We present an analysis of over 1,100 of the ∼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

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 Hl. 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.In the filamentous fungus Neurospora crassa, a high degree of genetic and metabolic regulation governs the expression of sets of enzymes within various global areas of metabolism such as nitrogen, phosphorus, and sulfur catabolism (7, 18). The sulfur control circuit of N. crassa consists of a set of unlinked structural genes which specify enzymes involved in sulfur metabolism. Synthesis of this entire family of sulfur-related enzymes, which includes aryl sulfatase, choline sulfatase, sulfate permease, a high-affinity methionine permease, and an extracellular protease occurs only when cellular levels of sulfur become limited (10,16,19,22). The expression of these sulfur-catabolic enzymes is controlled by two distinct regulatory genes. One of these, designated scon (for sulfur controller), appears to act in a negative fashion; scon mutants are insensitive to sulfur catabolite repression and thus express the sulfur-related enzymes in a constitutive fashion (4). The other sulfurregulatory gene, known as cys-3, acts in a positive manner to activate the expression of the various sulfur-related genes (17,21).Two different sulfate permease species, specified by distinct and unlinked structural genes, are both members of the sulfur circuit (16). The structural gene for sulfate permease II, cys-14, has been cloned and shown to encode an mRNA of approximately 3 kilobases (kb) whose content is highly regulated by cys-3, by scon, and by the sulfur status of the celis (11). Thus, it appears that both of the regulatory genes as well as sulfur repression act at the level of transcription or at a closely related step such as mRNA processing or stability. The cys-3 major sulfur control gene has been postulated to encode a regulatory protein which is needed to turn on the expression of the entire set of sulfur-related activities, presumably by binding at target DNA sequences adjacent to each structural gene (11). Mutants...

The ars-l+ gene of Neurospora crassa encodes the enzyme arylsulfatase. ars-l+ is in a group of highly regulated sulfur-related structural genes that are expressed under conditions of sulfur limitation and are under coordinate control of the cys-3+ and scon+ regulatory genes. The ars-l+ gene was cloned by chromosome walking from the qa gene cluster, using a lambda library. Cotransformation of an N. crassa ars-1 mutant with the isolated lambda clones and the benomyl resistance gene, followed by assay for arylsulfatase activity, was used to screen for the ars-l+ gene. Further confirmation that the cloned segment mapped to the ars-l+ locus was obtained by restriction-fragment-length polymorphism analysis. Northern (RNA) blot analysis showed that the ars-l + gene was transcribed to give an mRNA of 2.3 kilobases. In wild-type cells, the ars-1 + transcript was abundant under sulfur-derepressing conditions but absent under repressing conditions. Time course analysis showed that the appearance of ars-l+ message in sulfur-derepressed cultures paralleled the appearance of arylsulfatase enzyme activity. In addition, transcription of ars-l+ was detected only under derepressing conditions in a nuclear transcription assay. In a cys-3 regulatory mutant that was unable to synthesize arylsulfatase (or other sulfur-controlled enzymes), there was no ars-l+ transcript under repressing or derepressing conditions. In a temperature-sensitive cys-3 mutant, the ars-l + transcript was present only at the permissive growth temperature and under sulfur derepression. A negative regulatory mutant, sconC, displayed both constitutive expression of arylsulfatase enzyme activity and content of ars-l + message.In the filamentous fungus Neurospora crassa, the synthesis of sets of enzymes in global areas of metabolism such as nitrogen, phosphorus, and sulfur metabolism is highly regulated (24, 26). The sulfur regulatory circuit of N. crassa provides a model system for studying coordinate gene regulation and how a cell regulates its sulfur status. The sulfur control system is composed of a genetically defined set of trans-acting regulatory genes and a set of structural genes that encode enzymes used in the uptake and assimilation of a variety of sulfur compounds (6,26). When cultures of N. crassa are grown under conditions of sulfur limitation (i.e., derepressing conditions), then the set of sulfur-related genes is expressed in a coordinate manner. The structural genes involved encode for arylsulfatase, choline sulfatase, choline sulfate permease, methionine permease, sulfate permeases I and II, and an extracellular protease. None of the known genes in the sulfur regulatory circuit are linked (14,19,23,36).An important part of the sulfur control system involves the positive regulatory gene, cys-3+. In cys-3 mutants there is a pleiotropic loss of the entire set of sulfur-controlled enzymes (25). Paietta et al. (34) cloned the cys-3+ regulatory gene, and subsequent work (12) has shown that the encoded product is a 25.9-kilodalton protein that contains ...

The sulfur regulatory system of Neurospora crassa is composed of a set of structural genes involved in sulfur catabolism controlled by a genetically defined set of trans-acting regulatory genes. These sulfur regulatory genes include cys-3+, which encodes a basic region-leucine zipper transcriptional activator, and the negative regulatory gene scon-2+. We report here that the scon-2+ gene encodes a polypeptide of 650 amino acids belonging to the expanding ,8-transducin family of eukaryotic regulatory proteins. Specifically, SCON2 protein contains six repeated G,Uhomologous domains spanning the C-terminal half of the protein. SCON2 represents the initial filamentous fungal protein identified in the f3-transducin group. Additionally, SCON2 exhibits a specific amino-terminal domain that potentially defines another subfamily of .8-transducin homologs.Expression of the scon-2+ gene has been examined using RNA hybridization and gel mobility-shift analysis. The dependence of scon-2+ expression on CYS3 function and the binding of CYS3 to the scon-2+ promoter indicate the presence of an important control loop within the N. crassa sulfur regulatory circuit involving CYS3 activation of scon-2+ expression. On the basis of the presence of j8-transducin repeats, the crucial role of SCON2 in the signal-response pathway triggered by sulfur limitation may be mediated by protein-protein interactions.

The sulfur regulatory system of Neurospora crassa is composed of a group of highly regulated structural genes (e.g., the gene encoding arylsulfatase) that are under coordinate control of scon+ (sulfur controller) negative and cys-3+ positive regulatory genes. In scon-) (previously designated sconc) and scon-2 mutants, there is constitutive expression of sulfur structural genes regardless of the sulfur level available to the cells. The scon-2+ gene was cloned by sib selection screening of a cosmid-based gene library. The screening was based on the use of chromate, a toxic sulfate analog, which is transported into scon-2 cells grown on high sulfur but is not transported into cells that have regained normal sulfur regulation. Restriction fragment length polymorphism analysis was used to confirm that the cloned segment mapped to the proper chromosomal location. In wild-type cells, Northern (RNA) blot analysis showed that a 2.6-kilobase scon-2+ transcript was present at a substantial level only under sulfur-derepressing conditions. Kinetic analysis showed that scon-2+ mRNA content increased as the cells became sulfur starved. Further, scon-2+ RNA was detectable in a nuclear transcription assay only under derepressing conditions. In scon-l, the levels of scon-2+ mRNA were found to be constitutive. In the cys-3 regulatory mutant, there was a reduced level of scon-2+ transcript. cys-3+ and ars-l+ mRNAs were present under both derepressing and repressing conditions in the scon-2 mutant. Repeat-induced point mutation-generated scon-2 mutants were identical in phenotype to the known mutant.Sulfur uptake and assimilation in Neurospora crassa is carried out by a set of coordinately expressed structural genes. The structural genes are controlled by a set of genetically defined trans-acting regulatory genes and are expressed under conditions of sulfur limitation (i.e., derepressing conditions) (18). The unlinked structural genes encode arylsulfatase, choline sulfatase, sulfate permeases I and II, methionine permease, and an extracellular protease (12,14,19,28,31), which allow for the uptake and assimilation of a variety of sulfur compounds. (10,28). On the basis of heterokaryon studies with an electrophoretic variant of arylsulfatase, it was shown that scon-J + exerted only intranuclear control of arylsulfatase gene expression (i.e., nuclear limitation) (4, 20). Furthermore, in double-mutant studies, cys-3 was found to be epistatic to scon-J (8). On the basis of these results, scon-l+ can be placed in a regulatory hierarchy in which it is a negative effector of cys-3+ expression; cys-3+ then acts as a positive regulator of sulfur structural gene expression.A second gene which when mutant results in constitutive derepression of sulfur enzymes (e.g., arylsulfatase) was identified by P. S. Dietrich (M.S. thesis, University of Wisconsin, Madison, 1972). The mutation is designated here as scon-2. The scon-2 mutation is recessive and unlinked to other known loci in the system. scon-2 does not display the nuclear limitation effect s...