Analysis of fluG mutations that affect light-dependent conidiation in Aspergillus nidulans.

Conidiation in Aspergillus nidulans is induced by exposure to red light but can also be induced by blue light in certain mutant strains. We have isolated a mutation in the fluG gene that abolishes responsiveness to red light but does not affect the response to blue light. It has been shown that the veA1 (velvet) mutation allows conidiation to occur in the absence of light. We have identified three other fluG mutations that suppress the veA1 phenotype; these double mutants do not conidiate in the dark. The mutations described here define two new phenotypic classes of fluG alleles that display abnormal responses to light. We have characterized these mutations with respect to their molecular identity and to their effect on fluG transcription. Although it has been shown that fluG is required for the synthesis of an extracellular factor that directs conidiation, we do not detect this factor under conditions that promote conidiation in the veA1 suppressors. Furthermore, extracellular rescue is not observed in fluG deletion strains containing the wild-type veA allele. We propose that a genetic interaction between fluG and veA influences the production of the extracellular signal and regulates the initiation of conidiation.

Isolation of a gene required for programmed initiation of development by Aspergillus nidulans.

In contrast to many other cases in microbial development, Aspergillus nidulans conidiophore production initiates primarily as a programmed part of the life cycle rather than as a response to nutrient deprivation. Mutations in the acoD locus result in "fluffy" colonies that appear to grow faster than the wild type and proliferate as undifferentiated masses of vegetative cells. We show that unlike wild-type strains, acoD deletion mutants are unable to make conidiophores under optimal growth conditions but can be induced to conidiate when growth is nutritionally limited. The requirement for acoD in conidiophore development occurs prior to activation of brlA, a primary regulator of development. The acoD transcript is present both in vegetative hyphae prior to developmental induction and in developing cultures. However, the effects of acoD mutations are detectable only after developmental induction. We propose that acoD activity is primarily controlled at the posttranscriptional level and that it is required to direct developmentally specific changes that bring about growth inhibition and activation of brlA expression to result in conidiophore development.

Genetic analysis of suppressors of the veA1 mutation in Aspergillus nidulans.

Light-dependent conidiation in the filamentous ascomycete, Aspergillus nidulans, is contingent on the allelic state of the velvet (veA) gene. Light dependence is abolished by a mutation in this gene (veA1), which allows conidiation to occur in the absence of light. We have isolated and characterized six extragenic suppressors of veA1 that restore the light-dependent conidiation phenotype. Alleles of four genes, defined by complementation tests, were subjected to extensive genetic and phenotypic analysis. The results of light-dark shifting experiments and the phenotypes of double mutant combinations are consistent with the possibility that the expression of the light-dependent phenotype is regulated by specific interactions of the suppressor gene products with the velvet gene product and with each other.

Light is required for conidiation in Aspergillus nidulans.

Light is necessary for asexual sporulation in Aspergillus nidulans but will elicit conidiation only if irradiation occurs during a critical period of development. We show that conidiation is induced by red light and suppressed by an immediate shift to far red light. Conidiation-specific gene functions switch from light-independent to light-dependent activities coincident with the expression of brlA, a regulator of conidiophore development. We also show that light dependence is abolished by a mutation in the velvet gene, which allows conidiation to occur in the absence of light. We propose that the initiation of late gene expression is regulated by velvet and controlled by a red light photoreceptor, whose properties are reminiscent of phytochrome-mediated responses observed in higher plants.

Sea urchin early and late H4 histone genes bind a specific transcription factor in a stable preinitiation complex.

Early embryonic H4 (EH4) and H2B (EH2B) and late embryonic H4 (LH4) histone genes were transcribed in vitro in a nuclear extract from hatching blastula embryos of the sea urchin Strongylocentrotus purpuratus. The extract was prepared by slight modifications of the methods of Morris et al. (G. F. Morris, D. H. Price, and W. F. Marzluff, Proc. Natl. Acad. Sci. USA 83:3674-3678, 1986) that have been used to obtain a cell-free transcription system from embryos of the sea urchin Lytechinus variegatus. Achievement of maximum levels of transcription of the EH4 and LH4 genes required a 5- to 10-min preincubation of template with extract in the absence of ribonucleoside triphosphates. This preincubation allowed the formation of a stable complex which was preferentially transcribed compared with a second EH4 or LH4 template that was added 10 min later. Although the EH4 gene inhibited both EH4 and LH4 gene transcription in this assay and although the LH4 gene inhibited both EH4 and LH4 genes, neither of these genes inhibited transcription of the EH2B gene. Preincubation with the EH2B gene had no effect on the transcription of subsequently added EH4 or LH4 genes. Using this template commitment assay, we showed that the site of binding of at least one essential factor required for transcription of both EH4 and LH4 genes was located between positions -102 and -436 relative to the 5' terminus of the EH4 mRNA. Moreover, deletion of this region resulted in a reduction in EH4 gene transcription in vitro. The sea urchin gene-specific trans-acting factors, in the analysis of the cis-acting sequences with which they interact, and in biochemical studies on the formation of stable transcription complexes.

Insertion of an intermediate repetitive sequence into a sea urchin histone-gene spacer.

A common polymorphism of the early embryonic histone-gene repeat of Strongylocentrotus purpuratus is a 195-bp insertion within the H4-H2B spacer. The sequence, found as an insert in histone-gene repeats of 6 of 22 individuals screened, is also found at approximately 50 sites elsewhere in the genome of every individual. We compare the sequences of the histone-gene spacers that do and do not contain the insert. The insert is found not to have transposon-like features, and no sequence in the original spacer has been duplicated to flank the insert. There is, however, a hexanucleotide sequence that is repeated three times at one end of the insert, and the element has inserted between direct repeats of 5 bp that were present in the original spacer. One of the copies found outside the histone gene cluster was cloned and sequenced and is compared with the insert. Again, no transposon-like features are evident. Regions flanking the homologous sequence in this clone were used as hybridization probes in whole-genome blots. Results indicate that the 195-bp sequence insert is itself embedded within a larger element that is repeated within the genome. Therefore, only a portion of a larger repetitive sequence has integrated into the histone-gene spacer. The sequence features of the insert, although not typical of mobile elements, may be representative of other illegitimate recombination events.

Genetic analysis of mutants of Aspergillus nidulans blocked at an early stage of sporulation.

Three mutants of Aspergillus nidulans, selected to have a block at an early stage of conidiation (asexual sporulation), exhibit similar pleiotropic phenotypes. Each of these mutants, termed preinduction mutants, also are blocked in sexual sporulation and secrete a set of phenolic metabolites at level much higher than wild type or mutants blocked at later stages of conidiation. Backcrosses of these mutants to wild type showed that the three phenotypes always cosegregated. Diploids containing the mutant alleles in all pairwise combinations were normal for all phenotypes, showing that the three mutations are nonallelic. This conclusion was confirmed by the finding that the mutations map at three unlinked or distantly linked loci. Ten revertants of the two least leaky preinduction mutants, selected for ability to conidiate, were found in each case to arise by a second-site suppressor mutation. All of the revertants still showed accumulation of some of the phenolic metabolites but differed from each other in certain components. Three of the revertants retained the block in sexual sporulation. In these cases the suppressor has thus uncoupled the block in asexual sporulation from the block in sexual sporulation. These results are understandable in terms of a model in which preinduction mutations and their suppressors affect steps in a single metabolic pathway whose intermediates include an effector specific for asexual sporulation and a second effector specific for sexual sporulation.

Mutants of Aspergillus nidulans blocked at an early stage of sporulation secrete an unusual metabolite.

Mutants of Aspergillus nidulans defective in conidiation (asexual sporulation) can be classified according to whether they are blocked before or after induction of conidiation. Mutants blocked before induction (preinduction mutants) appear to be unable to respond to the inducing stimulus and thus are defective in one of the earliest events in the sporulation process. Three preinduction mutants have been isolated and characterized. Each was found to exhibit the same pleiotropic phenotype: they also were defective in sexual sporulation and secreted a set of phenolic metabolites at a level much higher than did wild type or mutants blocked at later stages of conidiation. One of the metabolites has been identified as the antibiotic diorcinal (3,3'-dihydroxy-5,5'-dimethyldiphenyl ether) which is known to be involved in the synthesis of certain farnesyl phenols of unknown function. These results suggest that preinduction mutants are blocked in a phenolic metabolic pathway, one or more product of which participates in the initiation of sporulation.

Evolving sea urchin histone genes--nucleotide polymorphisms in the H4 gene and spacers of Strongylocentrotus purpuratus.

We present a comparison of spacer and coding sequences of histone gene repeats from four Strongylocentrotus purpuratus individuals. Sequences of two previously cloned units (pCO2 and pSp2) were compared with three new histone gene clones, two of them from a single individual. Within a 1.7-kb region, 59 polymorphic sites were found in spacers, in mRNA nontranslated stretches, and at silent sites in codons of the H4 gene. The permitted silent-site changes were as frequent as in any other region studied. The most abundant polymorphisms were single-base substitutions. The ratio of transitions : transversions : single-base-pair insertions/deletions was 3:2:2. A number of larger insertions/deletions were found, as well as differences in the length of (CTA)n and (CT)n runs. Two of the five cloned repeats contained an insertion of a 195-bp element that is also present at many other sites in the genomes of every S. purpuratus individual studied. Pairwise comparisons of the different clones indicate that the variation is not uniformly divergent, but ranges from a difference of 0.34% to 3.0% of all nucleotide sites. A parsimonious tree of ancestry constructed from the pairwise comparisons indicates that recombination between the most distantly related repeats has not occurred in the 1-2 million years necessary for accumulation of the variation. The level of sequence variation found within the S. purpuratus population, for both tandemly repeated and single-copy genes, is 25%-50% of that found between S. purpuratus and S. drobachiensis.