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1.
Biochem Genet ; 42(1-2): 21-34, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15068336

RESUMO

The Neurospora crassa genome database was searched for sequence similarity to crnA, a nitrate transporter in Aspergillus nidulans. A 3.9-kb fragment (contig 3.416, subsequence 183190-187090) was cloned by PCR. The gene coding for this nitrate transporter was termed nit-10. The nit-10 gene specifies a predicted polypeptide containing 541 amino acids with a molecular mass of 57 kDa. In contrast to crnA, which is clustered together with niaD, encoding nitrate reductase, and niiA, encoding nitrite reductase, nit-10 is not linked to nit-3 (nitrate reductase), nit-6 (nitrite reductase), or to nit-2, nit-4 (both are positive regulators of nit-3), or nmr (negative regulator of nit-3) in Neurospora crassa. A nit-10 rip mutant failed to grow in the medium when nitrate (< 10 mM) was used as the sole nitrogen source, but grew similarly to wild type when nitrate concentration was 10 mM or higher. In addition, it showed strong sensitivity to cesium in the presence of nitrate and resistance to chlorate in the presence of alanine, proline, or hypoxanthine. The expression of nit-10 required nitrate induction and was subject to repression by nitrogen metabolites such as glutamine. Expression of nit-10 also required functional products of nit-2 and nit-4. The half-life of nit-10 mRNA was determined to be approximately 2.5 min.


Assuntos
Proteínas de Transporte de Ânions/genética , Proteínas Fúngicas/genética , Neurospora crassa/genética , Alanina/metabolismo , Alanina/farmacologia , Sequência de Aminoácidos , Proteínas de Transporte de Ânions/efeitos dos fármacos , Proteínas de Transporte de Ânions/metabolismo , Césio/metabolismo , Césio/farmacologia , Cloratos/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Glutamina/metabolismo , Glutamina/farmacologia , Meia-Vida , Hipoxantina/metabolismo , Hipoxantina/farmacologia , Dados de Sequência Molecular , Mutação , Neurospora crassa/efeitos dos fármacos , Transportadores de Nitrato , Polimorfismo de Fragmento de Restrição , Prolina/metabolismo , Prolina/farmacologia , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Microbiol Mol Biol Rev ; 68(1): 1-108, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15007097

RESUMO

We present an analysis of over 1,100 of the approximately 10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.


Assuntos
Proteínas Fúngicas/genética , Genoma Fúngico , Neurospora crassa , Animais , Biologia Computacional , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Humanos , Micoses/microbiologia , Neurospora crassa/química , Neurospora crassa/genética , Neurospora crassa/metabolismo , Neurospora crassa/patogenicidade , Doenças das Plantas/microbiologia
3.
Curr Genet ; 42(5): 260-7, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12589465

RESUMO

In Neurospora crassa, the nit-3 gene, which encodes nitrate reductase, an enzyme required for the utilization of inorganic nitrate, is subject to a high degree of genetic and metabolic regulation as a member of the nitrogen control circuit. The nit-3 gene promoter contains binding sites for a globally acting protein NIT2 and a pathway-specific protein NIT4. Expression of the nit-3 gene absolutely requires both the NIT2 and NIT4 transcription factors and only occurs under conditions of nitrogen source derepression and nitrate induction. In the sulfur control circuit, the cys-14 gene encodes sulfate permease II, which facilitates the assimilation of sulfate. Expression of cys-14 is strongly regulated by only a single positive-acting factor, CYS3. It was of interest to determine whether NIT2 or NIT4 alone was capable of turning on the expression of cys-14, since this structural gene is normally controlled by only one regulatory protein. NIT2- and/or NIT4-binding elements were introduced into the promoter of a wild-type cys-14 gene and these constructs were transformed into a cys-13(-) cys-14(-) mutant strain and into a nit-2(-) mutant host. We examined whether any of these cys-14 genes in these transformants could now be controlled as a nitrogen-regulated gene. Sulfate permease assays revealed that both NIT2 and NIT4 were required for cys-14 expression upon nitrate induction, while neither alone activated any detectable cys-14 expression. We thus conclude that neither NIT2 nor NIT4 is capable alone of activating gene expression in this context, but together they can cooperate to elicit strong activation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Neurospora crassa/genética , Transativadores , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Sequência de Bases , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Plasmídeos , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Fatores de Transcrição/genética
4.
Biochemistry ; 41(51): 15288-95, 2002 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-12484767

RESUMO

Several homologous genes encoding proteins involved in regulating siderophore synthesis in fungi have been isolated, including the sre gene from the filamentous fungus Neurospora crassa. We present data that further characterize SRE and provide new insights into the regulation of iron homeostasis in Neurospora. SRE is a member of the GATA factor family, which is comprised of transcription factors that contain either one or two zinc finger motifs that recognize and bind to GATA-containing DNA sequences. Results from electrophoretic mobility shift assays demonstrate that SRE binds with high affinity to a DNA probe containing the iron response element from the sid1 promoter from Ustilago. SRE binding to DNA was demonstrated to be zinc-dependent. Moreover, changes in the spacing between two GATA sites altered the DNA binding affinity of SRE. Mutants of highly conserved cysteine residues present in SRE and homologous proteins were created by site-directed mutagenesis. The combined results of mobility shift assays, siderophore synthesis assays, and ornithine oxygenase enzyme activity determinations demonstrate that these mutants with cysteine substitutions have a dominant repressor phenotype.


Assuntos
Cisteína/química , Proteínas de Ligação a DNA/química , Proteínas Fúngicas/química , Neurospora crassa/química , Neurospora crassa/metabolismo , Sideróforos/biossíntese , Cisteína/genética , Sondas de DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Fatores de Transcrição GATA , Mutagênese Sítio-Dirigida , Neurospora crassa/genética , Ligação Proteica/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Zinco/química , Dedos de Zinco/genética
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