Mitotic crossing over in chromosome I disomics of Aspergillus nidulans

Summary The frequency and pattern of homologous recombination in chromsome I disomics of Aspergillus nidulans is presented. Approximately 6% of randomly selected haploid breakdown sectors are recombinant. Most of these arise from double exchange events, one of which is located in the centromere region, the other distal on the left arm. Other marked regions are rarely involved in a recombination event. Reciprocal genotypes arise in approximately equal frequencies indicating that exchange results in reciprocally recombined non-sister chromatids at the four strand stage of mitosis. Possible theories for the extreme localisation of exchange events are discussed.     

Saccharomyces cerevisiae centromere CEN11 does not induce chromosome instability when integrated into the Aspergillus nidulans genome.

We constructed Aspergillus nidulans transformation plasmids containing the A. nidulans argB+ gene and either containing or lacking centromeric DNA from Saccharomyces cerevisiae chromosome XI (CEN11). The plasmids transformed an argB Aspergillus strain to arginine independence at indistinguishable frequencies. Stable haploid transformants were obtained with both plasmids, and strains were identified in which the plasmids had integrated into chromosome III by homologous recombination at the argB locus. Plasmid DNA was recovered from a transformant containing CEN11, and the sequence of the essential portion of CEN11 was determined to be unaltered. The transformants were further characterized by using them to construct heterozygous diploids and then testing the diploids for preferential loss of the plasmid-containing chromosomes. The CEN11 sequence had little or no effect on chromosome stability. Thus, CEN11 does not prevent chromosomal integration of plasmid DNA and probably lacks centromere activity in Aspergillus spp.     

Resistance and mitotic instability to chloroneb and 1,4-oxathiin in Aspergillus nidulans.

Mutants resistant to two fungicides, chloroneb (1,4-dichloro-2,5-dimethoxybenzene) and vitavax (2,3-dihydro-5-carboxanilido-6-methyl-1,4-oxathiin) were spontaneously obtained from a strain of Aspergillus nidulans with frequencies of 12.5 and 1.1 respectively, in 10(8) conidia. One chloroneb-resistant mutant (Chl 1) segregated as a single gene and was mapped in linkage group IV. It also caused a partial dependence of the strain on the fungicide and was semi-dominant. The mutant resistant to vitavax (Vit 1) also segregated as a single gene and was dominant. Both fungicides altered the instability of diploid and duplication strains. Chloroneb mainly increased haploidization, and vitavax reduced the mitotic recombination in diploids. Chloroneb increased the instability of duplication strains, and vitavax reduced such instability. The possible mode of action of such fungicides affecting stability is discussed.     

Tubulins in Aspergillus nidulans

The discovery and characterization of the tubulin superfamily in Aspergillus nidulans is described. Remarkably, the genes that encode alpha-, beta-, and gamma-tubulins were all identified first in A. nidulans. There are two alpha-tubulin genes, tubA and tubB, two beta-tubulin genes, benA and tubC, and one gamma-tubulin gene, mipA. Hyphal tubulin is encoded mainly by the essential genes tubA and benA. TubC is expressed during conidiation and tubB is required for the sexual cycle. Promoter swapping experiments indicate that the alpha-tubulins encoded by tubA and tubB are functionally interchangeable as are the beta-tubulins encoded by benA and tubC. BenA mutations that alter resistance to benzimidazole antimicrotubule agents are clustered and define a putative binding region for these compounds. gamma-Tubulin localizes to the spindle pole body and is essential for mitotic spindle formation. The phenotypes of mipA mutants suggest, moreover, that gamma-tubulin has essential functions in addition to microtubule nucleation.     

Genetic and physical mapping of two centromere-proximal regions of chromosome IV in Aspergillus nidulans

Chromosome IV is the smallest chromosome of Aspergillus nidulans. The centromere-proximal portion of the chromosome was mapped physically using overlapping clones of a cosmid genomic library. Two contiguous segments of a physical map, based on restriction mapping of cosmid clones, were generated, together covering more than 0.4 Mb DNA. A reverse genetic mapping approach was used to establish a correlation between physical and genetic maps; i.e., marker genes were integrated into physically mapped segments and subsequently mapped by mitotic and meiotic recombination. The resulting data, together with additional classical genetic mapping, lead to a substantial revision of the genetic map of the chromosome, including the position of the centromere. Comparison of physical and genetic maps indicates that meiotic recombination is low in subcentromeric DNA, its frequency being reduced from 1 crossover per 0.8 Mb to approximately 1 crossover per 5 Mb per meiosis. The portion of the chromosome containing the functional centromere was not mapped because repeat-rich regions hindered further chromosome walking. The size of the missing segment was estimated to be between 70 and 400 kb.     

Endo-exonuclease of Aspergillus nidulans

Endo-exonuclease (EE) has been found in both active and inactive, but trypsin-activatable, forms in Aspergillus nidulans. Active EE was present mainly in nuclei, mitochondria, and vacuoles, while trypsin-activatable EE was mainly in the cytosol. The active form accounts for over 90% of the neutral deoxyribonuclease activity extracted from mycelia. A single strand (ss) DNA-binding EE associated with a 28 kilodalton (kDa) polypeptide was partially purified and characterized. It was found to closely resemble, in size and enzymological properties, the ss-DNA-binding EE previously purified from Neurospora crassa. Aspergillus nidulans EE was also found to be immunochemically related to the N. crassa EE and, like that enzyme, was probably derived from a polypeptide of 90 kDa or larger through proteolysis during extraction and purification. It had divalent metal ion-dependent (Mg2+, Mn2+, or Zn2+) activity on both DNA and RNA, which ultimately produced small 5'-P-terminated oligonucleotides. The nuclease activity was mixed endo- and exo-nucleolytic with ss-DNA as substrate, but largely exonucleolytic with double strand (ds) DNA. Superhelical phi X-174 DNA was nicked by EE to form relaxed circular and then linear ds-DNA, which was rapidly degraded to shorter fragments. Linearized pBR322 DNA was extensively nicked internally under conditions where there was relatively low exonuclease activity, but this nicking required that 5'-P-termini be present on the linear ds-DNA. The levels of active EE found in extracts of two recombination-deficient mutants of A. nidulans, uvsC and uvsE, dit not differ significantly from those in extracts of the wild type.     

Supersuppressors in Aspergillus nidulans.

Summary Simultaneous reversion of mutations in two different Aspergillus nidulans loci adA and metG was found to be due monogenic suppressor mutations. Prelimirary evidence for the existance of supersuppressors in A. nidulans is presented.     

Selection arena in Aspergillus nidulans

The selection arena hypothesis states that overproduction of zygotes--a widespread phenomenon in animals and plants--can be explained as a mechanism of progeny choice. As a similar mechanism, the ascomycetous fungus Aspergillus nidulans may overproduce dikaryotic fruit initials, hereafter called dikaryons. Then, progeny choice might involve selection on which of these dikaryons will thrive to produce thousands of zygotes. These zygotes each produce eight sexual spores which together fill up one fruiting body. In this study, we test the selection arena hypothesis in this homothallic fungus that produces both sexual and asexual spores. We analyzed two mitochondrial and 15 auxotrophic mutations for consequences on sexual and asexual reproduction. We found that many of these mutations confer sexual self-sterility as a pleiotropic effect under conditions of normal asexual spore production. This confirms an important prediction of the selection arena, namely that dikaryons carrying a (slightly) deleterious mutation are not able to proliferate and produce sexual spores. The selection arena ensures that reproductive energy is invested mainly in dikaryons and thus sexual spores of good genetic quality.     

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.     

Apparent mRNA instability in Aspergillus nidulans and Aspergillus terreus of a heterologous cDNA encoding the major capsid antigen of Rotavirus

Two expression plasmids designed to produce the rotaviral VP6 protein in Aspergillus nidulans and Aspergillus terreus have been constructed. In one of these plasmids the inducible A. terreus Gla1 glucoamylase gene promoter and Gla1 signal sequence are fused to the VP6 cDNA to enable induction and extracellular secretion of the final protein product; in the other, the strong, constitutive A. nidulans gpdA gene promoter has been employed. A. nidulans and A. terreus transformants containing intact copies of these plasmids have been obtained but neither intra- nor extra-cellular VP6 protein was detectable. Northern analysis indicated specific degradation of the VP6 mRNA. This lack of VP6 mRNA stability may be related to fundamental differences between the general structure of Aspergillus mRNA and that of rotavirus, including codon usage and AU/GC ratio.