Induction of thermotolerance and mitotic recombination by heat-shock in Ustilago maydiss

Summary Wild-type cells of Ustilago maydis which have been briefly heated at 42 °C subsequently acquire resistance to the lethal effects of a higher temperature (48 °C). This induced thermotolerance develops over a 2 h period after treatment at 42 °C and is dependent on protein synthesis. It is distinct from the cellular response to DNA-damaging agents, since mutants deficient in this and other repair processes show the same induced thermotolerance as wild-type cells. However, UV-light treatment does induce some resistance to subsequent heat treatment. A single 48 °C heat treatment increases recombination in heteroallelic diploids, but the same treatment of thermotolerant cells does not stimulate recombination. This suggests that heat treatment can damage DNA, but thermotolerant cells are protected from such damage.     

Heat shock changes the response of the pso3 mutant of Saccharomyces cerevisiae to 8-methoxypsoralen photoaddition

A putative tolerance, induced by heat shock (HS), to the lethal and mutagenic effects of 8-methoxypsoralen (8-MOP) photoaddition and hyperthermia was analyzed in Saccharomyces cerevisiae using the wild-type strain N123 and the isogenic DNA repair-deficient mutant pso3-1. In wild-type cells, the HS (38°C for 1 h) did not modify either the survival or the mutation frequency observed after 8-MOP photoaddition, even though it conferred protection against the lethal effect of hyperthermia (50°C). In the pso3-1 mutant, HS induced an increase of the survival, and a decrease of the mutation frequency, after 8-MOP photoaddition and it also protected against the lethal effect of hyperthermia. The responses induced by HS were specific for 8-MOP photoaddition, since they were not observed after 254 nm ultraviolet-light damage. These results indicate that the protection conferred by HS depends of the type of lesion, and operates through the induction of different repair processes. In the pso3-1 mutant, HS could channel the repair intermediates to and error-free repair pathway.     

Effect of purine antagonists on the cAMP level in lymphocytes and bone marrow lymphoblasts

The effect of the purine receptor ligands N-ethylcarboxamide adenine and adenosine and of the purine antagonists mercaptopurine and azathioprine on the intracellular cAMP content in human peripheral blood lymphocytes and bone marrow lymphoblasts was studied. All preparations tested induced an increase in the cAMP level in peripheral blood lymphocytes. The selective immunosuppressive effect of adenosine antagonists may be due to their ability to modulate the activity of adenylate cyclase in lymphoid cells. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o . 3, pp. 294–296, March, 1995     

Sequence repeat-induced disruption of the major heat-inducible HSP70 gene of Neurospora crassa

The process of repeat-induced point mutation (RIP) was used to disrupt hsps-1, the gene encoding the major heat-inducible member of the HSP70 family of Neurospora crassa. A plasmid DNA, containing an incomplete copy of hsps-1 and the selectable marker qa-2⁺, was introduced into germinated conidia. The sexual progeny of transformants with ectopically integrated hsps-1 DNA was examined for RIP by Southern-blot analysis of MboI- and Sau3A-digested genomic DNA. Progeny strains, showing RIP, were tested for heat shock-responsive expression of hsps-1, by RNA-blot hybridization and Western-blot analysis, as well as for thermotolerance. Isolates with RIP showed low levels of hsps-1 mRNA and a lack of induction of HSP70 protein by heat shock, accompanied by only a marginal decrease in the acquisition of thermotolerance.     

Significance of adenylate cyclase system of the liver in its chronic diseases

The activity of adenylate cyclase in the hepatocyte plasma membranes, content of cAMP, and cAMP/cGMP ratio in the liver and blood plasma are decreased in patients with chronic liver diseases (fatty dystrophy, chronic hepatitis, and cirrhosis). This decrease depends on the disease severity and is most pronounced in cirrhosis. The sensitivity of liver adenylate cyclase to insulin and glucagon is changed. It is concluded that disorders in the adenylate cyclase system are an important molecular mechanism in the pathogenesis of chronic liver diseases. Translated fromByulleten' Eksperimental'noi Biologii i Medistiny, Vol. 124, No. 4, pp. 450–453, April 1998     

Mutations affecting cyclic phosphodiesterases and adenylate cyclase in Neurospora

Summary Two regulatory mutants in orthophosphateregulated cyclic phosphodiesterase (CPDase), cpd-3 and cpd-4, were isolated and mapped proximal to arg-1 on L.G. IC and distal to urg-12 on L.G. IIR, respectively. cpd-3 showed short aerial hyphae with dense formation of conidia. The morphology was very similar to that of cr-1, cpd-3 and cr-1 had reduced levels of cyclic 3,5-AMP, adenylate cyclase and cPDases (CPDase I, II and III in low phosphate condition) but had elevated levels of cyclic 3,5-GMP. Although cr-1 showed an enhanced level and enhanced activation of heat activated cyclic phosphodiesterase (ha-PDE), this enzyme was not activated in cpd-3. cpd-4, nuc-1 and nuc-2 produced neither CPDase I, II, III, alkaline phosphatase nor ribonuclease N1 in low phosphate media. These mutants did not produce aerial hypha or conidium when grown in low phosphate liquid medium. Activation of ha-PDE occurred in cpd-4, but not in nuc-1 and nuc-2.     

Mutations in the structural gene for cytochrome c result in deficiency of both cytochromes aa 3 and c in Neurospora crassa

The cyt-12-12 mutant of Neurospora crassa is characterized by slow growth and a deficiency of spectrophotometrically-detectable cytochromes aa ₃ and c. Using a sib-selection procedure we have isolated the cyt-12 ⁺ allele from a cosmid library of N. crassa genomic DNA. Characterization of the cyt-12 ⁺ allele reveals that it encodes the structural gene for cytochrome c. DNA sequence analysis of the cyt-12-12 allele revealed a mutation in the cytochrome c coding sequence that results in replacement of a glycine residue, which is invariant in the cytochrome c of other species, with an aspartic acid. Genetic analysis confirms that cyt-12-12 is allelic with the previously-characterized cyc-1-1 mutant, which was also shown to affect the single locus encoding cytochrome c in N. crassa. We suggest that the amount of functional cytochrome c present in mitochondria influences the level of cytochrome aa ₃ .     

Regulation of synthesis and secretion of acid and alkaline phosphatases in Neurospora crassa

Summary We show that N. crassa represses the production of acid phosphatase at pH higher than 8.0, irrespective of the carbon source used, whereas production was stimulated by sucrose at slightly acidic pH. The same profile of acid phosphatase production was observed in the pho-2A, pho-3A, nuc-1A, nuc-2A and preg ^c mutant strains. We also show that acid phosphatase synthesized by the preg ^c mutant strain grown on high phosphate medium has pronounced differences when compared to the enzyme synthesized by the wild-type strain grown on low phosphate medium in terms of heat stability, steady-state kinetic properties and DEAE-cellulose chromatography. In addition, the synthesis and/or secretion of only phosphate-repressible alkaline phosphatase is affected by mutations in acu-1, and acu-5 and acu-7 genes. These results, which indicate distinct pathways for the synthesis and secretion of acid and alkaline phosphatases in N. crassa, contradict the dosage titration model proposed by Metzenberg et al. (1974) whereby the synthesis of these enzymes should occur through a single hierarchical regulatory circuit as a response to phosphate starvation.     

Expression of tyrosinase in vegetative cultures of Neurospora crassa transformed with a metallothionein promoter/protyrosinase fusion gene

Summary Wild-type Neurospora crassa, strain Singapore, was transformed with a N. crassa metallothionein promoter/protyrosinase fusion gene. Transformants produced tyrosinase during vegetative growth, as determined by Western analyses and activity assays. This is in sharp contrast to wild-type strains, where this enzyme is only expressed in situations of starvation or sexual differentiation. Complete integration of a 400 bp metallothionein promoter-fragment leads to constitutive expression of protyrosinase, whereas a 3.6 kb promoter-fragment conferred copper inducibility on the reporter gene in four transformants. A transformant with high constitutive tyrosinase levels was able to produce melanin on complete medium agar plates supplemented with 1 mg/ml L-tyrosine.     

Inversions and recombinations in mitochondrial DNA of the (SG-1) cytoplasmic mutant in two Neurospora species

Summary The mitochondrial DNAs of [SG-1] cytoplasmically-mutant and wild-type strains of Neurospora crassa and Neurospora sitophila were examined by comparative restriction endonuclease analyses. The mtDNA of N. sitophila wild type of Whitehouse differs from type II mtDNA of N. crassa by insertions of 3.3 kb in EcoRI-9, and 1.2 kb in EcoRI-3, and a deletion of 1.1 kb in EcoRI-5. These DNA heteromorphisms provided convenient markers for tracing N. crassa [SG-1] mtDNA during and after its transfer into N. sitophila. The [SG-1] cytoplasmic mutant in both N. crassa and N. sitophila has a distinctive inversion that connects the fragment EcoRI-4 with HindIII-10a. The [SG-1] mtDNA from N. crassa remained essentially intact after it was transferred by crosses into N. sitophila. In each species, a unique second inversion occured in the [SG-1] mtDNA after the transfer was made. In N. sitophila, polar recombination in heteroplasmons between [SG-1] and wild-type preferentially yields strains with mtDNAs that contain the maximum possible number of insertions in the cob and co-1 loci of the EcoRI-3 region of the mitochondrial chromosome.     

Human platelets as the object of investigation of the molecular mechanisms underlying nongenomic effects of glucocorticoid hormones

The effect of hydrocortisone (100 nM–10 μM) on the major biochemical parameters of platelet activity (intracellular free calcium concentration, thromboxane B₂ content, and baseline and stimulated levels of cAMP and cGMP) is examined. The results obtained indicate that the inhibitory effect of glucocorticoids on platelet aggregation is mediated by activation of the adenylate cyclase system and suppression of the calcium response. Presumably, neither guanylate cyclase nor phospholipase A₂-dependent systems are the targets of nongenomic actions of glucocorticoid hormones. Platelets can serve as a convenient tool for the investigation of nongenomic effects of glucocorticoid hormones. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 9, pp. 285–287, September, 1996     

Adenylate cyclase activity of Pseudomonas aeruginosa ExoY can mediate bleb-niche formation in epithelial cells and contributes to virulence

We previously showed that ADP-ribosylation (ADP-r) activity of ExoS, a type III secreted toxin of Pseudomonas aeruginosa, enables bacterial replication in corneal and respiratory epithelial cells and correlates with bacterial trafficking to plasma membrane blebs (bleb-niche formation). Here, we explored another type III secreted toxin, ExoY, for its impact on intracellular trafficking and survival, and for virulence in vivo using a murine corneal infection model. Chromosomal or plasmid-mediated expression of exoY in invasive P. aeruginosa (strain PAO1) enabled bacteria to form and traffic to epithelial membrane blebs in the absence of other known effectors. In contrast, plasmid expression of any of four adenylate cyclase mutant forms of exoY did not enable bleb-niche formation, and bacteria localized to perinuclear vacuoles as for effector-null mutant controls. None of the plasmid-complemented bacteria used in this study showed ADP-r activity in the absence of ExoS and ExoT. In contrast to ADP-r activity of ExoS, bleb-niche formation induced by ExoY's adenylate cyclase activity was not accompanied by enhanced intracellular replication. In vivo results showed that ExoY-adenylate cyclase activity promoted P. aeruginosa corneal virulence in susceptible mice. Together the data show that adenylate cyclase activity of P. aeruginosa ExoY, similarly to the ADP-r activity of ExoS, can mediate bleb-niche formation in epithelial cells. While this activity did not promote intracellular replication in vitro, ExoY conferred increased virulence in vivo in susceptible mice. Mechanisms for bleb-niche formation and relationships to intracellular replication and virulence in vivo require further investigation for both ExoS and ExoY.     

Virulence of a Bordetella pertussis strain expressing a mutant adenylyl cyclase with decreased calmodulin affinity

Bordetella pertussis, the pathogen responsible for whooping cough, produces a toxic calmodulin-sensitive adenylyl cyclase which enters animal cells and increases intracellular cAMP. A point mutant of B. pertussis with abolished adenylyl cyclase catalytic activity was over 1000-fold less pathogenic to newborn mice than wild-type bacteria, demonstrating the importance of the adenylyl cyclase for B. pertussis virulence (Gross et al. ). The B. pertussis adenylyl cyclase is highly sensitive to calmodulin with an apparent K_d for calmodulin of approximately 1 nM. The importance of this high-affinity calmodulin binding for virulence in vivo was examined by the creation of a B. pertussis point mutant (Trp-242 to Glu-242) with 200-fold lower calmodulin affinity than the native enzyme. This mutant B. pertussis strain retained its virulence in a newborn mouse model of pertussis, but the time course for establishment of a lethal infection in vivo was significantly delayed for the mutant strain. These data illustrate that high-affinity calmodulin binding is not obligatory for the activity of this toxin but is important for the rate for establishment of a lethal infection.     

Effect of asphyxia on adenylate cyclase activity in cat brain cortex

Effect of 1–5-min asphyxia on adenylate cyclase activity in cat brain cortex is studied. Adenylate cyclase activity is measured in cortex specimens obtainedex vivo after 1, 2.5, and 5 min of asphyxia, and 30 and 60 min of reoxygenation by radioassay. Stimulating effects of norepinephrine and NaF on adenylate cyclase activity are assessed. Five-min asphyxia induces phasic changes in adenylate cyclase activity: on the 1st min basal activity of the enzyme increases by 97%, after 2.5 min it returns to the initial level, and increases again by 55% on the 5th min of asphyxia. On the 30th and 60th min of reoxygenation after 2.5- and 5-min asphyxia, basal adenylate cyclase activity does not differ from the initial activity. The stimulating effect of norepinephrine and NaF on enzyme activity is weakened after 5 min of asphyxia and 30 min of reoxygenation after 2.5- and 5-min asphyxia. Even short-term asphyxia affects adenylate cyclase activity and modifies the mechanisms of adrenergic signal transduction in the brain cortex in response to oxygen deficiency and probably to hypercapnia as well as during the early reoxygenation period. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 8, pp. 131–134, August, 1997     

Stimulating PACα Increases Miniature Excitatory Junction Potential Frequency at the Drosophila Neuromuscular Junction

Abstract: Photoactivated adenylate cyclase alpha (PACα) is a light-activated adenylate cyclase that was originally cloned from the eye spot of the protozoan Euglena gracilis. PACα has been shown to rapidly increase intracellular cyclic adenosine monophosphate (cAMP) in vivo in Xenopus oocytes and HEK293 cells, increase the spike width in Aplysia sensory neurons, and modify behavior in Drosophila. Using the GAL4 UAS system, we heterologously expressed PACα in motorneurons and quantified the effects of its activation at the neuromuscular junction of the Drosophila third instar wandering larva, a well-characterized model synapse. By recording from body-wall muscle 6, we show that the presynaptic activation of PACα with blue light significantly increased miniature excitatory junction potential (mEJP) frequency in the presence of calcium with a delay of about 1 minute. Similar effects have been observed in previous studies that utilized adenylate cyclase agonists (Forskolin) or membrane-permeable cAMP analogs [dibutyryl cAMP and 4-chlorophenylthio-(CPT)-cAMP] to increase presynaptic cAMP concentrations. PACα′s efficacy in combination with its specificity make it an invaluable tool for the rapid regulation of cAMP in vivo and for investigating the mechanisms by which cAMP can modulate synaptic transmission and neuronal plasticity in Drosophila.     

Basidiomycetousras cDNA functionally replaces its homolog genes in yeast

It was shown by a plasmid exchange procedure that the Ras-encoding cDNA of the basidiomyceteLentinus edodes (namedLeras cDNA) can functionally replace its homolog genes (ScRAS1 andScRAS2) in the yeastSaccharomyces cerevisiae to maintain the viability of an yeast strain containing genetic disruptions of bothRAS genes. The strain replaced by aLeras–cDNA-carrying plasmid, however, grew slower than the strains replaced by aScRAS1– or aScRAS2–carrying plasmid. The intracellular level of cAMP in the strain harboring theLeras–cDNA-carrying plasmid was clearly higher than that of a parental strain which maintains a plasmid carrying theS. cerevisiae cAMP-dependent protein kinase catalytic subunit C₁ gene,TPK1, but was lower than that in a strain harboring anScRAS2–carrying plasmid. These results suggest that theLeras cDNA can complement theras1 ^– ras2^– mutation of yeast by virture of the stimulation of adenylate cyclase activity, although the complementation is not as efficient as that obtained by expressing theScRAS2 gene.     

Isolation of mutants sensitive to 2-amiriopurine and alkylating agents and evidence for the role of DNA methylation in Penicillium chrysogenum

Summary Using high performance liquid chromatography, the presence of N6-methyladenine has been found at a level of 0.1 mol percent in DNA extracted from Penicillium chysogenum. No 5-methylcytosine was detected. A mutant strain HP547, which is sensitive to the lethal effects of N-methyl-N¹-nitro-N-nitrosoguanidine, methylmethane sulphonate and the base analogue 2-aminopurine shows an increased spontaneous mutation rate and no detectable DNA methylation. Comparison of restriction enzyme digests of wild type and undermethylated strains indicated that methylation was occurring at a different sequence to that of the Dam methylase system of E. coli.     

Identification and characterization of four new GCD genes in Saccharomyces cerevisiae

Summary Mutant strains, resistant against the amino acid analogues 5-methyltryptophan, 5-fluorotryptophan and canavanine were isolated, starting with a trp2 leaky auxotrophic strain. Of 10 such strains, only four turned out to be of the general control derepressed (gcd) mutant type. Three other isolates were shown to be defective in the general amino acid permease system, while the remaining three strains displayed low spore viability and were not further investigated. Complementation tests amongst the four new gcd-mutant strains, including strain RH558 gcd2-1 isolated earlier, yielded five complementation groups: GCD2, GCD3, GCD4, GCD5, and GCD6. All mutant strains showed a dual phenotype, which was not separable by wild type backcrosses: constitutive derepression and slow growth. Epistatis of all gcd mutations over gcn1-1, gcn2-1 and gcn3-1 was found with respect to both phenotypes, except for gcd5-1, which was lethal in these combinations. On the other hand gcn4-101 was found to be epistatic over all gcd mutations, but only with respect to the constitutive derepression phenotype, and not to slow growth; again the combination with gcd5-1 was lethal. Mutation gcd2-1 was mapped on chromosome VII, 50 cM from leu1 and 22 cM from ade6. A new model is discussed, in which GCD-genes are involved in the amino acid uptake into the vacuoles.     

Cloning and preliminary characterization of a molybdenum cofactor gene of Neurospora crassa

Summary A Neurospora crassa library, constructed in a derivative of the plasmid pBR322 (pRK9), was used to transform two E. coli ch1D molybdenum cofactor mutants (ch1D, ch1D::Mu). Subsequently, one transformant from each of three independent transformation experiments was restriction mapped. All three transformants had an identical N. crassa DNA insert (4.2 kb). Southern Blot analysis with one of the plasmids (pMoCo, 1:4) showed hybridization to a single band of N. crassa genomic DNA. When pMoCo plasmid (1:4) was used to transform various E. coli nitrate reductase mutants (ch1A, ch1B, ch1C, ch1D, ch1E, ch1G and ch1M), the pMoCo plasmid was capable of restoring E. coli nitrate reductase activity to only the ch1D mutant. In vitro reconstitution experiments using wild-type, ch1D and ch1D; pMoCo cell-free extracts as a source of molybdenum cofactor (MoCo) were performed with the N. crassa MoCo mutants nit-1, nit-7 and nit-8. MoCo from wild-type E. coli cell-free extracts was capable of reconstituting NADPH : nitrate reductase activity to all three N. crassa mutants. MoCo from ch1D; pMoCo cell-free extracts was capable of reconstituting more NADPH : nitrate reductase activity to the N. crassa mutants than cell-free extracts from the original ch1D mutant.     

Isolation,characterization and mapping of pyrimidine auxotrophs of Phycomyces blakesleeanus

A total of seven pyrimidine auxotrophs of Phycomyces were isolated from among 5-fluoroorotate acid (5-FOA)-resistant mutants. They were classified by complementation into two groups. A representative mutant strain belonging to one group was deficient in orotate phosphoribosyl transferase (OPRTase; EC 2.4.2.10) activity; the mutant strain belonging to the second group was deficient in orotidine-5-monophosphate decarboxylase (OMPdecase; EC 4.1.1.23). These mutants are defective in the genes pyrF and pyrG respectively. The results from random spore analysis, tetrad analysis, and gene-centromere distances showed that these two markers are located in linkage group VI, with pyrG being a proximal marker and pyrF a distal one.