invertase synthesis was partially repressed by glucose and mannose and was completely inhibited by 2-deoxyglucose. Acetate was found to inhibit invertase regeneration and the transport and incorporation of uracil and leucine. Several potential inhibitors of transcription, including alpha-amanitin, 5-fluorouracil, actinomycin D, and three derivatives of rifamycin, were ineffective in preventing invertase regeneration and in inhibiting the synthesis of ribonucleic acid. Conidia appeared to be very poorly permeable to these compounds.