Initial characterization of Aspergillus nidulans mutants blocked in the nuclear replication cycle.

Several hundred temperature-sensitive mutants of Aspergillus nidulans were screened for ability of their conidia to produce germ tubes at the nonpermissive temperature while still remaining with the original single conidial nucleus.     

Kinetics of the nuclear division cycle of Aspergillus nidulans.

We have analyzed the cell cycle kinetics of Aspergillus nidulans by using the DNA synthesis inhibitor hydroxyurea (HU) and a temperature-sensitive cell cycle mutant nimT that blocks in G2. HU rapidly inhibits DNA synthesis (S), and as a consequence progression beyond S to mitosis (M) is blocked. Upon removal of HU the inhibition is rapidly reversible. Conidia (asexual spores) of nimT were germinated at restrictive temperature to synchronize germlings in G2 and then downshifted to permissive temperature in the presence of HU. This procedure synchronizes the germlings at the beginning of S in the second cell cycle after spore germination. We have measured the total duration of S, G2, and M as the time required for these cells to recover from the HU block and undergo the next nuclear division. The duration of S was defined by the time course of sensitivity to reintroduction of HU during recovery from the initial HU block. The cell cycle time was measured as the nuclear doubling time, and the duration of mitosis was determined from the mitotic index. The duration of G1 was calculated by subtracting the combined durations of S, G2, and M from the nuclear doubling time, and the length of G2 was calculated by subtracting S and M from the aggregate length of S, G2, and M. We have also determined the duration of the phases of the cell cycle during the first cycle after spore germination. In these experiments spores were germinated directly in HU without first being blocked in G2. Because the durations of G1, S, G2, and M for the first cell cycle after spore germination were identical with those previously determined for spores presynchronized at the beginning of S in the second cell cycle, we conclude that dormant conidia of A. nidulans are arrested at, or before, the start of S.     

Conditionally lethal tubA alpha-tubulin mutations in Aspergillus nidulans

Summary We have mapped 17 extragenic suppressors of benA 33, a heat-sensitive -tubulin mutation of Aspergillus nidulans, to the tubA tubulin locus. Fifteen of these tubA mutations cause cold sensitivity in a genetic background with benA 33 and appear to cause lethality in a background with the wild-type benA allele. We examined the microtubule-mediated processes, nuclear division and nuclear migration, in seven different cold-sensitive double mutants, each carrying benA 33 and a different cold-sensitive tubA allele. Nuclear division and migration were inhibited at a restrictive temperature in each case, suggesting that cold sensitivity is due to the inhibition of microtubule function at low temperatures. A single allele, tubA4, suppressed the heat sensitivity conferred by benA33 but did not confer cold sensitivity in a benA33 background, however in a wildtype benA background, tubA4 conferred supersensitivity to antimicrotubule agents and weak cold sensitivity. TubA4 did not suppress the heat sensitivity conferred by two other benA alleles. The cold sensitivity conferred by tubA4 was suppressed by the microtubule stabilizing agent deuterium oxide, and the suppression of heat sensitivity conferred by four other tubA mutations was reversed by deuterium oxide. These results suggest that these mutations may affect hydrophobic interactions between -and -tubulin.     

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.     

Isolation of mip (microtubule-interacting protein) mutations of Aspergillus nidulans.

We identified four mutations in two previously undescribed loci involved in microtubule function in Aspergillus nidulans as extragenic suppressors of benA33, a heat-sensitive beta-tubulin mutation. Three of the four mutations map to a locus closely linked to riboB on linkage group VIII; we designated this locus mipA (for microtubule-interacting protein). We were not able to map the remaining suppressor because of chromosomal rearrangements. However, since it recombines with riboB at a significantly higher frequency than the mipA alleles, it is unlikely to be in mipA; thus, we designated it mipB1. The mip mutations are not allelic to the previously identified loci that encode alpha- and beta-tubulin, and it is likely that mipA and mipB encode previously unidentified nontubulin proteins involved in microtubule function. Each of the mip mutations suppresses the heat sensitivity conferred by benA33 and suppresses the blockage of nuclear division and movement conferred by this mutation at high temperatures. Interactions between mipA and benA are allele specific. All of the mipA mutations are cryptic in a wild-type benA background but cause cold sensitivity in combination with benA33. These mutations also confer cold sensitivity in combination with benA31 and benA32 and reduce the resistance conferred by these mutations to the antimicrotubule agent benomyl but do not suppress the heat sensitivity conferred by these alleles. Finally, the mipA alleles suppress the heat sensitivity conferred by benA11, benA17, and benA21 but do not confer cold sensitivity in combination with these alleles.     

On the mechanism of mitotic recombination in Aspergillus nidulans. I. Intragenic recombination and DNA replication

Summary Adenine or pABA starvation induce mitotic recombination within the ad9 and paba1 cistrons respectively. Adenine concentrations in the plating medium as low as 1×10^-8 M increase recombination frequency; the concentration optimal in respect to induced recombination frequency is 5×10^-7 M.Recombination within the paba1 cistron is stimulated by low pABA concentrations, or caseine hydrolysate, or methionine.Aminopterin applied for one or two hours before conidia of pABA-requiring diploid are plated on proper selective media, induces recombination within the pro1, ad9 and paba1 cistrons. Conclusion is drawn that it is adenine or thymine starvation which induce mitotic recombination.The implications of this and other similar evidence are discussed.     

The Aspergillus nidulans bncA1 mutation causes defects in the cell division cycle, nuclear movement and developmental morphogenesis

Wild-type Aspergillus nidulans conidia are uninucleate. The mutation bncA1 (binucleated conidia) was first described as a single mutation located on chromosome IV that caused formation of approximately 25% binucleate and 1% trinucleate conidia. In this study, we show that bncA1 conidia exit G1 arrest earlier than the wild type. Germlings have hyphal elements with abnormal morphology, elevated numbers of randomly distributed nuclei and an irregular septation pattern. Older hyphal elements undergo mitotic catastrophe, suggesting the nuclear division cycle of internal (nonterminal) elements is not arrested. The bncA1 mutation also causes aberrant morphogenesis of the asexual reproductive structure, the conidiophore. Metulae and phialides are elongated and have incorrect numbers of nuclei. Phialides also have internal septation that appears to delineate hyphal-like elements. Heterokaryon analysis using strains with contrasting auxotrophic markers showed that the bncA1 mutation resulted in a higher frequency of diploid and multinucleated prototrophic conidia than control heterokaryons. These results suggest that in bncA1 strains multiple nuclei can move from the conidiophore vesicle to the metulae and/or from the phialide to the conidium. The bncA1 mutant also showed hypersensitivity to the anti-microtubule drugs thiabendazole and nocodazole, which is consistent with the defects in cell cycle regulation and nuclear movement. We propose that bncA has an important role in correctly regulating both the cell division cycle and nuclear movement.     

L-histidine utilization in Aspergillus nidulans.

Histidase activity rather than uptake of L-histidine is the limiting factor for the utilization of histidine as the sole nitrogen source for Aspergillus nidulans. Histidine cannot act as the sole carbon source, and evidence is presented indicating that this is attributable to an inability to convert histidine to L-glutamate in vivo. It has been shown that this fungus lacks an active urocanase enzyme and that histidine is quantitatively converted to urocanate, which accumulates in the extracellular medium. The use of histidine as a nitrogen source is regulated by nitrogen metabolite repression control of histidase synthesis. In addition, evidence for a requirement for a carbon source for histidase synthesis and for a minor form of control by nitrate is presented. The activity of the histidase enzyme is inhibited by micromolar concentrations of the product urocanate and by physiological levels of L-glutamate and L-glutamine.     

Codon usage in Aspergillus nidulans.

Summary Synonymous codon usage in genes from the ascomycete (filamentous) fungus Aspergillus nidulans has been investigated. A total of 45 gene sequences has been analysed. Multivariate statistical analysis has been used to identify a single major trend among genes. At one end of this trend are lowly expressed genes, whereas at the other extreme lie genes known or expected to be highly expressed. The major trend is from nearly random codon usage (in the lowly expressed genes) to codon usage that is highly biased towards a set of 19–20 optimal codons. The G+C content of the A. nidulans genome is close to 50%, indicating little overall mutational bias, and so the codon usage of lowly expressed genes is as expected in the absence of selection pressure at silent sites. Most of the optimal codons are C- or G-ending, making highly expressed genes more G+C-rich at silent sites.     

Protoplasts from Aspergillus nidulans.

A very effective lytic enzyme system for massive micro/macro-scale production of protoplasts from the filamentous fungus Aspergillus nidulans is described. A striking coincidence was observed between maximal lytic activity towards Aspergillus mycelium and the presece of both chitinase and alpha-(1 leads to 3)-glucanase activities. The release of protoplasts was greatly enhanced by preincubating the mycelium with 2-deoxy-D-glucose. Furthermore, protoplast formation was influenced by fungal age, culture conditions, pH of incubation and the osmotic stabilizer used. From 40 mg of fresh mycelium, grown for 14--16 h on 1% glucose in a low phosphate-citrate medium, preincubated with 2-deoxy-D-glucose for 45 min, and then incubated with the lytic enzyme mixture at pH 6.5 in the presence of 0.3--0.4 M (NH4) SO4, 2.5 x 10(8) stable protoplasts were produced within 3 h of incubation at 30 degrees C. Comparable results were obtained with 40--50 g of mycelium. At low osmotic stabilizer concentrations a peculiar type of regeneration was observed in the presence of the lytic enzyme system; within 12 h of incubation aberrant hyphal structure emerged from the large vacuolated protoplasts.     

Betaine-homocysteine methyltransferase in the fungus Aspergillus nidulans.

A betaine:homocysteine methyltransferase activity was demonstrated in the cell-free extracts from the fungus $\text{Aspergillus}\text{nidulans}$. Among methionine-requiring mutants which do not grow on homocysteine one class responds to betaine indicating that this compound can serve as a methyl donor in methionine synthesis $\text{in vivo}$. Mutants of the second class which grow only on methionine were shown to have betaine: homocysteine — and methyltetrahydrofolate: homocysteine methyltransferases simultaneously impaired.     

Functional analysis of mutations in the human carnitine/acylcarnitine translocase in Aspergillus nidulans

Deficiency of the carnitine/acylcarnitine translocase (CACT), the most severe disorder of fatty acid beta-oxidation, is usually lethal in both humans and animals, precluding the development of animal models of the disease. In contrast, CACT deficiency is conditionally lethal in the fungus Aspergillus nidulans, since loss-of-function mutations in acuH, the translocase structural gene, do not prevent growth on carbon sources other than ketogenic compounds, such as fatty acids. Here, we describe the molecular characterization of extant acuH alleles and the development of a fungal model for CACT deficiency based on the ability of human CACT to fully complement, when expressed at physiological levels, the growth defect of an A. nidulans DeltaacuH strain on acetate and long-chain fatty acids. By using growth tests and in vitro assays this model enabled us to carry out a functional characterization of human CACT mutations showing that it may be useful for distinguishing potentially pathogenic human CACT missense mutations from neutral, single residue substitution-causing polymorphisms.