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.     

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.     

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     

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.     

A calcium binding protein from cell wall of Neurospora crassa

Isolated cell wall preparations of N. crassa bind significant levels of Ca, Mg and other divalent cations. Enzymatic treatment of the cell wall with β‐(1,3)‐glucanase, but not with chitinase, resulted in solubilization of only the calcium‐binding protein fraction. A calcium‐binding protein (CaBP) was purified by metal‐chelate affinity chromatography and reversed phase HPLC. CaBP has an Mr of around 6 kDa on SDS‐PAGE and mass spectrometry showed that it has a molecular mass of 5744 Da. One mole of CaBP binds 2 moles of calcium and is partially inhibited (15–50%) by other divalent cations (Mg, Ni and Cu). Quenching of tryptophan fluorescence was observed upon copper binding but not calcium binding. This is a first report of a calcium binding protein from the cell wall of fungi. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A regulatory phenotype associated with the en-am 1 mutant of Neurospora crassa

Summary We have investigated the nature of the en-am1 mutant of Neurospora crassa and have found that it affects the regulation of proline oxidase and utilisation of other nitrogen sources. This mutant is closely linked to the gln gene but not allelic with it. Data from crosses suggest that the two genes he on opposite sides of the in1 gene on linkage group VR.     

The mus-8 gene of Neurospora crassa encodes a structural and functional homolog of the Rad6 protein of Saccharomyces cerevisiae

We cloned a DNA repair gene, mus-8, of Neurospora crassa and sequenced the genomic DNA and cDNA. Nucleotide-sequence analysis indicated that the mus-8 gene contains an open reading frame (ORF) of 456 bp, interrupted by three small introns. The deduced amino-acid sequence showed that the mus-8 gene encodes a 17 kDa protein which has 77.5% and 83.3% identity to the Rad6 protein of Saccharomyces cerevisiae and the rhp6⁺ protein of Schizosaccharomyces pombe, respectively. The Rad6 protein is a ubiquitin-conjugating enzyme (E2) and is required for DNA repair, mutagenesis, and sporulation in yeast. Introduction of the mus-8 gene into a S. cerevisiae rad6 mutant resulted in significant recovery of DNA repair functions, especially UV-mutagenesis, and also sporulation, both of which are defective in the rad6 mutant. It is therefore postulated that mus-8 of Neurospora has a function very similar to that demonstrated for RAD6 of S. cerevisiae. Received: 27 February / 23 April 1996     

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.     

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     

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.     

Cloning and characterization of the yeast RAD1 homolog gene (mus-38) from Neurospora crassa: evidence for involvement in nucleotide excision repair

A Neurospora crassa gene encoding a product with homology to the Saccharomyces cerevisiae Rad1 nucleotide excision repair (NER) protein was isolated by degenerate PCR. The predicted protein consists of 892 amino acids with a molecular weight of 100.4 kDa, and 32–37% identity to the XPF/ERCC4 protein family. The homolog was mapped to the left arm of linkage group I, the location of the mus-38 gene. Subsequently, gene inactivation and complementation studies identified the RAD1 homolog as mus-38. Immunological assays showed that the mus-18 (UV-specific endonuclease) and mus-38 strains have partial and normal UV-damage excision activities, respectively, but removal of thymine dimers and TC (6-4) photoproducts is abolished in the mus-18 mus-38 double mutant. The double mutant also was synergistically more sensitive to UV than either single mutant. The data suggest that mus-38 may participate in a different NER pathway from that involving the mus-18 gene. Received: 27 November 1997 / 28 January 1998     

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.     

Smad泛素化调节因子-2及其mRNA在增生性瘢痕组织中的表达

目的研究烧伤后增生性瘢痕组织中Smad泛素化调节因子－2（Smurf2）及其mRNA的表达。方法选取烧伤后增生性瘢痕患者9例,取材于患者整形手术切除的瘢痕,同时取同一患者剩余正常皮肤作为对照。Western blotting方法检测smurf2蛋白水平,RT—PCR检测其mRNA表达;进一步分离培养人正常皮肤和增生性瘢痕成纤维细胞,加入外源件转化生长凶子β1（TGF—β1）刺激,观察Smurf2蛋白和mRNA水平的变化。结果增生性瘢痕组织中Smurf2蛋白水平和mRNA表达显著高于正常皮肤（P〈0．05）,而且,在外源性TGF—β1刺激下,瘢痕成纤维细胞中Smurf2蛋白和mRNA表达呈时间依赖性增加。结论烧伤后增生性搬痕组织中Smurf2及其mRNA表达增强;在TGF—β1刺激下,瘢痕成纤维细胞中Smurf2及儿mRNA表达逐渐增加。     

Genetic analysis of ad-3 mutants induced by AF-2 and two other nitrofurans in neurospora crassa

Our previous studies have shown that the nitrofurans AF-2, SQ18506, and FANFT are potent mutagens in Neurospora crassa. The genetic damage produced by these chemicals at the ad-3 region in N crassa has been characterized by a series of genetic tests. The results of these tests indicate that all three agents induce a high frequency of point mutations and probably a low frequency of multilocus deletions. A comparison of the complementation patterns among the AF-2-induced ad-3B mutants and those induced by other chemical agents indicates that the spectra of intragenic alterations induced by AF-2 in N crassa are similar to those induced by monofunctional alkylating agents.     

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.     

人脑胶质瘤组织中骨形成蛋白-2的表达及其临床病理学意义

目的:探讨骨形成蛋白-2(BMP-2)在人脑胶质瘤中的表达和临床意义.方法:随机选取脑胶质瘤患者88例, 采用SABC免疫组化方法和RT-PCR技术, 检测脑胶质瘤组织和正常脑组织中BMP-2蛋白及其mRNA的表达.并分析其与脑胶质瘤组织临床分级之间的关系.结果:BMP-2蛋白在脑胶质瘤组织中的阳性表达率为78.40% (69/88), 明显高于(P<0.01)其在正常脑组织中的阳性表达率[3% (6/20)].BMP-2蛋白与mRNA 在脑胶质瘤高级别组(Ⅲ～Ⅳ级) 中的表达均明显高于低级别组(Ⅰ～Ⅱ级), 说明随着肿瘤级别的升高BMP-2表达也增强.结论:BMP-2蛋白及其mRNA的异常表达可能与脑胶质瘤的恶性程度有关, 可以作为一个肿瘤标志物在临床中应用.