The genome of the filamentous fungus Neurospora crassa contains a single gene encoding a heterotrimeric G-protein  subunit, gnb-1. The predicted GNB-1 protein sequence is most identical to G proteins from the filamentous fungi Cryphonectria parasitica and Aspergillus nidulans. N. crassa GNB-1 is also 65% identical to the human GNB-1 protein but only 38 and 45% identical to G proteins from budding and fission yeasts. Previous studies in animal and fungal systems have elucidated phenotypes of G null mutants, but little is known about the effects of G loss on G␣ levels. In this study, we analyzed a gnb-1 deletion mutant for cellular phenotypes and levels of the three G␣ proteins. ⌬gnb-1 strains are femalesterile, with production of aberrant fertilized reproductive structures. ⌬gnb-1 strains conidiate more profusely and have altered mass on solid medium. Loss of gnb-1 leads to inappropriate conidiation and expression of a conidiation-specific gene during growth in submerged culture. Intracellular cyclic AMP levels are reduced by 60% in vegetative plate cultures of ⌬gnb-1 mutants. Loss of gnb-1 leads to lower levels of the three G␣ proteins under a variety of conditions. Analysis of transcript levels for the gna-1 and gna-2 G␣ genes in submerged cultures indicates that regulation of G␣ protein levels by gnb-1 is posttranscriptional. The results suggest that GNB-1 directly regulates apical extension rate and mass accumulation. In contrast, many other ⌬gnb-1 phenotypes, including female sterility and defective conidiation, can be explained by altered levels of the three N. crassa G␣ proteins.Heterotrimeric G proteins (G␣␥) transmit external signals sensed by seven-helix transmembrane receptors, leading to a variety of physiological responses (reviewed in references 12, 17, and 38). In the inactive state, G␣, G, and G␥ subunits are in association, with GDP bound to G␣. Ligand-induced conformational changes in its coupled receptor cause the G protein to dissociate into a GTP-bound G␣ and the G␥ heterodimer. Both of these complexes can activate or inhibit downstream effectors, thus triggering an array of cellular responses (reviewed in reference 17). Characterized G␥ effectors include adenylyl cyclases, phospholipase A2, phospholipase C, Na ϩ , Ca 2ϩ , and K ϩ channels, and tyrosine and serine/threonine protein kinases (reviewed in references 8 and 17). Hydrolysis of GTP by the G␣ subunit leads to reformation of the inactive heterotrimeric form.G proteins are important for environmental and cell-type signaling in yeasts and filamentous fungi. In the budding yeast Saccharomyces cerevisiae, the G subunit Ste4p functions as a positive regulator of the pheromone response in haploid cells by recruiting the Ste20p mitogen-activated protein kinase kinase kinase kinase (MAPKKKK) to the Ste11p MAPKKK on the Ste5p scaffold (reviewed in reference 14). The Git5 G protein from Schizosaccharomyces pombe was originally thought to participate in the mating pathway through its association with the G␣ Gpa1 (25). However, accum...
Heterotrimeric G proteins, consisting of α, β, and γ subunits, transduce environmental signals through coupling to plasma membrane-localized receptors. We previously reported that the filamentous fungus Neurospora crassa possesses a Gα protein, GNA-1, that is a member of the Gαi superfamily. Deletion of gna-1 leads to defects in apical extension, differentiation of asexual spores, sensitivity to hyperosmotic media, and female fertility. In addition, Δgna-1 strains have lower intracellular cAMP levels under conditions that promote morphological abnormalities. To further define the function of GNA-1 in signal transduction in N. crassa, we examined properties of strains with mutationally activated gna-1 alleles (R178C or Q204L) as the only source of GNA-1 protein. These mutations are predicted to inhibit the GTPase activity of GNA-1 and lead to constitutive signaling. In the sexual cycle, gna-1R178C and gna-1Q204L strains are female-fertile, but produce fewer and larger perithecia than wild type. During asexual development, gna-1R178C and gna-1Q204L strains elaborate abundant, long aerial hyphae, produce less conidia, and possess lower levels of carotenoid pigments in comparison to wild-type controls. Furthermore, gna-1R178C and gna-1Q204L strains are more sensitive to heat shock and exposure to hydrogen peroxide than wild-type strains, while Δgna-1 mutants are more resistant. In contrast to Δgna-1 mutants, gna-1R178C and gna-1Q204L strains have higher steady-state levels of cAMP than wild type. The results suggest that GNA-1 possesses several Gβγ-independent functions in N. crassa. We propose that GNA-1 mediates signal transduction pathway(s) that regulate aerial hyphae development and sensitivity to heat and oxidative stresses, possibly through modulation of cAMP levels.
Heterotrimeric (abc) G proteins interact with sensory receptors to transduce signals to downstream eectors in eukaryotes. We previously reported that GNA-1 from Neurospora crassa is a microbial member of the Ga i family found in higher organisms. Deletion of gna-1 leads to female sterility, slower growth rates on normal and hyperosmotic solid medium, and increased resistance to heat and oxidative stress. In this study we compare mammalian genes for proteins of the Ga i subfamily (Ga i , Ga o , Ga t and Ga z ), and Ga s (which is not a member of the Ga i family) with the N. crassa gna-1 gene with respect to their ability to complement Dgna-1 phenotypes. Northern analysis detected full-length transcripts of all these genes, except that for Ga i , in N. crassa transformants. Measurements of pertussis toxin-catalyzed ADP-ribosylation and Western analysis showed that the GNA-1, Ga z , Ga o and Ga s proteins were present in the respective transformed strains. Strains in which the mammalian Ga protein could be detected were subjected to phenotypic testing. During the vegetative cycle, none of the mammalian Ga genes complemented the thermotolerance phenotype of Dgna-1. However, the three expressed mammalian Ga genes achieved at least partial complementation of the defects in vegetative apical extension rate. cAMP levels did not correlate with restoration of vegetative growth rate by the mammalian genes. During the sexual cycle, Ga o was the only mammalian Ga gene that rescued the defect in female fertility characteristic of Dgna-1 strains. Alignment of GNA-1, Ga z , Ga o and Ga s protein sequences revealed correlations between the observed complementation pattern and the degree of identity to GNA-1 in various functional motifs. The ®nding that Ga z gave the best restoration of vegetative growth but could not restore normal female fertility implies that GNA-1 regulates dierent pathways that are important for vegetative and sexual growth in N. crassa.
Hemophilia B is an X-linked recessive bleeding disorder caused by diverse mutations throughout the F9 gene. The same F9 mutation may result in different degrees of clotting factor deficiency. The aim of this study was to investigate the pathogenesis of two hemophilia B patients with different severity in a family. A family with two hemophilia B patients was recruited in this study. Coagulation assays, activities of FVIII (FVIII:C) and FIX (FIX:C) were evaluated. All of the exons and intron exon boundaries of the F9 gene were amplified by PCR and analyzed by direct sequencing. The proband, 12-year-old boy with moderate bleeding history, had manifest prolonged activated partial thromboplastin time (98.1 s) and markedly decreased FIX activity (1%). His maternal uncle presented slightly prolonged activated partial thromboplastin time (48.2 s) and mildly decreased FIX activity (15.2%). Molecular genetic analysis of F9 revealed that they were hemizygous for a novel missense mutation, c.157G>C (p.Glu53Gln). Our study widens the mutation spectrum of the FIX gene. In addition, this report provides a specific case associated with genotype and phenotype heterogeneity of hemophilia B.
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