Possible role of a regulatory gene product upon the myo-inositol-1-phosphate synthase production in Neurospora crassa.

The regulatory effect of inositol on inositol-1-phosphate synthase in Neurospora crassa strains was studied. Inositol represses enzyme production in the cultures of the wild type and that of the thermosensitive inositol-requiring mutant grown at 22 degrees C. Enzyme activity as well as the quantity of enzyme protein decreased sharply in both strains by increasing concentrations of inositol in the medium. Inositol-requiring strains used in our experiments can be divided into two groups. The first group produces a protein related immunologically to inositol phosphate synthase, but which is enzymatically inactive. The synthesis of this defective enzyme was also repressed by inositol. In the second group, this protein was found to be completely lacking, in both the thermosensitive mutant grown at 37 degrees C, and in a strain requiring inositol due to a reciprocal translocation. The thermostability and the cross immunoelectrophoresis of the enzyme suggest that in the case of the thermosensitive inositol-requiring mutant, the mutation did not occur in the structural gene of the enzyme, but its regulation was probably affected.

A new cyclitol derivative influences inositol metabolism in Neurospora crassa.

Cyclitol derivatives have been synthesized and screened for growth inhibitory effect upon prokaryotic and eukaryotic organisms. One derivative, (2S,3R,5R)-3-azido-2-benzoyloxy-5-hydroxycyclohexanone, was studied in detail: it has no effect upon bacteria, but it is inhibitory to Neurospora crassa. In Neurospora crassa it increased the amount of myo-inositol-1-phosphate synthase and inhibited the activity of myo-inositol-monophosphatase. The enhanced synthesis of myo-inositol-1-phosphate synthase was the consequence of lowering the intracellular inositol concentration. Li+ treatment of Neurospora crassa has effects similar to those of P.I.-658.

Inheritance of DNA-induced inositol independence of inositolless Neurospora crassa.

Neurospora crassa (N. crassa) inositolless (inl) strain gives inositol-independent colonies (inl+) if treated with DNA from wild type N. crassa. There is an increased number of unusual non-Mendelian tetrads (24%) among the progeny. In sexual crosses of these strains with inl alleles ascospores of complete ordered tetrads were isolated. Clones of these aberrant tetrads were examined for the inheritance of the inl+ and inl markers respectively. Several inl+ clones of single ascospores of these tetrads were often pseudowild types. These clones segregate inl+ and inl nuclei if growing on inositol-containing (complete) medium and the inl+ character shows somatic segregation, sometimes disappears very quickly. The transformed inl+ chromosomes is occasionally passed on to the next generation in sexual cross, but the ascospores are of two kinds: inl+ and pseudowild type inl+. The inl+ trait is transmitted to further generations though the ascospores of the next generation have often lower germination rate. The pseudowild types in sexual cross are sterile on minimal medium and segregate very quickly inl nuclei. The mechanism of the origin of pseudowild types is assumed to be diploidization and consequently disomy of chromosome 2.

Behaviour of DNA-induced inositol-independent transformants of Neurospora crassa in sexual crosses.

Treatment of inositolless (inl) strains of Neurospora crassa with DNA from the wild type (allo-DNA) gives rise to inositol-independent (inl (+)) colonies. Some of these DNA-induced inl (+) strains (transformants) are sterile in sexual crosses on minimal medium that selects for the maintaining of the inl (+) character. The same inl (+) transformants, when crossed with an inl standard strain, are fertile on complete (inositol-containing) medium. There are, however, an increased number of unusual non-Mendelian tetrads (24%) among the progeny. The inl (+) and inl progeny from these complete non-Mendelian tetrads were further examined for the inheritance of the inl (+) trait. Several inl (+) progeny of these tetrads segregate inl conidia if growing on inositol-containing medium. The number of inl (+) conidia in certain inl (+) cultures decreases quickly under non-selective conditions. In transformants carrying mutant markers in linkage groups III, IV and VI non-Mendelian segregation of these traits can also be detected.The mechanism of the development of sterility and of the aberrant segregation is discussed.

Hybridization of DNA's from Neurospora crassa strains may indicate base sequence alterations as a consequence of genetic transformation.

Deoxyribonucleic acids of Neurospora crassa strains involved in genetic transformation experiments were studied by means of DNA-DNA hybridization. No significant difference was detected in the extent of hybridization reassociating 32P-DNA of an inositol-requiring recipient strain with an excess amount of unlabelled homologous DNA and that of the transformed, spontaneous revertant and wild-type strains. Studies on the thermal stability of hybrids revealed 1.2-1.7% heterology between the recipient and transformant DNA's. The spontaneous revertant and wild-type strains proved to be homologous with the recipient strain. We suppose that the heterology we measured is the result of the alteration of the nucleotide sequences caused by the multilocal integration of transforming DNA into the recipient genome.

A comparative study of DNA-induced transformants and spontaneous revertants of inositolless Neurospora crassa.

Inositolless (inl-) Neurospora crassa strains were treated with DNA (allo-DNA) of wild type N. Crassa. Hyphal fragments of a mycelial suspension of the N. Crassa ragged inl- strain used as recipient in our transformation experiments were found to consist of units containing 100--1000 nuclei. In this strain the inositol-independent (inl+) nuclei appearing after DNA treatment or by spontaneous reversion are present in the cytoplasm together with a large number of inl- nuclei. Thus, both transformation and reversion initially must result in heterokaryosis. Under appropriate conditions the inl- nuclei can be detected in the transformed and spontaneous inl+ phenotype revertant strains. Spontaneous revertants are usually characterized by the loss of their inl+ nuclei after transfers on inositol-supplemented medium. On minimal medium, the growth rate of transformed strains is significantly lower than that of spontaneous revertants. The inl+ gene appearing after DNA treatment or by spontaneous reversion is inherited as a trait bound to chromosomes. In crosses with the transformed strains, there is a significant increase in the number of non-Mendelian (6:2 and 5:3) tetrads in the inl locus.

Isolation and characterization of a DNA-uptake-stimulating protein from the culture medium of Neurospora crassa slime strain.

A protein fraction was purified to homogeneity from the culture medium of the wall-less (slime) strain of Neurospora crassa (FGSC 1118), which proved to be identical with DNA-uptake-stimulating factor (designated DUSF), which has been described earlier [Schablik, M. and Szabó, G. (1981) FEMS Microbiol. Lett. 10, 395-397]. The quantity of DUSF is measured by the amount of [3H]DNA uptake by Neurospora cells at standard conditions. Its relative molecular mass was 230,000. It has an isoelectric point of pH 5.5. This protein consists of two identical subunits, relative molecular mass 110,000.

Characterization of a mutation that causes overproduction of inositol in Neurospora crassa.

Slow-growing (inl+/-) spontaneous mutants have been isolated from an inositol requiring (inl) strain of Neurospora crassa that produces defective myo-inositol-1-phosphate synthase (MIPS), the enzyme responsible for the production of inositol-1-phosphate from glucose-6-phosphate. The defective enzyme has some residual activity. In the inl+/- strain the synthesis of the defective enzyme is enhanced, which enables the strain to grow slowly on minimal medium. The mutation (opi1) responsible for the partial inositol independence segregates independently from the inositol locus, and suppresses the inositolless character by overproduction of defective MIPS. opi1 acting upon the wild type (inl+) allele increases MIPS production and causes inositol excretion.

Defective myo-inositol-1-phosphate synthase production in an inositolless double mutant Neurospora crassa strain.

A slow growing inl+/- mutant was isolated from an inositol dependent (inl) Neurospora crassa strain. The latter strain produces defective myo-inositol-1-phosphate synthase which has residual activity. Inositol, similarly to that found in wild and inl mutant strains, represses the enzyme production in the inl+/- strain as well. Withdrawing inositol from the medium results in derepression of the enzyme synthesis. Derepression is hindered by cycloheximide. Inl+/- character in the double mutant is brought about by overproduction of the defective myo-inositol-1-phosphate synthase.

Inositol derivatives down-regulate c-myc inducing growth arrest without differentiation.

Cyclitol derivatives that are structurally related to myo-inositol induce growth arrest without differentiation in human promyelocytic leukemia (HL60) cells. An early effect is the rapid down-regulation of c-myc mRNA levels. This was observed also in several mouse and human lines carrying either normal or rearranged myc. The mRNA levels of a constitutive mouse myc construct transfected into HL60 were not affected at the same time. Uridine and thymidine incorporation were significantly decreased by the cyclitol treatment. These effects partly resemble those of certain differentiation inducers and those of hexachlorcyclohexane, another myo-inositol analogue. This new group of agents offers a novel approach to studying control mechanisms involving c-myc.

Separation of Neurospora crassa myo-inositol-1-phosphate synthase from glucose-6-phosphate dehydrogenase by affinity chromatography.

The purification of Neurospora crassa myo-inositol-1-phosphate synthase (EC 5.5.1.4) was studied by affinity chromatography using the substrate (glucose-6-phosphate), the inhibitor (pyrophosphate), the coenzyme (NAD+) and the coenzyme analogues (5'AMP and Cibacron Blue F3G-A) of the enzyme as adsorbents attached to agarose gel. Myo-inositol-1-phosphate synthase could be separated completely from the contaminating substance, glucose-6-phosphate dehydrogenase (EC 1.1.1.49), on Blue Sepharose CL-6B and on pyrophosphate-Sepharose. The purified enzyme had a specific activity of 16 400 U/mg. The sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the 60 micrograms of this purified enzyme gave a homogenous band. The enzyme was found to be composed of four identical subunits having a molecular weight of 65 000.

Characterization of inl+ transformants of Neurospora crassa obtained with a recombinant cosmid-pool.

We constructed a Neurospora crassa gene library in a cosmid vector and used the cosmid-pool DNA to transform an inl, rg Neurospora crassa strain to inositol prototrophy. The inl+ colonies obtained in this experiment proved to be integrative type transformants. Genetic analysis revealed that the integration event occurred at or near the inl locus. In one of the transformants the inl+ trait exhibited mitotic and meiotic instability. In hybridization experiments free plasmids were detected in the F1 progeny of the transformants. We were able to recover eleven different plasmids from the F1 progeny of the transformants. None of these plasmids proved to carry a functional copy of the inl+ gene as judged by its transforming ability. Possible explanations for the observed phenomena are discussed.

Demonstration of myo-inositol-1-phosphate synthase and its assumed defective variant in various Neurospora crassa strains by immunological methods.

Immunological experiments were performed to demonstrate myo-inositol-1-phosphate synthase (EC 5.5.1.4) and its assumed defective variant in various Neurospora crassa stains. An enzymatically inactive protein fraction was isolated from the inl-mutant by the same procedure as that of the enzyme. It consisted of several components by gel electrophoresis, and produced a positive immune reaction demonstrated by immunodiffusion using immune sera produced against the enzyme. Using immunodisc gel electrophoresis it produced an immunoprecipitate of slightly lower mobility than the enzyme itself. Similarly, positive immune reactions were obtained with the enzyme using immune sera produced against the protein fraction isolated from the inl- mutant. Enzyme activity was demonstrated both in a strain transformed by wild-type DNA and in a spontaneous revertant. The enzymes were subsequently isolated from both strains, and some properties were compared with those of the wild-type enzyme. The specific activities were lower but the Michaelis constants were nearly the same. The immunodisc gel electrophoretic patterns of these enzymes were similar to that of the protein fraction from the inositol requiring mutant.

Factors influencing the uptake of DNA by Neurospora crassa.

Under optimal conditions intact Neurospora crassa cells incorporated nearly the same amount of 3H-labelled DNA as that of the endogenous DNA content of cells. After 18 h of incorporation more than 80 per cent of the radioactivity was retained in the cells. A maximum uptake of exogenous DNA occurred at 28 degrees C, pH 6.35, in the presence of 100 mM calcium when the concentration of donor DNA was 150 micrograms/ml. Denatured DNA was incorporated at a higher rate than native DNA. The present findings suggest that the uptake of DNA by Neurospora crassa is a biologically active process.

Conditions of transformation by DNA of Neurospora crassa.

The DNA uptake and transformation of inositol-requiring recipient Neurospora strains were investigated. Exponentially growing cultures can accumulate 5-10 fold quantities of donor DNA than older ones. The rate of DNA uptake depends on the physiological state of the recipient cell, and on the molecular weight of donor DNA. The exocellular DNase activity of the recipient culture may influence the DNA uptake and the transformation process. "Young" inositol-requiring Neurospora crassa cultures can be transformed by wild type DNA reproducibly, but with low efficiency.

Investigations on myo-inositol-1-phosphate synthase from the wild type and the inositol-dependent mutant of Neurospora crassa.

The inositol-dependent mutant of Neurospora crassa lacks inositol-1-phosphate synthetase activity. This defect can be revorted by the addition of high-molecular DNA isolated from the wild type. To elucidate the biochemical background of inositol dependence, inositol-1-phosphate synthetase was studied. A method has been developed fro the isolation of the enzyme from the wild type strain in 10 mg scale by salt fractionation, gel filtration and ion-exchange chromatography. The specific activity of the purified enzyme is 4750 U/mg protein and its purity has increased about 100-fold. Polyacrylamide gel electrophoresis indicated that, in addition to the main enzymatically active band, several accompanying proteins occur in very small amount. The molecular weight of the enzyme is 225,000 daltons. Probably it consists of four subunits, two with a molecular weight of 64,000 daltons and another two of 50,000 daltons. An enzymatically inactive protein has been isolated from the mutant with the same procedure as that of the enzyme; it migrated at gel electrophoreis similarly to the enzyme. It may be supposed that the isolated protein is the defective enzyme molecule.