Structural organization of the Z-line protein, alpha-actinin, in developing skeletal muscle cells

The vegetative growth of the adenylyl cyclase-deficient mutant cr-1 (crisp), of Neurospora crassa, resembled a conidiogenic microcycle. It was demonstrated that an enzyme which is exclusively confined to conidia in wild-type strains, i.e., nicotinamide adenine dinucleotide (phosphate) glycohydrolase (NAD(P)ase; EC 3.2.2.6), was continuously released in the culture medium by the mutant. NAD(P)ase activity of agitated cr-1 cultures was much lower than that of standing cultures; nevertheless, the enzyme was actively produced immediately after agitation was stopped. Supplementation of the growth medium with cyclic AMP normalized the morphological phenotype of the cr-1 mutant and drastically reduced NAD(P)ase production. These results suggest that NAD(P)ase regulation is somehow dependent on cyclic AMP metabolism. However, the effect of the nucleotide over the enzyme does not appear to be direct, since other crisp mutants, cr-2 and cr-3, which also overproduced NAD(P)ase, were completely unresponsive to cyclic AMP. These strains possess normal adenylyl cyclase activity.     

Regulation of fructose 2,6-bisphosphate levels in Neurospora crassa

Both wild type and cr-1 mutant (adenylate cyclase and cyclic AMP-deficient) strains of Neurospora crassa contain fructose 2,6-bisphosphate at levels of 27 nmol/g dry tissue weight. This level decreases by about 50% in both strains upon depriving the cells of carbon or nitrogen sources for 3 h. An increase in cyclic AMP levels produced by addition of lysine to nitrogen-starved cells produced no increase in fructose 2,6-bisphosphate levels. Both strains respond to short-term addition of salicylate, acetate, or 2,4-dinitrophenol with an increase in fructose 2,6-bisphosphate. Thus, the above-described regulation of fructose 2,6-bisphosphate levels is cyclic AMP-independent. A suspension of the wild type produces a transient increase of fructose 2,6-bisphosphate in response to administration of glucose, whereas the mutant strain does not respond unless it is fed exogenous cyclic AMP. Substitution of acetate for sucrose as a sole carbon source for growth leads to a differential decrease in fructose 2,6-bisphosphate levels between the two strains: the wild type strain has 63% and the cr-1 mutant strain has 37% of the levels of fructose 2,6-bisphosphate on acetate as compared to sucrose-grown controls. This may be the basis for an advantage of cr-1 over wild type in growth on acetate. Thus, although most regulation of fructose 2,6-bisphosphate is cyclic AMP-independent, the levels can be regulated by a combination of carbon source and cyclic AMP levels.     

In vivo control of gluconeogenesis in wild-type Neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp) mutant.

The rate of cycloheximide-resistant incorporation of carbon from [14C]alanine and [14C]acetate into polysaccharidic material was used to study gluconeogenic activity in wild-type Neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp-1) mutant. The wild-type efficiently utilized alanine and acetate as gluconeogenic substrates, whereas the mutant used acetate efficiently but was unable to use alanine. Cycloheximide-resistant 14C-incorporating activity was sensitive to carbon catabolite effects (repression and inactivation) in the two strains, which suggested that cyclic AMP metabolism was not involved in these regulatory responses. In the wild type, gluconeogenesis was induced by incubation of the cells in the absence of a carbon source. In contrast, cr-1 required supplementation with acetate. This finding suggested that induction of gluconeogenesis in N. crassa could be mediated by metabolites formed in carbon-starved cells. The cr-1 mutant seemed to be deficient in this process and to depend on an exogenous effector to induce gluconeogenesis. Incubation of cr-1 with cyclic AMP partially overcame the acetate requirement for induction of gluconeogenesis.     

Regulation of lactate/pyruvate ratios by cyclic AMP in Neurospora crassa

Cyclic AMP is thought to have a general role in stimulating the breakdown of carbohydrate reserves and subsequent glycolytic activity. This would be expected to increase the availability of reducing equivalents in the form of cytoplasmic NADH. The current study examines another potential reaction controlling cytoplasmic NADH in the fungus Neurospora crassa, that of lactate dehydrogenase, to determine whether it is also regulated by cyclic AMP. The cr-1, adenylate cyclase and cyclic AMP-deficient mutant, grown with and without exogenous cyclic AMP was compared with an isogenic wild type. The results show that cyclic AMP raises pyruvic acid pools and lowers both lactic acid pools and lactate/pyruvate ratios. It does that, in part or in whole, by lowering lactate dehydrogenase activity. The possibility that cytoplasmic NAD+/NADH is a major target of cyclic AMP control is discussed. The high performance liquid chromatography procedures used in these studies are applicable to the measurement of intracellular pools of tricarboxylic acid cycle and other organic acids.     

Properties of two cyclic nucleotide-deficient mutants of Neurospora crassa.

Studies on the crisp-1 (cr-1), cyclic adenosine 3',5'-monophosphate (cAMP)-deficient mutants of Neurospora crassa were undertaken to characterize the response of these mutants to exogenous cyclic nucleotides and cyclic nucleotide analogs. A growth tube bioassay and a radioimmune assay for cyclic nucleotides yielded the following results. (i) 8-Bromo cAMP and N6-monobutyryl cAMP but not dibutyryl cAMP are efficient cAMP analogs in Neurospora, stimulating mycelial elongation of the cr-1 mutants. Exogenous cyclic guanosine 3'5'-monophosphate (cGMP) also stimulates such mycelial elongation. (ii) Both cAMP levels and cGMP levels found in cr-1 mycelia are lower than those in wild type. However, the levels of both cyclic nucleotides are normal in conidia of cr-1. The data on cr-1 mycelia and those reported earlier in Escherichia coli (M. Shibuya, Y. Takebe, and Y. Kaziro (Cell 12:528-528, 1977) show a previously unexpected relationship between cAMP and cGMP metabolism in microorganisms. The semicolonial morphology of another adenylate cyclase-deficient mutant of Neurospora, frost, was not corrected by exogenous cyclic nucleotides or by phosphodiesterase inhibitors indicating that the frost morphology is probably not caused by low endogenous cAMP levels. The low adenylate cyclase activity and the abnormal morphology of frost may be related separately to the linolenate deficiency reported in the mutant.     

Control of nucleotide and erythroascorbic acid pools by cyclic AMP in Neurospora crassa

UDPglucuronic acid and erythroascorbic acid were identified in extracts of the fungus Neurospora crassa. The concentrations of these two compounds are estimated, in growing wild type N. crassa, to be about 0.10 and 0.28 mumol/ml of cell water, respectively. The pools of these two compounds are regulated by cyclic AMP in Neurospora, both being elevated in the cr-1, adenylate cyclase deficient mutant and both being lowered by exogenous cyclic AMP. The pools of these two compounds are also elevated on nitrogen deprivation. The pools of a large number of other nucleotides are not influenced by cyclic AMP. Possible relationships between the metabolism of UDPglucuronic acid and erythroascorbic acid are discussed. It was found that exogenous cyclic AMP was much more effective in influencing cultures grown at 30-37 degrees C than those grown at 25 degrees C. We suggest that higher temperatures may render Neurospora more permeable to a variety of different compounds.     

Properties of a germination mutant of Phycomyces blakesleeanus

Spores of the Phycomyces blakesleeanus strain S440 germinated only for some 4 to 7% when activated with a heat treatment or with ammonium acetate. Contrary to wild type spores, they showed no increase in trehalase activity during or after the activating treatment. This was not due to a variant trehalase or a defective protein kinase but rather to the absence of an increase in cellular cyclic AMP which normally occurs in the wild type. Phosphodiesterase activity in the mutant was comparable to wild type activity and in both strains phosphodiesterase was inactivated by a heat treatment. The phosphodiesterase inhibitor 1-isobutyl, 3-methyl xanthine caused germination and trehalase activation in the wild type but not in the mutant. The results corroborate the importance of cyclic AMP in the breaking of dormancy and the activation of trehalase in this fungus.     

Control of Neurospora crassa morphology by cyclic adenosine 3'', 5''-monophosphate and dibutyryl cyclic adenosine 3'', 5''-monophosphate.

The role of cyclic adenosine 3', 5'-monophosphate (cyclic AMP) in the control of the Neurospora asexual life cycle was studied. Endogenous cyclic AMP levels were 10 to 20 times higher in strains having the wild-type cr-1 allele than in those carrying the mutated allele. In a wild-type strain these levels remained constant throughtout the entire growth period in shaken liquid cultures, except during a short period at the beginning of the stationary growth phase. In this period a marked increase in the cyclic nucleotide level was observed. The culture of cr-1 mutant strains in the presence of cyclic AMP or its dibutyryl derivative restores some morphological properties characteristic of wild-type strains. Specifically these cyclic nucleotides stimulated the rate of mycelial elongation, as well as the differentiation of aerial hyphae.     

Cell biology of conidial anastomosis tubes in Neurospora crassa

Although hyphal fusion has been well documented in mature colonies of filamentous fungi, it has been little studied during colony establishment. Here we show that specialized hyphae, called conidial anastomosis tubes (CATs), are produced by all types of conidia and by conidial germ tubes of Neurospora crassa. The CAT is shown to be a cellular element that is morphologically and physiologically distinct from a germ tube and under separate genetic control. In contrast to germ tubes, CATs are thinner, shorter, lack branches, exhibit determinate growth, and home toward each other. Evidence for an extracellular CAT inducer derived from conidia was obtained because CAT formation was reduced at low conidial concentrations. A cr-1 mutant lacking cyclic AMP (cAMP) produced CATs, indicating that the inducer is not cAMP. Evidence that the transduction of the CAT inducer signal involves a putative transmembrane protein (HAM-2) and the MAK-2 and NRC-1 proteins of a mitogen-activated protein kinase signaling pathway was obtained because ham-2, mak-2, and nrc-1 mutants lacked CATs. Optical tweezers were used in a novel experimental assay to micromanipulate whole conidia and germlings to analyze chemoattraction between CATs during homing. Strains of the same and opposite mating type were shown to home toward each other. The cr-1 mutant also underwent normal homing, indicating that cAMP is not the chemoattractant. ham-2, mak-2, and nrc-1 macroconidia did not attract CATs of the wild type. Fusion between CATs of opposite mating types was partially inhibited, providing evidence of non-self-recognition prior to fusion. Microtubules and nuclei passed through fused CATs.     

Shared and independent roles for a Galpha(i) protein and adenylyl cyclase in regulating development and stress responses in Neurospora crassa

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 Gα protein GNA-1 is a direct positive regulator of adenylyl cyclase. Δgna-1 strains are female-sterile, and Δgna-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 Δgna-1 cr-1 double mutant was analyzed to differentiate cAMP-dependent and -independent signaling pathways regulated by GNA-1. Δgna-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 Δgna-1 cr-1, defects, which is consistent with previous results demonstrating that Δgna-1 mutants do not respond to exogenous cAMP. The females of all strains carrying the Δgna-1 mutation are sterile; however, unlike cr-1 and Δgna-1 strains, the Δgna-1 cr-1 mutant does not produce protoperithecia. The Δgna-1 and cr-1 mutations were synergistic with respect to inappropriate conidiation during growth in submerged culture. Thermotolerance followed the order wild type < Δgna-1 < cr-1 = Δgna-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.     

Coexpression of mutant and wild type protein kinase in lymphoma cells resistant to dibutyryl cyclic AMP.

A mutant clone resistant to dibutyryl cyclic AMP was isolated from S49 mouse lymphoma cells. The mutant expressed a form of cyclic AMP-dependent protein kinase distinguishable from wild type kinase by its decreased sensitivity to activation by cyclic AMP and its increased thermal lability. Hybrids formed between mutant and wild type cells were resistant to dibutyryl cyclic AMP and expressed both mutant and wild type activities in about equal amount. The parent mutant cells also appeared to express wild type kinase activity, but at a lower level. We conclude that wild type S49 cells have and express two identical alleles for the regulatory subunit of protein kinase, one of which has undergone mutation in the mutant cells.     

Replacing the general energy-coupling proteins of the phospho-enol-pyruvate: sugar phosphotransferase system of Salmonella typhimurium with fructose-inducible counterparts results in the inability to utilize nonphosphotransferase system sugars

A Salmonella typhimurium mutant lacking Enzyme I and HPr, general proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), but producing homologues EI(Fructose) and FPr constitutively, did not grow in minimal medium supplemented with non-PTS sugars (melibiose, glycerol, and maltose) in the absence of any trace of Luria-Bertani broth; adding cyclic AMP allowed growth. On melibiose, rapid growth began only when melibiose permease activity had reached a threshold level. Wild-type cultures reached this level within about 2 h, but the mutant only after a 12-14 h lag period, and then only when cyclic AMP had been added to the medium. On a mixture of melibiose and a PTS sugar, permease was undetectable in either the wild type or mutant until the PTS sugar had been exhausted. Permease then appeared, increasing with time, but in the mutant it never reached the threshold allowing rapid growth on melibiose unless cyclic AMP had been added. On rich medium supplemented with melibiose or glycerol, the mutant produced lower (30%) levels of melibiose permease or glycerol kinase compared with the wild type. We propose that poor phosphorylation of the regulatory protein Enzyme IIA(Glucose), leading to constitutive inducer exclusion and catabolite repression in this strain, accounts for these results.     

Regulation of luminescence by cyclic AMP in cya-like and crp-like mutants of Vibrio fischeri.

Mutants of Vibrio fischeri MJ-1 (wild type) apparently deficient in adenylate cyclase (cya-like) or cyclic AMP receptor protein (crp-like) were isolated and characterized. Compared with MJ-1, the mutants produced low levels of luminescence and luciferase. Addition of cyclic AMP restored wild-type levels of luminescence and luciferase in the cya-like mutant but not in the crp-like mutant. The results are consistent with the hypothesis that in V. fischeri cyclic AMP and cyclic AMP receptor protein are required for induction of the luminescence system.     

On the control mechanism of bacterial growth by cyclic adenosine 3',5'-monophosphate

Inhibition of E. coli growth by cyclic adenosine monophosphate is observed in wild type strains cultured in glucose as carbon source, but not in a cyclic AMP receptor protein deficient mutant. A deletion mutant of the adenylate cyclase gene requires cyclic adenosine monophosphate for optimal growth. Using glucose as carbon source, 2 mM cyclic AMP promotes maximal rates of cell multiplication in this mutant; however higher concentrations of the nucleotide inhibit growth. Cell multiplication of wild type strains grown in glycerol is not affected by cyclic adenosine monophosphate. Nevertheless, in this carbon source the growth rate of the adenylate cyclase mutant is strongly inhibited by concentrations of this nucleotide beyond 0.1 mM. This suggests that growth inhibition by exogenous cyclic adenosine monophosphate is highly dependent on the intracellular levels of the nucleotide.     

A mutant strain of Polysphondylium pallidum deficient in production of cyclic AMP.

A mutant strain (PN507) of the cellular slime mold Polysphondylium pallidum is described which: (a) is morphogenetically abnormal in stalk formation; (b) secretes unusually low quantities of cyclic AMP; (c) responds to exogenous cyclic AMP in the same manner as wild type, by differentiating stalk cells and synthesizing several specific proteins; (d) complements with other morphogenetic mutants secreting normal amounts of cyclic AMP to produce fruiting structures resembling wild type. The tentative conclusion is that the critical defect of PN507 is low production of cyclic AMP.     

Effect of exogenous cycllc amp on catabolite repression of cellulase formation in aspergillus nidulans

The addition of 1 mM cyclic AMP to induced and repressed cultures of Aspergillus nidulans and its mutant strain (CRR 141) resistant to catabolite repression was fully capable of releasing the wild type from catabolite repression while it caused hyperproduction of cellulases in glycerol repressed cultures. The relief of the catabolite repression was also accompanied by a dramatic drop in enhanced protease levels, thereby indicating that the synthesis of proteases (during the catabolite repression) is under the control of cyclic AMP.     

A catalytic subunit of cyclic AMP-dependent protein kinase, PKAC-1, regulates asexual differentiation in Neurospora crassa

A cyclic AMP (cAMP)-dependent protein kinase pathway has been shown to regulate growth, morphogenesis and virulence in filamentous fungi. However, the precise mechanisms of regulation through the pathway remain poorly understood. In Neurospora crassa, the cr-1 adenylate cyclase mutant exhibits colonial growth with short aerial hyphae bearing conidia, and the mcb mutant, a mutant of the regulatory subunit of cAMP-dependent protein kinase (PKA), shows the loss of growth polarity at the restrictive temperature. In the present study, we isolated mutants of the catalytic subunit of the PKA gene pkac-1 through the process of repeat-induced point mutation (RIP). PKA activity of the mutants obtained through RIP was undetectable. The genome sequence predicts two distinct catalytic subunit genes of PKA, named pkac-1 (NCU06240.1, AAF75276) and pkac-2 (NCU00682.1), as is the case in most filamentous fungi. The results suggest that PKAC-1 works as the major PKA in N. crassa. The phenotype of the pkac-1 mutants included colonial growth, short aerial hyphae, premature conidiation on solid medium, inappropriate conidiation in submerged culture, and increased thermotolerance. This phenotype of pkac-1 mutants resembled to that of cr-1 mutants, except that the addition of cAMP did not rescue the abnormal morphology of pkac-1 mutants. The loss of growth polarity at the restrictive temperature in the mcb mutant was suppressed by pkac-1 mutation. These results suggest that the signal transduction pathway mediated by PKAC-1 plays an important role in regulation of aerial hyphae formation, conidiation, and hyphal growth with polarity.     

Phosphate inhibition of secondary metabolism in Streptomyces hygroscopicus and its reversal by cyclic AMP

Inorganic phosphate inhibited the biosynthesis of the macrolide antibiotic turimycin in different strains of Streptomyces hygroscopicus. In the wild type strain a depression was observed with increasing phosphate concentrations. A total inhibition was found at 0.1 M phosphate. In a high producing mutant a minimum of turimycin production occured when the phosphate concentration was between 5 mM and 10 mM. Above this concentration the antibiotic synthesis increased again but the production period shifted to a later period of cultivation. Addition of inorganic phosphate resulted in an initial increase of intracellular cyclic AMP content. But a second elevation characterizing the normal level of cyclic AMP throughout the growth phase was prevented by phosphate. Exogenous cyclic AMP as well as positive effectors of the adenylyl cyclase system were able to overcome the phosphate suppression. Cyclic AMP abolished the reduction of protein synthesis following phosphate addition and caused the reappearance of a protein band which may be responsible for the turimycin biosynthesis.     

Control of nucleotide and erythroascorbic acid pools by cyclic AMP in Neurospora crassa

UDPglucuronic acid and erythroascorbic acid were identified in extracts of the fungus Neurospora crassa. The concentrations of these two compounds are estimated, in growing wild type N. crassa, to be about 0.10 and 0.28 μmol/ml of cell water, respectively. The pools of these two compounds are regulated by cyclic AMP in Neurospora, both being elevated in the cr-1, adenylate cyclase deficient mutant and both being lowered by exogenous cyclic AMP. The pools of these two compounds are also elevated on nitrogen deprivation. The pools of a large number of other nucleotides are not influenced by cyclic AMP. Possible relationships between the metabolism of UDPglucuronic acid and erythroascorbic acid are discussed. It was found that exogenous cyclic AMP was much more effective in influencing cultures grown at 30–37°C than those grown at 25°C. We suggest that higher temperatures may render Neurospora more permeable to a variety of different compounds.     

An adenosine 3′,5′-monophosphate-requiring mutant of the luminous bacteria Beneckea harveyi

We have isolated a mutant of the luminous bacterium Beneckea harveyi, which requires exogenous adenosine 3′,5′-monphosphate (cyclic AMP) to snnthesize luciferase and emit light. The mutant was pleiotropic, lacking not only the ability to luminesce, but also the capacities to form flagella and the ability to utilize a variety of carbohydrates for growth. All these deficiencies could be corrected by added cyclic AMP. The cyclic AMP-induced de novo synthesis of luciferase was possible only ffter autoinduction had occurred. The induction time by cyclic AMP ranged between 6 and 10 min at 27°C.