Transformants of Neurospora crassa with the nit-4 nitrogen regulatory gene: copy number,growth rate and enzyme activity

Summary nit-4 is a pathway-specific regulatory gene which controls nitrate assimilation in Neurospora crassa, and appears to mediate nitrate induction of nitrate and nitrite reductase. The NIT4 protein consists of 1090 amino-acid residues and possesses a single GAL4-like putative DNA-binding domain plus acidic, glutaminerich, and polyglutamine regions. Several mutants with amino-acid substitutions in the putative DNA-binding domain and a nit-4 deletion mutant, which encodes a truncated NIT4 protein lacking the polyglutamine region, are functional, i.e., they are capable of transforming a nit-4 mutant strain. However, transformants obtained with most of these nit-4 mutant genes possess a markedly reduced level of nitrate reductase and grow only slowly on nitrate, emphasizing the need to examine quantitatively the affects of in vitro-manipulated genes. The possibility that some mutant genes could yield transformants only if multiple copies were integrated was examined. The presence of multiple copies of wild-type or mutant nit-4 genes did not generally lead to increased enzyme activity or growth rate, but instead frequently appeared to be detrimental to nit-4 function. A hybrid nit-4-nirA gene transforms nit-4 mutants but only allows slow growth on nitrate and has a very low level of nitrate reductase.     

NIT2, the nitrogen regulatory protein of Neurospora crassa,binds upstream of nia,the tomato nitrate reductase gene,in vitro

Summary The nit-2 gene of Neurospora crassa encodes a trans-acting regulatory protein that activates the expression of a number of structural genes which code for nitrogen catabolic enzymes, including nitrate reductase. The NIT2 protein contains a Cys2/Cys2-type zinc-finger DNA-binding domain that recognizes promoter regions of the Neurospora nitrogen-related genes. The NIT2 zincfinger domain/-Gal fusion protein was shown to recognize and bind in a specific manner to two upstream fragments of the nia gene of Lycopersicon esculentum (tomato) in vitro, whereas two mutant NIT2 proteins failed to bind to the same fragments. The dissociation kinetics of the complexes formed between the NIT2 protein and the Neurospora nit-3 and the tomato nia gene promoters were examined; NIT2 binds more strongly to the nit-3 promoter DNA fragment than it does to fragments derived from the plant nitrate reductase gene itself. The observed specificity of the binding suggests the existence of a NIT2-like homolog which regulates the expression of the nitrate assimilation pathway of higher plants.     

Amino-acid substitutions in the zinc finger of NIT2, the nitrogen regulatory protein of Neurospora crassa,alter promoter element recognition

NIT2, the major nitrogen regulatory protein of Neurospora crassa mediates nitrogen catabolite derepression of the structural genes which specify enzymes of nitrogen catabolism. The promoter of the structural gene for L-amino acid oxidase, a nitrogen-regulated enzyme, was found to contain two NIT2 binding sites, each with two copies of a GATA core consensus sequence. Site-directed mutagenesis was employed to create amino-acid substitutions within the single zinc-finger region of NIT2, which serves as the DNA-binding domain. The affect of those mutations upon NIT2 function in vivo in the activation of three separate structural genes was examined by transformation assays and relevant enzyme activities, and DNA-binding activity in vitro was determined by gel band mobility-shift assays. It was shown that specific amino-acid residues within the zinc-finger loop region of NIT2 are important for DNA-binding activity, whereas other residues influence the specificity of DNA binding. Mutant NIT2 proteins were obtained which retain DNA-binding activity and alter the specificity of DNA recognition, thus allowing a distinction between related DNA elements.     

Amber nonsense mutations in regulatory and structural genes of the nitrogen control circuit of Neurospora crassa

Summary Neurospora crassa possesses a set of nitrogen-regulated enzymes whose expression requires a lifting of nitrogen catabolite repression and specific induction. The nit-2 gene is a major regulatory locus which appears to act in a positive way to turn on the expression of these nitrogen-related enzymes whereas the nit-4 gene appears to mediate nitrate induction of nitrate and nitrite reductase. The nit-3 gene specifies nitrate reductase and is subject to control by both nit-2 and nit-4. Many new nit-2, nit-3, and nit-4 mutants were isolated in order to obtain amber nonsense mutations in these loci which were suppressible by the suppressor gene, Ssu-1. A nit-2 nonsense mutant was isolated which has altered regulatory properties for control of nitrate reductase, L-amino acid oxidase, and uricase, and which may encode a truncated regulatory protein. Four nit-3 nonsense mutations were isolated, each of which completely lacks nitrate reductase activity, which is restored to markedly different levels by suppression with Ssu-1. Studies of heat activation and thermal lability of nitrate reductase suggest a qualitative alteration of the enzyme occurs in two of the Ssu-1 nit-3 strains.     

Characterization of the Aspergillus parasiticus niaD and niiA gene cluster

 The nitrate reductase gene (niaD) and nitrite reductase gene (niiA) of Aspergillus parasiticus are clustered and are divergently transcribed from a 1.6-kb intergenic region (niaD-niiA). The deduced aminoacid sequence of the A. parasiticus nitrate reductase demonstrated a high degree of homology to those of other Aspergillus species, as well as to Leptosphaeria maculans, Fusarium oxysporum, Gibberella fujikuroi and Neurospora crassa, particularly in the cofactor-binding domains for molybdenum, heme and FAD. A portion of the deduced nitrite reductase sequence was homologous to those of A. nidulans and N. crassa. The nucleotide sequences in niaD-niiA of A. parasiticus and of A. oryzae were 95% identical, indicating that these two species are closely related. Several GATA motifs, the recognition sites for the N. crassa positive-acting global regulatory protein NIT2 in nitrogen metabolism, were found in A. parasiticus niaD-niiA. Two copies of the palindrome TCCGCGGA and other partial palindromic sequences similar to the target sites for the pathway specific regulatory proteins, N. crassa NIT4 and A. nidulans NirA, in nitrate assimilation, were also identified. A recombinant protein containing the A. nidulans AreA (the NIT2 equivalent) zinc finger and an adjacent basic region was able to bind to segments of niaD-niiA encompassing the GATA motifs. These results suggest that the catalytic and regulatory mechanisms of nitrate assimilation are well conserved in Aspergillus. Received: 17 November 1995/16 January 1996     

Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa

In the filamentous fungus Neurospora crassa during conditions of sulfur limitation, CYS3, a major positive-acting regulatory protein, turns on the expression of an entire set of genes which encode permeases and enzymes involved in the acquisition of sulfur from environmental sources. CYS3 functions as a homodimeric protein and possesses a b-Zip domain that confers sequence-specific DNA binding. Expression of various hybrid GAL4-CYS3 fusion proteins in yeast was used to detect regions involved in gene activation. An amino-terminal serine/threonine-rich domain of CYS3 alone strongly activated expression of β-galactosidase, the yeast reporter. Moreover, mutant CYS3 proteins with amino-acid substitutions in this region that showed increased expression in Neurospora also displayed an enhanced activation potential in yeast. The cys-3 gene of the exotic N. crassa Mauriceville strain and of N. intermedia were cloned and demonstrated to be functional for gene activation and for sulfur-mediated regulation by complementation of a loss-of-function cys-3 mutation. The amino-terminal serine/threonine-rich region is highly conserved in these two CYS3 proteins, in agreement with the possibility that it serves as the activation domain. Surprisingly, an extended promoter region of the cys-3 gene in the Mauriceville strain and in N. intermedia was very well conserved with that of the standard N. crassa gene, including the presence of three CYS3-binding sites possibly involved in autogenous control. Results are presented which indicate that synthesis of the CYS3 regulatory protein is highly regulated and can be detected in the nucleus of cells subjected to sulfur de-repression, but is not found in the nucleus or the cytoplasm of S-repressed cells. The amino-acid substitutions of the CYS3 protein present in a temperature-sensitive cys-3 mutant and in a second-site revertant of a cys-3 null mutation are presented and are shown to affect their DNA-binding activities. Received: 9 January / 5 March 1998     

Cloning and preliminary characterization of a molybdenum cofactor gene of Neurospora crassa

Summary A Neurospora crassa library, constructed in a derivative of the plasmid pBR322 (pRK9), was used to transform two E. coli ch1D molybdenum cofactor mutants (ch1D, ch1D::Mu). Subsequently, one transformant from each of three independent transformation experiments was restriction mapped. All three transformants had an identical N. crassa DNA insert (4.2 kb). Southern Blot analysis with one of the plasmids (pMoCo, 1:4) showed hybridization to a single band of N. crassa genomic DNA. When pMoCo plasmid (1:4) was used to transform various E. coli nitrate reductase mutants (ch1A, ch1B, ch1C, ch1D, ch1E, ch1G and ch1M), the pMoCo plasmid was capable of restoring E. coli nitrate reductase activity to only the ch1D mutant. In vitro reconstitution experiments using wild-type, ch1D and ch1D; pMoCo cell-free extracts as a source of molybdenum cofactor (MoCo) were performed with the N. crassa MoCo mutants nit-1, nit-7 and nit-8. MoCo from wild-type E. coli cell-free extracts was capable of reconstituting NADPH : nitrate reductase activity to all three N. crassa mutants. MoCo from ch1D; pMoCo cell-free extracts was capable of reconstituting more NADPH : nitrate reductase activity to the N. crassa mutants than cell-free extracts from the original ch1D mutant.     

Cloning and sequence analysis of the glucoamylase gene of Neurospora crassa

A 1.0-kb DNA fragment, corresponding to an internal region of the Neurospora crassa glucoamylase gene, gla-1, was generated from genomic DNA by the polymerase chain reaction, using oligonucleotide primers which had been deduced from the known N-terminal amino-acid sequence or from consensus regions within the aligned amino-acid sequences of other fungal glucoamylases. The fragment was used to screen an N. crassa genomic DNA library. One clone contained the gene together with flanking regions and its sequence was determined. The gene was found to code for a preproprotein of 626 amino acids, 35 of which constitute a signal and propeptide region. The protein and the gene are compared with corresponding sequences in other fungi.     

NRE,the major nitrogen regulatory protein of Penicillium chrysogenum,binds specifically to elements in the intergenic promoter regions of nitrate assimilation and penicillin biosynthetic gene clusters

NRE, the nitrogen regulatory protein of Penicillium chrysogenum, contains a single Cys2/Cys2-type zinc-finger motif followed immediately by a highly basic region. The zinc-finger domain was expressed to Escherichia coli as a fusion protein with -galactosidase. In order to test the putative DNA-binding ability of NRE, the intergenic promoter region of the nitrate reductase/nitrite reductase gene cluster (niiA-niaD) of Penicillium was sequenced. Our results show that NRE is a DNA-binding protein and binds to the intergenic promoter regions of the P. chrysogenum niiA-niaD and acvA-pcbC gene cluster, encoding the first two enzymes in penicillin biosynthesis. Three of the four high-affinity NRE-binding sites contained two GATA core elements. In one of the recognition sites for NRE, one GATA motif was replaced by GATT. The two GATA elements showed all possible orientations, head-to-head, head-to-tail and tail-to-tail, and were separated by between 4 and 27 bp. Missing-contact analysis showed that all three purines in both of the GATA core sequences and the single adenine residue in each of the complementary TATC sequences were involved in the binding of NRE. Moreover, loss of purines in the flanking regions of the GATA elements also affect binding of NRE, as their loss causes reduced affinity.     

Biochemical characterization of the molybdenum cofactor mutants of Neurospora crassa: in vivo and in vitro reconstitution of NADPH-nitrate reductase activity

Summary Molybdenum cofactor (MoCo) mutants of Neurospora crassa lack both NADPH-nitrate reductase and xanthine dehydrogenase activity. In vivo and in vitro studies to further characterize these mutants are now reported. The MoCo mutants nit-9A and nit-9B are capable of growing, albeit poorly, with nitrate as the sole nitrogen source, provided high levels of molybdate are present. The MoCo mutants nit-9A, nit-9B and nit-9C, but not nit-1, nit-7 or nit-8, have significant levels of NADPH-nitrate reductase when grown in nitrate medium containing 30 mM molybdate. In vitro reconstitution experiments using cell free extracts of the N. crassa MoCo mutants and E. coli HB101 as a source of wild-type MoCo were performed. MoCo from E. coli was capable of reconstituting NADPH-nitrate reductase activity to nit-1, nit-7 and nit-8. Molybdate is required for the in vitro reconstitution of NADPH-nitrate reductase activity. It was not possible to in vitro reconstitute NADPH-nitrate reductase activity in the MoCo mutants nit-9A, nit-9B or nit-9C.     

Nuclear DNA-binding proteins which recognize the intergenic control region of penicillin biosynthetic genes

The biosynthesis of penicillin, a secondary metabolite produced by Penicillium chrysogenum, is subject to sophisticated genetic and metabolic regulation. The structural genes, pcbC and pcbAB, which encode two of the penicillin biosynthetic enzymes are separated by a 1.16-kb intergenic region and transcribed divergently from one another. To identify and characterize nuclear proteins which interact with the pcbAB-pcbC intergenic promoter region, crude and partially purified nuclear extracts were used in mobility shift and DNA footprinting assays. Multiple DNA-binding proteins appear to bind to different regions of this DNA segment. An abundant nuclear protein, nuclear factor A (NF-A), binds at a single site in the intergenic promoter region and recognizes an 8-bp sequence, GCCAAGCC. Penicillin production is sensitive to nitrogen catabolite repression. The global-acting nitrogen regulatory protein NIT2 of Neurospora crassa binds strongly to the intergenic promoter region of the pcbAB and pcbC genes at a single site that contains two closely spaced GATA sequences.     

Cloning of the nitrate reductase gene of Stagonospora (Septoria) nodorum and its use as a selectable marker for targeted transformation

The nitrate reductase gene (NIA1) of the phytopathogenic fungus Stagonospora (Septoria) nodorum has been cloned from a cosmid library by homologous hybridisation with a PCR-generated probe. A 6.7-kb fragment carrying the NIA1 gene was subcloned and partially characterised by restriction mapping. Sequencing of the gene indicated a high degree of homology, both at the nucleotide and amino-acid levels, with nitrate reductase genes of other filamentous fungi. Furthermore, consensus regulatory signals thought to be involved in the control of nitrogen metabolism are present in the 5′ flanking region. The cloned NIA1 gene has been used to develop a gene-transfer system based on nitrate assimilation. Stable nia1 mutants of S. nodorum defective in nitrate reductase were isolated by virtue of their resistance to chlorate. These were transformed back to nitrate utilisation with the wild-type S. nodorum NIA1 gene. Southern analyses revealed that transformation occurred as a result of the integration of transforming DNA into the fungal genome; in all cases examined, integration was targeted to the homologous sequence. Received: 30 March / 9 June 1998     

Isolation and characterization of Pioneer1, a novel Chlamydomonas transposable element

During the course of this study a novel family of Chlamydomonas mobile elements has been identified in natural isolate strain 224. The first member of this class to be characterized, a 2.8-kb element named Pioneer1, was trapped in an intron of the nitrate reductase structural gene, NIT1. This element has been cloned and completely sequenced and found to be unusual in structure. Pioneer elements are present in a very low-copy number of three per genome in strain 224. The copy number increased by one upon transposition of Pioneer1. Hybridization of Pioneer1 to a variety of Chlamydomonas strains confirmed that this element differed from previously described Chlamydomonas transposons. It also indicated that related elements are present in low-copy number in natural isolate strains 356 and S1D2, but not in the most commonly used laboratory strains 137c and 21 gr. For these reasons, members of the Pioneer family might prove useful as insertional mutagens.     

Domains of Rinderpest virus phosphoprotein involved in interaction with itself and the nucleocapsid protein.

The yeast two-hybrid system was used to identify domains involved in specific in vivo interactions between the Rinderpest virus (RPV) phosphoprotein (P) and nucleocapsid protein (N). N and P genes were cloned in both the yeast GAL4 DNA-binding and GAL4 activation domain vectors, which enabled analysis of self and interprotein interactions. Mapping of the domain of P protein involved in its association with itself revealed that the COOH-terminal 32 amino acids (316-347) that forms a part of the highly conserved coiled coil region is important for interaction. In addition, just the coiled coil region of RPV P protein fused to the DNA-binding domain and activation domain of GAL4 was found to be sufficient to bring about activation of the beta-galactosidase reporter. Similarly, mapping of the domains of P protein involved in its interaction with N protein revealed that NH2-terminal 59 amino acids and COOH-terminal 32 amino acids (316-347) involved in P-P interaction are simultaneously required for association with N protein. Interestingly, a P protein mutant with just the NH2-terminal 59 amino acids and the coiled coil domain with all other P protein regions deleted retained its ability to interact with N protein. Furthermore, we were able to show N and P protein interaction in vitro using recombinant N and P proteins expressed in Escherichia coli, demonstrating the existence of direct physical interaction between the two proteins.     

Isolation and characterization of a methylammonium resistant mutant of Neurospora crassa

Summary A mutant of Neurospora crassa has been isolated which is resistant to methylammonium, a structural analog of ammonium. In contrast to wild type, this mutant, mea-1, has derepressed nitrate reductase and nitrite reductase activities in the presence of ammonium. However, glutamine still represses these nitrate assimilation enzymes in mea-1. The nit-2 mutant was epistatic to mea-1 since the mea-1; nit-2 double mutant has the nit-2 mutant phenotype. In addition, mea-1; nit-2 double mutants cannot utilize ammonium as a nitrogen source. We suggest therefore that nit-2 and mea-1 loci play a role in ammonia/methylamine uptake.