Isolation of a sexual sporulation hormone from Aspergillus nidulans.

Psi factor is a substance produced by Aspergillus nidulans that induces premature sexual sporulation. Chromatographic analysis of psi-active extracts showed that psi activity resides in several different forms. Two of the forms, psiA1 and psiB1, have been isolated and have been shown to have closely similar compositions. The most abundant form, psiA1, reacts with alcohols in acidic solution by the addition of one entire molecule of the alcohol. This reaction, which is reversible, suggests that psiA1 may be a lactone whose ring is opened by alcohol addition. At high concentration, psiA1 is antagonistic to the response exhibited by the other forms of psi, but this antagonism is lost by the alcoholic derivatives. At least one unpurified psi species can be converted to psiA1 by acid catalysis. We suggest that psiA1 may be the metabolic precursor of at least some of the other more active psi components and that this conversion during Aspergillus development may be part of the process that triggers sexual sporulation.

High level of complexity of small nuclear RNAs in fungi and plants.

The complexity of the trimethylguanosine-capped, small nuclear RNA (snRNA) populations in a number of organisms has been examined using immunoprecipitation and two-dimensional gels. From the fungi Aspergillus nidulans and Schizosaccharomyces pombe, over 30 major snRNAs can be resolved. The most abundant of these correspond to the putative analogues of vertebrate U1, U2, U4 and U5, which have been reported to be precipitated by anti-Sm antibodies, but other snRNAs are little less abundant than the major Sm-precipitable species. A similarly high level of complexity of snRNAs is detected in pea plants. In Candida albicans, the snRNAs are somewhat less numerous (about 22 major species) and are substantially less abundant than those of the above fungi, features shared with another budding yeast, Saccharomyces cerevisiae. Ten species of human snRNA have been reported; on two-dimensional gels, a number of additional snRNAs can be resolved from human cells. Each fungus, as well as pea plants, contains snRNAs substantially larger than any reported from vertebrates or detected in the human RNA used here. It appears that many eukaryotes contain substantially more species of snRNA than was previously believed.

Osmotic adjustment in the filamentous fungus Aspergillus nidulans.

Aspergillus nidulans was shown to be xerotolerant, with optimal radial growth on basal medium amended with 0.5 M NaCl (osmotic potential [psi s] of medium, -3 MPa), 50% optimal growth on medium amended with 1.6 M NaCl (psi s of medium, -8.7 MPa), and little growth on medium amended with 3.4 M NaCl (psi s of medium, -21 MPa). The intracellular content of soluble carbohydrates and of selected cations was measured after growth on basal medium, on this medium osmotically amended with NaCl, KCl, glucose, or glycerol, and also after hyperosmotic and hypoosmotic transfer. The results implicate glycerol and erythritol as the major osmoregulatory solutes. They both accumulated during growth on osmotically amended media, as well as after hyperosmotic transfer, except on glycerol-amended media, in which erythritol did not accumulate. Furthermore, they both decreased in amount after hypoosmotic transfer. With the exception of glycerol, the extracellular osmotic solute did not accumulate intracellularly when mycelium was grown in osmotically amended media, but it accumulated after hyperosmotic transfer. It was concluded that the extracellular solute usually plays only a transient role in osmotic adaptation. The intracellular content of soluble carbohydrates and cations measured could reasonably account for the intracellular osmotic potential of mycelium growing on osmotically amended media.

The fatty acid composition of the conidia and mycelia of the fungus Aspergillus nidulans.

The total fatty acids were characterized from conidia, exponential phase, and stationary phase Aspergillus nidulans. Several quantitative and qualitative variations were observed. Most notable was the approximately 15-fold increase in linolenate observed during the 1st day of incubation and its subsequent total disappearance by day 4.

[Fatty acid composition of the lipids in fungi of the genus Aspergillus developing on mineral media with different carbon sources]. / Zhirnokislotnyi sostav lipidov gribov roda Aspergillus pri razvitii ikh na mineral'nykh sredakh s razlichnymi istochnikami ugleroda.

This work was aimed at studying the effect of different carbon sources in the composition of mineral media on the growth of fungi belonging to the Aspergillus genus and on the fatty acid composition of their lipids. A chemically-defined medium with glucose was shown to be optimal for the growth of 18 Aspergillus strains and for the synthesis of lipids by them. The fatty acid composition of lipids was studied when the fungi grew in media with different carbon sources. The lipids were shown to contain saturated and unsaturated fatty acids with the prevalence of oleic, linoleic and linolenic acids.

Structure and regulated expression of the SpoC1 gene cluster from Aspergillus nidulans.

We have previously described the organization of a 13.3 kb region of the Aspergillus nidulans genome, designated SpoC1, coding for multiple poly(A)+ RNAs that accumulate in asexual spores but not in somatic cells. We have determined the limits of the SpoC1 gene cluster by investigating the transcriptional features of 53 kb of chromosomal DNA. This segment of the genome codes for at least 19 poly(A)+ RNAs, some of which are transcribed from overlapping regions. The area of developmental regulation is approximately 38 kb in length and is delimited by 1.1-kb direct repeats. With one exception, RNAs transcribed from the central part of the cluster appear late during conidiophore development and accumulate specifically in spores. The exceptional transcript appears earlier during development and accumulates specifically in cells of the conidiophore. In contrast, RNAs encoded at the borders of the cluster occur in both somatic cells and spores. The results indicate that if a chromatin-level control mechanism operates to regulate expression of the SpoC1 gene cluster, as previously suggested by us, additional levels of regulation must also exist.

Ergosterol and lanosterol from Aspergillus nidulans.

Ergosterol was identified as the major free sterol of Aspergillus nidulans by thin-layer chromatography, alumina column chromatography, gas-liquid chromatography, high-performance liquid chromatography, UV spectroscopy, proton magnetic resonance spectroscopy and mass spectral analysis. Lanosterol, the initial cyclized precursor of ergosterol, was identified as a minor component of the free sterols. In the steryl ester material, however, lanosterol was usually more abundant than ergosterol, suggesting that the esters serve as storage compounds for the membrane sterol precursors.

Extracellular siderophores of rapidly growing Aspergillus nidulans and Penicillium chrysogenum.

The highly active extracellular siderophores previously detected in young cultures of Aspergillus nidulans and Penicillium chrysogenum have been identified as the cyclic ester fusigen (fusarinine C), and its open-chain form, fusigen B (fusarinine B).

Selection for increased penicillin titre following hybridization of divergent lines of Aspergillus nidulans.

Sexual hybridization of two divergent lines of Aspergillus nidulans, which had been selected for increased penicillin titre through successive cycles of mutagenesis, released considerable variation for this character. The recovery of segregants with titres equivalent to that of the unselected ancestor suggested that mutations in different genes had been selected in the two lines. However, complementary segregants with substantially improved titres were not found, indicating interactions, probably of a duplicate nature, among the induced mutations. All the genetic variation released by hybridization was fixed following two generations of selection for high titre, but only a small gain over the initial selection lines was achieved. Hybridization of divergent strains has been widely advocated as a means of strain development. The failure to achieve the anticipated gains in this programme is attributed primarily to the unfavourable interactions amongst the induced mutations. Whether similar interactions occur generally in crosses between strains selected by mutagenesis remains to be established and will be an important factor in determining the contribution of recombinational approaches to yield improvement.

Lipid components variation among the temperature-sensitive mutants and wild strain of Aspergillus nidulans.

The fungus Aspergillus nidulans and its two temperature-sensitive mutants were grown in synthetic media at permissive and restrictive temperatures. After seven days of growth, total lipids were extracted and characterised from the mycelia. In the case of the wild strain, an increase in the incubation temperature resulted in an increase in the ratio of unsaturated to saturated fatty acids and was inversely proportional to the ratio of short chain to long chain fatty acids. It was interesting to note that the ts mutants tested showed a decreased ratio of unsaturated to saturated fatty acids at the higher temperature (restrictive). The ratio of short chain to long chain fatty acids of these two mutants was also found to be varied at the higher temperature. Variations were also found in the case of individual lipid components.

Nucleotide sequence of 5S ribosomal RNA from Aspergillus nidulans and Neurospora crassa.

The nucleotide sequences of 5S rRNA molecules isolated from the cytosol and the mitochondria of the ascomycetes A. nidulans and N. crassa were determined by partial chemical cleavage of 3'-terminally labelled RNA. The sequence identity of the cytosolic and mitochondrial RNA preparations confirms the absence of mitochondrion-specific 5S rRNA in these fungi. The sequences of the two organisms differ in 35 positions, and each sequence differs from yeast 5S rRNA in 44 positions. Both molecules contain the sequence GCUC in place of GAAC or GAUY found in all other 5S rRNAs, indicating that this region is not universally involved in base-pairing to the invariant GTpsiC sequence of tRNAs.

Nuclear and mitochondrial suppression of a mitochondrially inherited cold-sensitive mutation in Aspergillus nidulans.

Partial suppressors of a mitochondrially inherited mutation, [cs-67], conferring cold-sensitivity at 20 degrees C were identified. These mapped at one mitochondrial and four unlinked nuclear loci. Most suppressors partially restored the cytochrome aa3 deficiency of the cold-sensitive strain at 20 degrees C. Strains carrying two or more suppressors and [cs-67] showed considerably impaired growth. This effect was temperature-dependent, being more severe at 37 degrees C, and was not expressed in the presence of the [cs-67+] allele. The cytochrome oxidase activity of one of these strains was no more heat-sensitive than that of the wild-type implying that these mutations did not directly modify cytochrome oxidase. The wild-type strain grown in the presence of chloramphenicol and the cold-sensitive strain grown at 20 degrees C had similar cytochrome spectra and mitochondrial membrane protein profiles on sodium dodecyl sulphate gradient acrylamide gels. [cs-67] conferred pleiotropically a low level of resistance to paramomycin at 37 degrees C. It is suggested that [cs-67] and the suppressors act at the level of the mitochondrial ribosome.

A hydroxamic acid from Aspergillus nidulans with antibiotic activity against Proteus species.

An iron-complexing antibiotic with a narrow spectrum of biological activity was produced by several strains of Aspergillus nidulans when grown in a low-iron, chemically defined medium. Its chemical and biological properties closely resembled those of desferritriacetylfusigen, a metabolite of several other Aspergilli and Penicillia.

Isolation of DNA from Aspergillus nidulans.

A procedure for isolation of DNA from Aspergillus nidulans on a preparative scale is described. Mechanical disruption of lyophilized material in high-salt medium and treatment with proteinase K, followed by sedimentation of the lysate into saturated CsC1 solution yielded pure, highly polymerized DNA.

Nucleosome structure in Aspergillus nidulans.

The structure of chromatin from Aspergillus nidulans was studied using micrococcal nuclease and DNAase I. Limited digestion with micrococcal nuclease revealed a nucleosomal repeat of 154 base pairs for Aspergillus and 198 base pairs for rat liver. With more extensive digestion, both types of chromatin gave a similar quasi-limit product with a prominent fragment at 140 base pairs. The similarity of the two limit digests suggests that the structure of the 140 base pair nucleosome core is conserved. This implies that the difference in nucleosome repeat lengths between Aspergillus and rat liver is caused by a difference in the length of the DNA between two nucleosome cores. Digestion of Aspergillus chromatin with DNAase I produced a pattern of single-stranded fragments at intervals of 10 bases which was similar to that produced from rat liver chromatin.

The isolation of nuclei from the filamentous fungus Aspergillus nidulans.

A method has been developed for isolating nuclei from the filamentous fungus Aspergillus nidulans. In this procedure, the mycelia from 14 to 16 h spore-derived cultures of A. nidulans NRI, a stable diploid strain, were frozen with liquid nitrogen and homogenized in a Waring blender. After homogenization, the mycelia were warmed to 4 degrees C and the nuclei were purified from the homogenate by differential centrifugation followed by sedimentation through 2.1 M-sucrose. The final nuclear yield was 15 to 20%, based on DNA estimations. The purified nuclear pellet was free of whole cells. The morphology of the isolated nuclei resembled that of the in situ nuclei; they contained a nucleolus, chromatin, and had a surrounding double membrane. The purified nuclei were characterized by a DNA:RNA:protein ratio of 1:2.8:8.7.

Presence of histones in Aspergillus nidulans.

Five major histone proteins have been extracted from chromatin isolated from purified nuclei of the fungus, Aspergillus nidulans. These proteins had chromatographic properties which were similar to reference calf thymus histones and were purified to electrophoretic homegeneity by gel chromatography of Bio-Gel P10, Bio-Gel P60, and Sephadex G-100. Electrophoresis of these proteins in three different systems (urea-starch, urea-acetic acid polyacrylamide, and discontinuous SDS polyacrylamide) showed that the A. nidulans histones H3 and H4 were nearly identical to calf thymus H3 and H4 with respect to net charge and molecular weight criteria, whereas the fungal histones H1, H2a and H2b were similar but not identical to the corresponding calf thymus histones. Amino acid analysis of A. nidulans histones H2a, H2b, and H4 showed them to be closely related to the homologous calf thymus histones. The mobility patterns of A. nidulans ribosomal basic proteins in three different electrophoretic systems were distinctly different from those of the fungal histones.