We have identified a new gene encoding the G protein ␣ subunit, gna-3, from the filamentous fungus Neurospora crassa. The predicted amino acid sequence of GNA-3 is most similar to the G␣ proteins MOD-D, MAGA, and CPG-2 from the saprophytic fungus Podospora anserina and the pathogenic fungi Magnaporthe grisea and Cryphonectria parasitica, respectively. Deletion of gna-3 leads to shorter aerial hyphae and premature, dense conidiation during growth on solid medium or in standing liquid cultures and to inappropriate conidiation in submerged culture. The conidiation and aerial hypha defects of the ⌬gna-3 strain are similar to those of a previously characterized adenylyl cyclase mutant, cr-1. Supplementation with cyclic AMP (cAMP) restores wild-type morphology to ⌬gna-3 strains in standing liquid cultures. Solid medium augmented with exogenous cAMP suppresses the premature conidiation defect, but aerial hypha formation is still reduced. Submergedculture conidiation is refractory to cAMP but is suppressed by peptone. In addition, ⌬gna-3 submerged cultures express the glucose-repressible gene, qa-2, to levels greatly exceeding those observed in the wild type under carbon-starved conditions. ⌬gna-3 strains exhibit reduced fertility in homozygous crosses during the sexual cycle; exogenous cAMP has no effect on this phenotype. Intracellular steady-state cAMP levels of ⌬gna-3 strains are decreased 90% relative to the wild type under a variety of growth conditions. Reduced intracellular cAMP levels in the ⌬gna-3 strain correlate with lower adenylyl cyclase activity and protein levels. These results demonstrate that GNA-3 modulates conidiation and adenylyl cyclase levels in N. crassa.G-protein-coupled receptors (GPCRs) are a family of seven transmembrane helix receptors that bind ligands such as neurotransmitters, pheromones, and odorants. GPCRs are associated with heterotrimeric G proteins, consisting of ␣, , and ␥ subunits (for a review, see reference 82). In the inactive state, the heterotrimer is docked at the receptor and GDP is bound to the G␣ subunit. Binding of a ligand activates the receptor, leading to the exchange of GDP for GTP on G␣ and the subsequent dissociation of G␣-GTP from the G␥ moiety. G␣-GTP and the G␥ dimer can activate downstream effectors, such as enzymes and ion channels, to produce a response to the signal (for a review, see reference 7). The response is terminated and the cycle is completed with hydrolysis of GTP by G␣. The GDP-bound G␣ protein reassociates with G␥, and the heterotrimer is then able to bind to the receptor to await the next cycle of activation (for reviews, see references 10 and 20).Neurospora crassa is a filamentous fungus that has a complex life cycle due to its ability to produce both asexual and sexual spores (for a review, see reference 71). Grown with adequate nitrogen, N. crassa remains in the asexual cycle and extends basal hyphae to form a complex, intertwined network called a mycelium. Asexual spores, or conidia, are produced by two pathways: macroconidiation and micr...
Growth and development are regulated using cyclic AMP (cAMP)-dependent and -independent pathways in Neurospora crassa. The cr-1 adenylyl cyclase mutant lacks detectable cAMP and exhibits numerous defects, including colonial growth habit, short aerial hyphae, premature conidiation on plates, inappropriate conidiation in submerged culture, and increased thermotolerance. Evidence suggests that the heterotrimeric G␣ protein GNA-1 is a direct positive regulator of adenylyl cyclase. ⌬gna-1 strains are female-sterile, and ⌬gna-1 strains have reduced apical extension rates on normal and hyperosmotic medium, greater resistance to oxidative and heat stress, and stunted aerial hyphae compared to the wild-type strain. In this study, a ⌬gna-1 cr-1 double mutant was analyzed to differentiate cAMP-dependent and -independent signaling pathways regulated by GNA-1. ⌬gna-1 cr-1 mutants have severely restricted colonial growth and do not produce aerial hyphae on plates or in standing liquid cultures. Addition of cAMP to plates or standing liquid cultures rescues cr-1, but not ⌬gna-1 cr-1, defects, which is consistent with previous results demonstrating that ⌬gna-1 mutants do not respond to exogenous cAMP. The females of all strains carrying the ⌬gna-1 mutation are sterile; however, unlike cr-1 and ⌬gna-1 strains, the ⌬gna-1 cr-1 mutant does not produce protoperithecia. The ⌬gna-1 and cr-1 mutations were synergistic with respect to inappropriate conidiation during growth in submerged culture. Thermotolerance followed the order wild type < ⌬gna-1 < cr-1 ؍ ⌬gna-1 cr-1, consistent with a cAMP-dependent process. Taken together, the results suggest that in general, GNA-1 and CR-1 regulate N. crassa growth and development using parallel pathways, while thermotolerance is largely dependent on cAMP.Development in fungal systems frequently occurs in response to specific environmental cues and stressors. In the presence of abundant nutrients, the filamentous fungus Neurospora crassa extends hyphae that elongate and fuse to form the multicellular mycelium (for a review, see reference 51). Desiccation or nutrient deprivation causes the mycelium to differentiate aerial hyphae that give rise to conidiophores and multinucleate asexual spores, macroconidia (referred to here as conidia). Elaboration of conidiophores and production of conidia require an air-water interface; however, submerged cultures can be induced to undergo conidiation by carbon or nitrogen starvation or exposure to high temperatures (7,18,43,55). Nitrogen limitation initiates the sexual cycle by stimulating production of female reproductive structures, or protoperithecia (reviewed in reference 44). Fertilization by a conidium or hypha of the opposite mating type results in formation of the fertilized structure (perithecium), within which sexual spores (ascospores) develop. Mature ascospores are subsequently ejected from the perithecium in the direction of blue light.Heterotrimeric GTP-binding proteins, consisting of ␣, , and ␥ subunits, transduce various environmental signals ...
Heterotrimeric G␣ proteins play a critical role in regulating growth and differentiation in filamentous fungi. No systematic analysis of functional relationships between subunits has been investigated. This study explores the relative contributions of Neurospora crassa G␣ subunits, gna-1, gna-2, and gna-3, in directing development by analyzing strains deleted for various combinations of these genes. Although viable, mutants lacking all G␣ subunits or gna-1 and gna-3 are severely restricted in apical growth, forming small colonies. These strains form little aerial hyphae during asexual development on solid medium and exhibit inappropriate sporulation in submerged cultures. Similar to all strains carrying the ⌬gna-1 mutation, these mutants are female sterile. Defects attributed to gna-2 are observed only in conjunction with the loss of gna-1 or gna-3, suggesting a minor role for this G␣ in N. crassa biology. Results from analysis of adenylyl cyclase and epistatic studies with the cAMP-dependent protein kinase regulatory subunit (mcb) indicate separate functions for GNA-1 and GNA-3 in cAMP metabolism and additional cAMP-independent roles for GNA-1. These studies indicate that although G␣ subunits are not essential for viability in filamentous fungi, their loss results in an organism that cannot effectively forage for nutrients or undergo asexual or sexual reproduction.
Two human genes were assigned to chromosomes. Cartilage-derived morphogenic protein 1 (CDMPl-PEN) was assigned to chromosome 20 and asialoglycoprotein receptor 2 (ASGR2) was assigned to one of six different chromosomes using the polymerase chain reaction (PCR) and gel electrophoresis (Chang, Spiess). PCR is a DNA amplification technique in which a specific sequence is exponentially amplified through the action of a thermostable enzyme that polymerizes complementary nucleotides to a single stranded DNA template to extend primers used to locate the gene by complementary base pairing. Detection is made by analyzing the PCR product using gel electrophoresis, a separation technique in which DNA is caused to migrate through a semisolid matrix in an electrical current. Chromosomal assignment was made if the primers located the unassigned gene in the chromosomal DNA; exponentially amplified the gene; and produced discemable bands using gel electrophoresis. Data collected in this research project will be beneficial to the Human Genome Project. The Human Genome Project is a worldwide , collaborative research initiative to produce physical, genetic, and cytogenetic maps of the human genome and, ultimately, identify, sequence, and assign all 50,000 to 100,000 human genes (Collins). Genes successfully assigned to chromosomes will provide researchers with the necessary data to determine the specific location of the gene on the chromosome (Adams).
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