Requirements of delta 9 and delta 12 fatty acid desaturation in Neurospora

Microsomes prepared from the wild-type strain and lipid auxotrophs of Neurospora were analyzed for delta 9 - (stearoyl-CoA) and delta 12 - (oleoyl-CoA) desaturase activities. The wild-type delta 9-desaturase was found to have a 20-fold higher specific activity and 2-fold lower activation energy than the delta 12-desaturase. In addition, delta 12-desaturase had higher Km app values for oleoyl-CoA and for NADH than the equivalent values for delta 9-desaturase. These properties were correlated with a rate-limiting role of delta 12-desaturase in the production of 18:2, the major fatty acid of Neurospora. The delta 12-desaturase also exhibited a higher tolerance to pH changes and to cyanide than did the delta 9-desaturase. Both activities could be measured in the same reaction mixture using stearoyl-CoA as the substrate, indicating a coupling of the two enzymes. Enrichment of cellular membranes of the wild-type Neurospora with 18:0 and 18:1, 18:2, 18:3 fatty acids led to the conclusion that the presence of excess substrate in the membrane induces activation of the appropriate desaturase. These experiments also suggested that the membrane fluidity, as determined by the degree of unsaturation of membrane fatty acids, may influence the activities of the desaturating enzymes. Perturbation of the polar head groups of the membrane phospholipids indicated that the correct composition of anionic phospholipids is an absolute requirement for the function of both desaturases. These studies show that the activities of the delta 9-desaturase and the delta 12-desaturase are regulated by a variety of factors and that the delta 12-desaturase is subjected to less stringent controls than the delta 9-desaturase.     

Genes acting in longevity-related pathways in the endoparasitoid,Pteromalus puparum

Among insects, lifespans vary over a broad range, from the short-lived mayflies to the 17-year periodical cicadas. Generally, lifespans are determined by a phase in life, the reproductive lifespan, which varies among species. Numerous pathways, such as the insulin/insulin-like growth factor signaling pathway, the target of rapamycin pathway and the mitogen-activated protein kinase/extracellular signal-regulated kinases pathways, influence aging and lifespan. Components of these pathways were identified as lifespan-related genes, including genes mediating growth, metabolism, development, resistance, and other processes. Many age-related genes have been discovered in fruit flies, honeybees, and ants among other insect species. Studies of insect aging and longevity can help understand insect biology and develop new pest management technologies. In this paper, we interrogated the new Pteromalus puparum genome, from which we predicted 133 putative lifespan-related genes based on their homology with known lifespan-related genes of Drosophila melanogaster. These genes function in five signaling pathways and three physiological processes. The conserved domain structures of these genes were predicted and their expression patterns were analyzed. Amino acid sequence alignments and domain structure analysis indicate that most components remain conserved across at least six insect orders. The data in this paper will facilitate future work on parasitoid lifespans, which may have economic value in biocontrol programs.     

Selection and analysis of superoxide dismutase mutants of Neurospora.

A survey of 12 genetically distinct, heat-sensitive mutants of Neurospora revealed three (un-1, un-3, and un-17) that are specifically deficient in the superoxide dismutase (SOD) isozymes SOD-2 (mitochondrial), SOD-3 (mitochondrial), SOD-4 (exocellular), respectively. Genetic analysis of the three mutants indicates that the enzyme deficiencies are probably the cause of the heat-sensitive phenotype. The phenotypes of the mutants are (1) no growth at the normally optimal temperature 35 degrees C and comparatively inferior growth at 15-30 degrees C; (2) inferior resistance to the oxidants paraquat or oxygen; (3) female sterility; and (4) inferior conidial viability and longevity. Paraquat or O2 inhibition is alleviated respectively by desferrioxamine-Mn (a SOD mimic) and tocopherol. Diverse antioxidants, including tocopherol, are therapeutic for the heat-sensitive and female-sterile phenotypes, and for inferior growth of wild type at stressfully high temperatures. The results support previous theories that heat stress is a form of oxyradical/oxidant stress and that antioxidant enzymes such as SOD are essential for normal growth, development, and longevity. Since the three genes may encode the three enzymes and are not closely linked to either one another or the family of antioxidant-enzyme regulatory genes Age-1, the latter apparently trans-regulate their expression.     

Cloning and expression of genes related to the sucrose-metabolizing enzymes and carbohydrate changes in peach

To shed light on the relationship between sucrose metabolism and expression of genes related to sucrose-metabolizing enzymes, six genes encoding sucrose-metabolizing enzymes were isolated, and the levels of four main carbohydrates and related enzyme activities as well as the expression of these six genes were determined in fruits, leaves and phloem-enriched fraction throughout peach fruit development. Sucrose content in mature fruit ranked first followed by glucose, fructose and sorbitol in that order, while sorbitol was the highest and sucrose lowest in phloem-enriched fraction and leaves. Glucose and fructose had similar change patterns throughout fruit development. Cloning results reveal that the nucleotide sequences of the six genes have high similarity to corresponding genes isolated from other plants. In addition, the expression of these genes and the levels of related enzyme activities varied with tissue and stage of fruit development, suggesting a complexity in relationships between carbohydrates, enzymes activities and related gene expression. Sucrose phosphate synthase maybe a key enzyme involved in sucrose synthesis while sucrose synthase may mainly be responsible for sucrose synthesis in peach fruits at later stages of development. Further studies are needed to genetically and physiologically characterize these genes and enzymes in peach and to gain a better understanding of their functions and relationship with carbohydrate metabolism.     

Gene flow simulations demonstrate resistance of long-lived species to genetic erosion from habitat fragmentation

Habitat fragmentation restricts the movement of individuals across a landscape. In terrestrial and aquatic systems, barriers to movement can modify population and community dynamics at local or regional scales. This study contrasted life history traits related to lifespan with habitat fragmentation to determine impacts on species population genetic structure in the Neuse River Basin, USA. For this, we simulated gene flow among evenly-spaced populations in a river network and tracked individual and population genetics for 200 years. The modeled scenarios represent a full cross between five life history strategies and four riverscapes representing varying degrees of fragmentation. The five life history strategies include species (based on freshwater mussels) with average lifespans ranging from 10 to 50 years and age at maturity from 2 to 6 years. The movement landscapes included a (1) panmictic, (2) stepping-stone landscape allowing movement to only neighboring populations during each dispersal event, (3) partially-fragmented landscape divided by dams currently in the network, and (4) fully-fragmented landscape. Results suggest species with shorter lifespans have higher population genetic structure in fragmented landscapes than species with longer lifespans. Furthermore, species with shorter lifespans in highly fragmented landscapes may be harboring genetic degradation or decline as allele fixation and loss. Although anthropogenic fragmentation of many river systems is only 100–200 years old, the simulation indicates that species can respond genetically in that period of time. Additionally, the time frame of the simulation suggests that genetic impacts of habitat fragmentation in some species present in the Neuse River Basin may not yet be manifesting and restoration activities could be successful.     

Functional analysis of pathogenicity genes in a genomics world

Genome-wide mutational and expression analyses have been performed in yeast and provide a model for large-scale analysis of gene function in filamentous fungi. The recent completion of the Neurospora crassa genome offers a resource for comparative analysis with plant pathogenic filamentous fungi. These advances have important implications for molecular genetic studies of pathogenicity genes.     

Regulation of amino acid utilization in Neurospora crassa: effect of nmr-1 and ms-5 mutations.

The effect of the nmr-1 and ms-5 mutations, which lead to insensitivity to glutamine-mediated nitrogen metabolite repression, was examined with respect to extracellular deaminase production by Neurospora crassa. Deaminase production normally requires nitrogen limitation, but these mutations eliminated this requirement and allowed production of deaminase activity under nitrogen metabolite repressing conditions. Demonstration of normal glutamine transport by both strains eliminated the possibility that these mutations exerted their effects through repressor exclusion. We have proposed a new working model for nitrogen regulation in Neurospora based on the findings that these mutations affected a nitrogen-regulated activity in addition to those activities originally reported and that the mutations are genetically very closely linked and likely allelic.     

The rhythms of life: circadian output pathways in Neurospora

Research in Neurospora crassa pioneered the isolation of clock-controlled genes (ccgs), and more than 180 ccgs have been identified that function in various aspects of the fungal life cycle. Many clock-controlled genes are associated with damage repair, stress responses, intermediary metabolism, protein synthesis, and development. The expression of most of these genes peaks just before dawn and appears to prepare the cells for the desiccation, mutagenesis, and stress caused by sunlight. Progress on characterization of the output signaling pathways from the circadian oscillator mechanism to the ccgs is discussed. The authors also review evidence suggesting that, similar to other clock model organisms, a connection exists between the redox state of the cell and the Neurospora clock. The authors speculate that the clock system may sense not only light but also the redox potential of the cell through one of the PAS domains of the core clock components WC-1 or WC-2.     

Comparison of the mitochondrial endonucleases from Neurospora crassa and Saccharomyces cerevisiae

The endonucleases from Neurospora crassa and Saccharomyces cerevisiae are not closely related antigenically. They also differ with respect to their activity at pH 8, their degree of hydrophobicity, and their sensitivity to elevated temperatures. However, the two nucleases have similar specific activities, are inhibited by EDTA, and have nearly identical substrate specificities. Since the enzymes also have the same mode of action and intracellular location, these similarities may indicate that they have the same physiological role despite their structural differences.     

Human-Like Eukaryotic Translation Initiation Factor 3 from Neurospora crassa

Eukaryotic translation initiation factor 3 (eIF3) is a key regulator of translation initiation, but its in vivo assembly and molecular functions remain unclear. Here we show that eIF3 from Neurospora crassa is structurally and compositionally similar to human eIF3. N. crassa eIF3 forms a stable 12-subunit complex linked genetically and biochemically to the 13^th subunit, eIF3j, which in humans modulates mRNA start codon selection. Based on N. crassa genetic analysis, most subunits in eIF3 are essential. Subunits that can be deleted (e, h, k and l) map to the right side of the eIF3 complex, suggesting that they may coordinately regulate eIF3 function. Consistent with this model, subunits eIF3k and eIF3l are incorporated into the eIF3 complex as a pair, and their insertion depends on the presence of subunit eIF3h, a key regulator of vertebrate development. Comparisons to other eIF3 complexes suggest that eIF3 assembles around an eIF3a and eIF3c dimer, which may explain the coordinated regulation of human eIF3 levels. Taken together, these results show that Neurospora crassa eIF3 provides a tractable system for probing the structure and function of human-like eIF3 in the context of living cells.     

Genetic Determinants of Circadian Rhythmicity in Neurospora

Timex, a strain of Neurospora crassa which exhibits a circadian rhythm of conidia formation in growth-tube cultures, has been found to differ from wild-type strains by two genes. One gene, inv, is responsible for an invertase deficiency, whereas the second gene, bd, is of unknown function. Both genes map independently from other genes known to induce Neurospora rhythmicity. The inv gene is not essential for the timex phenotype because bd strains express that phenotype on certain media. Although inv strains do exhibit some rhythmicity of their own, the rhythmicity apparently is not a direct result of the invertase deficiency, since there is no correlation between invertase level and rhymicity in 29 strains tested. Of the 29 strains tested, 20 exhibited some rhythmicity in growth-tube cultures, suggesting that morphological manifestations of rhythmicity in Neurospora may result from the function or the loss of function of numerous genes, or both. There was no correlation in these strains between rhythmicity and (i) genetic background; (ii) geographical origin; or (iii) nutritional requirements.     

Understanding circadian rhythmicity in Neurospora crassa: from behavior to genes and back again

Circadian clocks have been described in organisms ranging in complexity from unicells to mammals, in which they function to control daily rhythms in cellular activities and behavior. The significance of a detailed understanding of the clock can be appreciated by its ubiquity and its established involvement in human physiology, including endocrine function, sleep/wake cycles, psychiatric illness, and drug tolerances and effectiveness. Because the clock in all organisms is assembled within the cell and clock mechanisms are evolutionarily conserved, simple eukaryotes provide appropriate experimental systems for dissecting the clock. Significant progress has been made in deciphering the circadian system in Neurospora crassa using both genetic and molecular approaches, and Neurospora has contributed greatly to our understanding of (1) the feedback cycle that comprises a circadian oscillator, (2) the mechanisms by which the clock is kept in synchrony with the environment, and (3) the genes that reside in rhythmic output pathways. Importantly, the lessons learned in Neurospora are relevant to our understanding of clocks in higher eukaryotes.     

What's in the genome of a filamentous fungus? Analysis of the Neurospora genome sequence

The German Neurospora Genome Project has assembled sequences from ordered cosmid and BAC clones of linkage groups II and V of the genome of Neurospora crassa in 13 and 12 contigs, respectively. Including additional sequences located on other linkage groups a total of 12 Mb were subjected to a manual gene extraction and annotation process. The genome comprises a small number of repetitive elements, a low degree of segmental duplications and very few paralogous genes. The analysis of the 3218 identified open reading frames provides a first overview of the protein equipment of a filamentous fungus. Significantly, N.crassa possesses a large variety of metabolic enzymes including a substantial number of enzymes involved in the degradation of complex substrates as well as secondary metabolism. While several of these enzymes are specific for filamentous fungi many are shared exclusively with prokaryotes.     

Induction of drug metabolizing enzymes in the liver of diabetic mice

The effects of two classical inducers, phenobarbital and 3-methylcholanthrene, have been tested on some liver microsomal drug-metabolizing enzymes (monooxygenases and phase II enzymes) and on benzo(a)pyrene metabolism in genetically (ob/ob) and chemically (streptozotocin) diabetic mice. 1) In ob/ob mice, the basal activities and the inducibility of phase I and phase II enzymes, as well as the electrophoretic pattern of microsomal proteins, were not notably different from those of similarly treated lean mice. 2) A possibly common form of cytochrome P 450 present both in microsomes from steptozotocin-diabetic non-induced mice and in those from phenobarbital-treated non-diabetic mice could explain the increased "phenobarbital-like" enzyme activities in chemically diabetic animals. 3) The increase of monooxygenase activities produced by streptozotocin treatment is partially depressed by 3-methylcholanthrene, probably as a result of the dilution of "phenobarbital-like" cytochrome P 450 forms by 3-methylcholanthrene-induced cytochrome P 448. 4) The increased formation of the most carcinogenic metabolites of benzo(a)pyrene, and the slight decrease of phase II conjugation enzyme activities, may add their deleterious effects in 3-methylcholanthrene-induced streptozotocin-diabetic animals.     

Metagenomic analysis of novel lignocellulose-degrading enzymes from higher termite guts inhabiting microbes

A metagenomic fosmid library was constructed from genomic DNA isolated from the microbial community residing in hindguts of a wood-feeding higher termite (Microcerotermes sp.) collected in Thailand. The library was screened for clones expressing lignocellulolytic activities. Fourteen independent active clones (2 cellulases and 12 xylanases) were obtained by functional screening at pH 10.0. Analysis of shotgun-cloning and pyrosequencing data revealed six ORFs, which shared less than 59% identity and 73% similarity of their amino acid sequences with known cellulases and xylanases. Conserved domain analysis of these ORFs revealed a cellulase belonging to the glycoside hydrolase family 5, whereas the other five xylanases showed significant identity to diverse families including families 8, 10, and 11. Interestingly, one fosmid clone was isolated carrying three contiguous xylanase genes that may comprise a xylanosome operon. The enzymes with the highest activities at alkaline pH from the initial activity screening were characterized biochemically. These enzymes showed a broad range of enzyme activities from pH 5.0 to 10.0, with pH optimal of 8.0 retaining more than 70% of their respective activities at pH 9.0. The optimal temperatures of these enzymes ranged from 50 degrees C to 55 degrees C. This study provides evidence for the diversity and function of lignocellulose-degrading enzymes in the termite gut microbial community, which could be of potential use for industrial processes such as pulp biobleaching and denim biostoning.