Enhancers of Conidiation Mutants in Aspergillus Nidulans

Mutants at a number of loci, designated sthenyo, have been isolated as enhancers of the oligoconidial mutations at the medA locus. Two loci have been mapped: sthA on linkage group I, and sthB on linkage group V. Two probable alleles have been identified at each locus but two further mutants were unlinked to either sthA or sthB. Neither sthA nor sthB mutants have conspicuous effects on morphology on their own, nor could the sthA1 sthB2 double mutant be distinguished from wild type. Mutants at both loci also interact with the temperature-sensitive brlA42 mutant at the permissive temperature to give a phenotype described as ``Abacoid.' sthA1 also induces a slight modification of the phenotype of an abaA mutant. We conclude that sthenyo genes act mainly at the phialide stage of conidiation. We also describe the isolation of new medA mutants arising spontaneously as outgrowths on brlA42 colonies.     

N-acetyl-6-hydroxytryptophan oxidase, a developmentally controlled phenol oxidase from Aspergillus nidulans

We have purified a specific phenol oxidase which is produced during conidiophore development in the fungus Aspergillus nidulans. Two active forms (A and B) have molecular masses of 50 and 48 kDa respectively; they have identical N-termini (24 residues). We have analysed the metal ion content of the B form; it is unusual in consisting of one zinc and two copper atoms per molecule. A temperature-sensitive mutant (ivoB192) produces a thermolabile enzyme, implying that ivoB is the structural locus. The natural substrate of the enzyme is N-acetyl-6-hydroxytryptophan, but it can be assayed colorimetrically or polarographically using hydroquinone monomethyl ether (HME) as substrate. It will also oxidize p-cresol, but not tyrosine, 3,4-dihydroxyphenylalanine or o-methoxyphenol. Colour development with HME substrate is strongly enhanced by high ammonium ion concentrations. Activity against HME is inhibited by 2,3-dihydroxynaphthalene, phenylhydrazine, diethyl dithiocarbamate and 8-hydroxyquinolene.     

Isolation of Aspergillus nidulans Mutants That Overcome brlA-Induced Growth Arrest

The Aspergillus nidulans brlA gene is a primary regulator of development-specific gene expression during conidiation. Forced activation of brlA in vegetative cells leads to inappropriate induction of conidiophore formation and causes growth to stop. In fact, when conidia containing a nutritionally inducible brlA gene fusion are placed on inducing medium, they fail to germinate. We used this phenotype to select 174 mutants that continue growing following such forced brlA activation. Forty-six of these mutants also produced abnormal developmental structures during air-induced conidiation as expected if the mutations resulted in an altered response to BrlA (designated sbr mutants for suppressors of brlA response). The predominant mutant class identified was defective in a known developmental regulatory gene, abaA. We also identified mutants with defects in the previously characterized early acting developmental regulatory genes flbB and flbD and in four previously undescribed loci designated sbrA-D. sbrA mutants represent the second largest group and are characterized by production of conidiophore stalks that lack a normal vesicle and form branching sterigmata that rarely make spores. Because abaA expression could not be detected in sbrA mutants following brlA activation we propose that sbrA functions as a developmental modifier, participating in brlA-dependent activation of other developmental regulators.     

A Diphenol Oxidase Gene Is Part of a Cluster of Genes Involved in Catecholamine Metabolism and Sclerotization in Drosophila. I. Identification of the Biochemical Defect in Dox-A2 [l(2)37Bf] Mutants

Phenol oxidase, a complex enzyme, plays a major role in the processes of sclerotization and melanization of cuticle in insects. Several loci have been reported to affect levels of phenol oxidase activity, but to date only one structural locus has been identified [Dox-3F (2-53.1+)]. Recently isolated Dox-A2 mutations (2-53.9) are recessive, early larval lethals, which as heterozygotes reduce phenol oxidase activity. A homozygous mutant escaper had weak, completely unpigmented cuticle and unpigmented bristles. Enzyme assays show that Dox-A2 heterozygotes have diphenol oxidase activity reduced to 47-79% of wild type, whereas monophenol oxidase activity, at 94-106% of wild type, is normal. Elevated pool sizes of the diphenol oxidase substrates DOPA, dopamine, and N-acetyldopamine are observed in the mutant, confirming the enzyme assay results. Separation of the three phenol oxidase A component activities on polyacrylamide gels shows that Dox-A2 mutations reduce the activity of only the A2 component. Dox-A2 may identify a structural locus for the A2 component of the diphenol oxidase enzyme system. The Dox-A2 locus is one of 18 loci in the dopa decarboxylase, Df (2L)TW130 region of the second chromosome, at least 14 of which affect the formation, melanization or sclerotization of cuticle in some way. These loci form an apparent cluster of functionally related genes.     

Terminal synthesis of xanthommatin in Drosophila melanogaster. I. Roles of phenol oxidase and substrate availability

Eye color mutants of Drosophila melanogaster are known which block the conversion of 3-hydroxykynurenine to xanthommatin. It has been proposed that this reaction depends on the presence of 3-hydroxykynurenine and a redox system maintained by phenol oxidase activity. The mutants st and ltd lack throughout development detectable amounts of 3-hydroxykynurenine or its metabolic derivatives. When the substrate is fed or injected, these mutants fail to form xanthommatin even though phenol oxidase activity is normal. The mutant cd accummulates excessive amounts of 3-hydroxykynurenine, has normal phenol oxidase activity, but is also deficient in xanthommatin formation. Mutants are also known which lack phenol oxidase activity but nevertheless form xanthommatin. It is concluded that the proposed relationship between 3-hydroxy-kynurenine and phenol oxidase activity is not sufficient to explain the in vivo synthesis and regulation of synthesis of xanthommatin in Drosophila. The bearing of these findings on the actual mode of synthesis is discussed.Supported by PHS 1029 and NSF GB-4539.     

Aspergillus Fabm Encodes an Essential Product That Is Related to Poly(a)-Binding Proteins and Activates Development When Overexpressed

Overexpression of several different Aspergillus nidulans developmental regulatory genes has been shown to cause inappropriate developmental activation and growth inhibition. We previously exploited this observation that induced development caused growth inhibition in designing a screen to identify other genes that could activate development when overexpressed. We identified 16 mutants in which induced expression of different random genomic DNA sequences caused growth inhibition, accumulation of mRNA corresponding to the brlA developmental regulatory locus, and in several cases sporulation. This phenotype was designated FAB for Forced expression Activation of brlA and the genes were called fabA through fabP. Here we describe one of these genes, fabM, which is predicted to encode a poly(A)-binding protein (PABP) that is constitutively expressed and is essential for viability. While it is unclear why overexpression of the fabM caused sporulation, we showed that this activity required other known early developmental regulators including brlAβ, flbA, flbB, flbC, and fluG. We propose that fabM is an example of a gene that is not only required for growth, but also has specific functions early in development that assist developmental induction, presumably by allowing translation of specific mRNAs like brlA.     

Evidence that the Aspergillus nidulans class I and class II chitin synthase genes, chsC and chsA, share critical roles in hyphal wall integrity and conidiophore development

Although many chitin synthase genes have been identified in a broad range of fungal species, there have been only a few reports about their role in fungal morphogenesis. In most cases, single gene disruption or replacement did not reveal their function, possibly because of functional redundancy among them. We obtained null mutants of Aspergillus nidulans chsA and chsC genes encoding non-essential class II and class I chitin synthases, respectively. The DeltachsA DeltachsC mutant exhibited growth defects on media supplemented with sodium dodecyl sulfate (SDS), high concentration of salts, chitin-binding dyes, or chitin synthase competitive inhibitors, suggesting loss of integrity of hyphal wall. Moreover, remarkable abnormalities of the double mutant were observed microscopically during its asexual development. The conidiophore population was drastically reduced. Interestingly, secondary conidiophores were occasionally produced from vesicles of the primary ones. The morphology of these conidiophores was similar to those of the A. nidulans developmental mutants, medusa (medA), abacus (abaA), and some kinds of bristle (brlA). In situ staining patterns suggested that chsA was mainly expressed in the metulae, phialides, and conidia, whereas chsC was expressed in hyphae as well as conidiophores. These results suggest that ChsA and ChsC share critical functions in hyphal wall integrity and differentiation.     

Formal Relations between Om Mutants and Their Suppressors in Drosophila Ananassae

Optic morphology (Om) mutants associated with insertions of the tom transposable element at each of three tested loci are neomorphs as defined by the phenotypic equivalence of +/+/Om with +/Om and of +/Om/Om with Om/Om. Mutants behaving as suppressors of Om mutants and mapping to at least six loci are recovered from the same source and in similar frequency as Om mutants. The semidominant and nonpleiotropic suppressors at four of the six loci display defective eye phenes themselves, and the phenotypically normal mutants at a fifth locus are suspected alleles of a gene represented by recessive furrowed eye mutants. These and other properties imply that the suppressors, like suppressible Om mutants, are neomorphic due to insertion of the tom element into a hypothetical sequence they share with other members of a set of genes involved in development of the eye. Concurrently premature expression of both the suppressor and suppressed mutants would allow interaction of their products just as in normal development.     

Pigmentation and lysosomal phenotypes in mice doubly homozygous for both light-ear and pale-ear mutant alleles

We have developed a new strain of mice homozygous for mutant alleles at both the light-ear locus on chromosome 5 and the pale-ear locus on chromosome 19. The pigmentation pattern of the double mutants, designated light-pale, is indistinguishable from the parental type. Elevated concentrations of lysosomal enzymes observed in certain tissues of the light-ear and pale-ear singly homozygous mice also are present in the double mutants, and are quantitatively indistinguishable from either parent. Although both mutations have pleiotropic effects on organelles in several tissues, neither locus influences the secretion of pancreatic zymogen granules. The close similarity in phenotypes of light ear, pale ear, and light-pale mice suggest that the le and ep loci encode different subunits of a multimeric protein, and that mutations affecting either subunit result in comparable losses of function.     

acon-3, the Neurospora crassa ortholog of the developmental modifier, medA, complements the conidiation defect of the Aspergillus nidulans mutant

Aspergillus nidulans and Neurospora crassa are ascomycetes that produce asexual spores through morphologically distinct processes. MedA, a protein with unknown function, is required for normal asexual and sexual development in A. nidulans. We determined that the N. crassa ortholog of medA is acon-3, a gene required for early conidiophore development and female fertility. To test hypotheses about the evolutionary origins of asexual development in distinct fungal lineages it is important to understand the degree of conservation of developmental regulators. The amino acid sequences of A. nidulans MedA and N. crassa ACON-3 shared 37% identity and 51% similarity. acon-3 is induced at late time points of conidiation. In contrast, medA is constitutively expressed and MedA protein localizes to nuclei in all tissue types. Nonetheless, expression of acon-3 using its native promoter complemented the conidiation defects of the A. nidulans ΔmedA and medA15 mutants. We conclude that the biochemical activity of the medA orthologs is conserved for conidiation.     

Insertion of the LINE retrotransposon MGL causes a conidiophore pattern mutation in Magnaporthe grisea

We obtained three Magnaporthe grisea morphological mutants that had the LINE transposon MGL inserted into the ACR1 locus. Sequence analysis revealed that ACR1 is homologous to medA, a developmental regulator of Aspergillus nidulans conidiation. These results demonstrated that MGL elements could transpose and cause insertional mutagenesis in M. grisea.     

Identification of Developmental Regulatory Genes in Aspergillus Nidulans by Overexpression

Overexpression of several Aspergillus nidulans developmental regulatory genes has been shown to cause growth inhibition and development at inappropriate times. We set out to identify previously unknown developmental regulators by constructing a nutritionally inducible A. nidulans expression library containing small, random genomic DNA fragments inserted next to the alcA promoter [ alcA (p) ] in an A. nidulans transformation vector. Among 20,000 transformants containing random alcA (p) genomic DNA fusion constructs, we identified 66 distinct mutant strains in which alcA (p) induction resulted in growth inhibition as well as causing other detectable phenotypic changes. These growth inhibited mutants were divided into 52 FIG (Forced expression Inhibition of Growth) and 14 FAB (Forced expression Activation of brlA) mutants based on whether or not alcA (p) induction resulted in accumulation of mRNA for the developmental regulatory gene brlA. In four FAB mutants, alcA (p) induction not only activated brlA expression but also caused hyphae to differentiate into reduced conidiophores that produced viable spores from the tips as is observed after alcA (p) :: brlA induction. Sequence analyses of the DNA fragments under alcA (p) control in three of these four sporulating strains showed that in two cases developmental activation resulted from overexpression of previously uncharacterized genes, whereas in the third strain, the alcA (p) was fused to brlA. The potential uses for this strategy in identifying genes whose overexpression results in specific phenotypic changes like developmental induction are discussed.     

Genetic and cytological characterization of a developmental mutant of Aspergillus nidulans induced by 5-azacytidine.

An analysis of a new medusa mutant of Aspergillus nidulans obtained by 5-azacytidine-treatment and named B116 is provided. The B116 mutant was phenotypically characterized by the production of conidiophores with reduced pigmentation and vesicles bearing multiple tiers of sterigmata. A single nuclear gene located on chromosome I is responsible for phenotypical changes in the mutant. The 5-azacytidine-altered locus, designated medA102, is recessive in heterozygous diploid and the medusa mutant is a Dp(II,I) duplication bearer that renders the strain mitotically unstable.     

w4-Mutable line in soybean

An unstable mutation for anthocyanin pigmentation in soybean (Giycine max [L.] Merr.) was identified in 1983. The mutability is conditioned by an allele at the w4 locus that is recessive to wild type. The population containing the mutable allele is known as the w4-mutable line. Most plants in the line have chimeric flowers with purple sectors on a near-white background. The mutable allele yields germinal revertants at a rate that varies from 5 to 10% per generation, and the revertant alleles are stable. Approximately 1% of the progenies derived from germinal revertant plants contain mutations at other loci These features, as well as the occurrence of pale flower phenotypes and changes of state, suggest that a transposable element system is producing the unstable phenotype. Several new mutants were isolated in an experiment designed to tag loci. The first three chlorophyll-deficient mutants found (CD-1, CD-2, and CD-3) are inherited as single-gene recessives. Each of the mutants lacks the same two mitochondrial malate dehydrogenase (MDH) bands. No recombination has been detected between the MDH phenotype and the chlorophyll-deficient phenotype. Genetic data indicate that the three mutants are allelic, and additional evidence suggests that each of the CD mutants is the result of a deletion. In the CD-1, CD-2, and CD-3 mutants, the deletions result in the silencing of an MDH locus, atypical chloroplast development, and an altered chlorophyll composition. Additional mutants for root necrosis, partial and near sterility, chlorophyll deficiency, and flower color isolated from the transposon tagging study have provided material for future research.