The regulatory protein NIT4 that mediates nitrate induction inNeurospora crassa contains a complex tripartite activation domain with a novel leucine-rich,acidic motif

Expression ofnit-3 andnit-6, the structural genes which encode nitrate reductase and nitrite reductase inNeurospora crassa, requires the global-acting NIT2 and the pathway specific NIT4 regulatory proteins. NIT4, which consists of 1090 amino-acid residues, possesses a Cys₆/Zn₂ zinc cluster DNA-binding-domain. NIT4 was dissected to localize transactivation domains by fusion of various segments of NIT4 to the DNA-binding domain of GAL4 for in vivo analysis in yeast. Three separate activation subdomains, and one negative-acting region, which function in yeast were located in the carboxyl-terminal region of NIT4. The C-terminal tail of 28 amino-acid residues was identified as a minimal activation domain and consists of a novel leucine-rich, acidic region. Most deletions which removed even small segments of the NIT4 protein were found to lead to the loss of NIT4 function in vivo inN. crassa, implying that the central region of the protein which lies between the DNA-binding and activation domains is essential for function. The yeast two-hybrid system was employed to identify regions of NIT4 responsible for dimer formation. A short isoleucine-rich segment downstream from the zinc cluster, predicted to form a coiled coil, allowed dimerization in vivo; this same isoleucine-rich region also showed dimerization in vitro when examined via chemical cross linking. The enzyme nitrate reductase has been postulated to exert autogenous regulation by directly interacting with the NIT4 protein. This possible nitrate reductase-NIT4 interaction was investigated with the yeast two-hybrid system and by direct in vitro binding assays; both assays failed to identify such a protein-protein interaction.     

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.     

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.     

Inducible responses to DNA damaging or stress inducing agents in Neurospora crassa

Summary Two-dimensional polyacrylamide gel electrophoresis has been used to analyze proteins from wild type and mutagen sensitive mutants of Neurospora crassa under constitutive conditions and after treatment with mutagens and other stress inducing agents. Several proteins have been detected that are either induced or show changes in electrophoretic mobility in response to UV irradiation, 4-NQO, X-ray, paraquat and heat shock. Ten proteins were found to respond to more than one of the stress inducing agents, demonstrating a complex pattern of polypeptide inductions. The significance of these findings and the possible effects of some of these proteins on the DNA repair process and stress management are discussed.     

Alteration of the cytochrome c oxidase subunit 2 gene in the [exn-5] mutant of Neurospora crassa

Summary The maternally inherited [exn-5] mutant of Neurospora crassa is characterized by its slow-growth rate and deficiency of cytochrome aa ₃ relative to wildtype strains. We have determined the DNA sequence of the COXI and COXII genes of the mutant, which encode subunits 1 and 2 of cytochrome c oxidase, respectively. No changes in the DNA sequence of the COXI gene relative to the corresponding wild-type gene were found. In the region of the COXII gene we found two alterations, one a C to T transition eight base pairs upstream of the coding sequence and the second within the coding sequence for subunit 2 affecting amino acid 27 of the precursor polypeptide (amino acid 15 of the mature polypeptide). The altered codon in [exn-5] specifies an isoleucine residue rather than the wild-type threonine residue. The corresponding position in subunit 2 sequences of all other organisms examined is conserved either as a threonine or a serine residue. Thus, we consider it likely that the mutation directly affecting the coding sequence of the polypeptide is responsible for the [exn-5] phenotype. Analysis of serially passaged heterokaryons constructed between wild-type and [exn-5] shows that both mutations segregate with the [exn-5] phenotype. Examination of mitochondrial translation products in [exn-5] revealed a deficiency of subunit 2, as well as the presence of a polypeptide that corresponds to a previously described precursor of subunit 1 that accumulates in a COXI mutant of N. crassa, [mi-3]. We propose possible relationships between [exn-5], [mi-3], and the nuclear su-1 ^[mi-3] allele, which suppresses both mutations.     

Genetic analysis of transformation in a microconidiating strain of Neurospora crassa

Summary We have characterized Neurospora crassa transformants obtained with plasmid pDV1001 bearing the cloned catabolic dehydroquinase (qa-2 ⁺) gene (Hughes et al. 1983) and fluffy 268 host strain producing only uninucleate microconidia allowing to isolate individual transformation products. The percentage of transformed nuclei in the mycelium and their stability were determined by genetic analysis of microconidia produced on selective or non-selective medium. About half of the transformants originating from mycelial spheroplasts were apparently homokaryotic. Catabolic dehydroquinase activity was in agreement with the proportion of transformed nuclei. The DNAs from four transformants analyzed by Southern hybridization showed restriction fragments expected for integration of pDV1001 into genomic DNA by non-homologous recombination. No plasmids could be rescued from the undigested DNAs of the transformants by transformation of E. coli. One transformant, 8268-6, was unstable and generated a high proportion of segregants. Plasmid pDV1001 sequences were absent in their DNA. Colonies originating from microconidia of strain fl268-6 on selective plates often lost the transformed character. These results suggest that instability in this transformant is due to the loss of integrated plasmid sequences during vegetative growth.     

Generation of new mutants of nmr,the negative-acting nitrogen regulatory gene of Neurospora crassa,by repeat induced mutation

Summary The repeat induced point mutation (RIP) phenomenon has been used to generate new mutants of nmr, the negative nitrogen regulatory gene in Neurospora crassa. The wild-type nmr gene was cotransformed along with the hygromycin B resistance gene into wild-type cells by selecting for hygromycin B resistance. Following purification of primary transformants using microconidia, crosses to wild-type. Detailed analyses of some of the progeny revealed that we had generated authentic nmr mutants at high frequency. The polymerase chain reaction was used to amplify and clone a fragment of a mutagenized nmr copy from one of the mutants. The nucleotide sequence analysis showed that 14% of the guanine residues have been converted into adenines, resulting in numerous missense and nonsense mutations. The newly created nmr mutants were found suitable for use as host strains in transformation experiments.     

Characterization of a variant Neurospora crassa mitochondrial DNA which contains tandem reiterations of a 1.9 kb sequence

Summary We have identified a second variant of Neurospora mtDNA which contains tandem, head-to-tail repeats of sequences at the boundary of Eco RI-4 and 6. This region may contain a. major replication origin of Neurospora mtDNA.     

Regulation of synthesis and secretion of acid and alkaline phosphatases in Neurospora crassa

Summary We show that N. crassa represses the production of acid phosphatase at pH higher than 8.0, irrespective of the carbon source used, whereas production was stimulated by sucrose at slightly acidic pH. The same profile of acid phosphatase production was observed in the pho-2A, pho-3A, nuc-1A, nuc-2A and preg ^c mutant strains. We also show that acid phosphatase synthesized by the preg ^c mutant strain grown on high phosphate medium has pronounced differences when compared to the enzyme synthesized by the wild-type strain grown on low phosphate medium in terms of heat stability, steady-state kinetic properties and DEAE-cellulose chromatography. In addition, the synthesis and/or secretion of only phosphate-repressible alkaline phosphatase is affected by mutations in acu-1, and acu-5 and acu-7 genes. These results, which indicate distinct pathways for the synthesis and secretion of acid and alkaline phosphatases in N. crassa, contradict the dosage titration model proposed by Metzenberg et al. (1974) whereby the synthesis of these enzymes should occur through a single hierarchical regulatory circuit as a response to phosphate starvation.     

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.     

Transformation of Neurospora crassa with the trp-1 gene and the effect of host strain upon the fate of the transforming DNA

Summary Neurospora trp-1 ⁺ transformants, obtained by transforming a trp-1 inl strain with plasmid DNA containing the wild type trp1 ⁺ gene, were characterized by genetic and Southern blot analyses. The transforming trp-1 gene integrated at or near the resident site in all of the trp-1 ⁺ transformants obtained with circular DNA or DNA cut within the trp-1 coding region. The frequency of homologous integration decreased substantially when the donor DNA was cleaved outside the trp-1 coding region. The transformants were very stable mitotically and, in general, also showed meiotic stability. Analysis of trp-1 ⁺ transformants obtained with another recipient strain, trp-1 ⁺ ga-2 aro-9 inl, showed that homologous integration of donor DNA occurred in only 20% of the transformants, whether circular or linear DNA was used. Thus, the host strain employed for transformation appears to be a major factor in determining the fate of transforming DNA. Southern blot analysis of transformants showed that integration of the transforming DNA at the homologous site occurred by double crossover or gene conversion events rather than by insertion of the entire plasmid DNA. Multiple and apparently non functional integration events were observed in some transformants.     

The orientation of gene maps by recombination of flanking markers for the am locus of Neurospora crassa

Fincham (1967), Smyth (1973b) and Rambosek and Kinsey (1983) have each generated fine-structure maps of the am gene of Neurospora crassa. Each map had a consistent linear order of alleles but the assignment of an orientation with respect to other linkage group-V loci differed. Fincham found the end marked by the am ₆ allele to be at the distal end of the locus, Smyth found am ₆ to be at the proximal end while the data of Rambosek and Kinsey did not suggest an orientation. Smyth's orientation has been adopted as the standard, but not unreservedly. We have aligned the genetic and physical maps of the am gene, showing that am ₆ is at the distal end, supporting Fincham's orientation. However, we suggest that an assumption used to orient fine structure genetic maps is flawed and that the conflicting orientation between these three studies follows from the different choice of flanking markers.     

Plasmid recovery from transformants and the isolation of chromosomal DNA segments improving plasmid replication in Neurospora crassa

Summary The efficient recovery of plasmid DNA from Neurospora crassa transformants is described. Lithium acetate-treated spores were transformed with plasmid DNA and grown in mass in liquid culture. The resulting mycelial growth was harvested and plasmid DNA was extracted and used to transform E. coli to ampicillin resistance. Although at low frequency, routine recovery of plasmid pSD3 which carries the Neurospora qa-2 ⁺ gene and pBR322 sequences has been demonstrated. About 10% of the recovered plasmids carried deletions and transformed Neurospora at a higher frequency. The liquid culture procedure was also used in attempts to isolate autonomously replicating sequences (ars). In order to select for a stable vector which contains an ars sequence, a clone bank containing a selectable marker (qa-2 ⁺) and Neurospora chromosomal BamHI fragments was constructed and used to transform Neurospora. Several plasmid isolates resulting from a screening of the clone bank showed an improvement in the efficiency of recovery from Neurospora transformants. The properties of one such isolated plasmid, pJP102, suggest that it may contain an ars sequence. Some potential applications of these results for cloning in Neurospora and other filamentous fungi are discussed.     

Determination of the Neurospora crassa CYS 3 sulfur regulatory protein consensus DNA-binding site: amino-acid substitutions in the CYS3 bZIP domain that alter DNA-binding specificity

CYS3 is the positive-acting global regulatory protein involved in the sulfur control circuit in Neurospora crassa and belongs to the family of bZIP DNA-binding proteins. Here we report a characterization of native DNA-binding sites recognized by CYS3. DNA footprinting experiments and systematic mutational analysis were used to define the consensus CYS3-binding sequence, 5′-ATGPuPyPuPyCAT, a 10-bp palindrome. The sequence 5′-ATGACGTCAT acts as a strong binding site, and all single nucleotide changes within this sequence resulted in a reduction, or even complete loss, of CYS3 DNA-binding. Site-directed mutagenesis was employed to study two uncharged residues, serine 113 and phenylalanine 116, in the basic region of the CYS3 protein bZip DNA-binding domain. Ser¹¹³ appears to be directly involved in a specific interaction with nucleotide 2 of the binding site, possibly by making a direct contact with this base, and Phe¹¹⁶ contributes significantly to DNA-binding affinity. Received: 21 April / 9 June 1996     

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.     

Characterization of the glyoxysomal isocitrate lyase genes of Aspergillus nidulans (acuD) and Neurospora crassa (acu-3)

Summary The nucleotide sequences of the genes encoding the acetate-inducible glyoxylate cycle enzyme isocitrate lyase from the ascomycete fungi Aspergillus nidulans (acuD) and Neurospora crassa (acu-3) are presented. The respective A. nidulans and N. crassa genes are interrupted at identical positions by two introns and encode proteins of 538 and 543 amino acids, which have 75% identity. The predicted protein sequences do not demonstrate the C-terminal tripeptide S-K-L that has been implicated in peroxisomal targeting and found in the glyoxysomally located enzyme malate synthase from the same species. However, the protein sequences do exhibit a partial repeat which, in common with malate synthase, is located in regions that are absent from, or non-homologous with, the E. coli enzyme, which is not compartmentalized.     

Disruption of the gene encoding the 78-kilodalton subunit of the peripheral arm of complex I in Neurospora crassa by repeat induced point mutation (RIP)

We have used the procedure of sheltered RIP to generate mutants of the 78-kDa protein of the peripheral arm of Neurospora crassa complex I. The nuclei containing the mutations were initially isolated as one component of a heterokaryon but subsequent analysis showed that nuclei containing null alleles of the gene could be propagated as homokaryons. This demonstrates that the gene does not serve an essential function. Sequence analysis of one allele shows that 61 transition mutations were created resulting in 39 amino-acid changes including the introduction of four stop codons. Mutant strains grow at a slower rate than wild-type and exhibit a decrease in the production of conidia. Electron paramagnetic spectroscopy of mutant mitochondria suggest that they are deficient in Fe–S clusters N-1, N-3, and N-4.     

Heterokaryotic transmission of senescence plasmid DNA in Neurospora

Summary Heterokaryotic transmission is one of the major techniques for the study of cytoplasmic inheritance and here we have applied it to the senescence-determining plasmids kalilo (Hawaiian) and maranhar (Indian). We have shown that kalilo-based senescence is effectively transmitted by cytoplasmic contact, both in N. crassa and in N. intermedia. In the first place, the heterokaryons themselves are senescent, confirming the suppressivity of the senescence phenotype in mixtures of normal and senescent cytoplasms. Second, senescence is found in new nuclear associations, as shown by analysis of conidial isolates and meiocytes stemming from the heterokaryons. In addition, the free plasmid AR-kalDNA, and its form that is inserted into mtDNA, (mtIS-kalDNA), are both transmitted to new nuclear associations. In a transient fusion between senescent N. intermedia and nonsenescent N. crassa cells, AR-kalDNA was transmitted to N. crassa and mtIS-kalDNA was transmitted to N. crassa mtDNA. A cryptic mitochondrial plasmid, not associated with senescence, was also transmitted very efficiently to N. crassa mitochondria. In mixed kalilo/maranhar fusions, both plasmids coexisted, approximately equally, in the heterokaryons themselves, and in conidial isolates. However, in sexual derivatives, AR-marDNA was in an excess and AR-kalDNA was sometimes absent. The efficient heterokaryotic transmission of these elements suggests that this is one of their natural modes of spread in populations.