Characterization of the acuN356 mutation. (A) Schematic of the acuN gene corresponding to AN5746.3 in the A. nidulans database. The positions of exons are shown as thick lines and the coordinates are relative to the position of the ATG start codon. An expanded view of the 5′-end of the gene shows the position of the HindIII and HaeIII restriction sites used to map the acuN356 translocation breakpoint by Southern blotting of genomic DNA. The position of the intron in the 5′ untranslated region is shown (open box) and the position of 5′ endpoints of sequenced cDNAs is indicated, as are the positions of primers (inverse PCR-1 and -2) used for cloning the acuN356 translocation region. (B) Schematic of the acuN356 translocation breakpoint showing the contig coordinates on chromosomes 1 and 5 and the position of the breakpoint relative to the genes AN10770.3 and AN5746.3 (acuN). (C) Deletion of the acuN gene by replacement with the A. nidulans riboB gene in a riboB2 homozygous diploid strain. The relevant genotypes on linkage groups V and VIII of the resulting diploid are shown and phenotypes of haploids are recovered by haploidizing this diploid. All haploids have the riboB2 mutation (linkage group VIII) but none were phenotypically Ribo⁺ and all were FacA⁺, indicating that no haploids with the acuNΔ were recovered.

Regulation of expression of the acuG∷lacZ fusion gene. (A) Expression in the presence of various carbon sources and in the creA204 mutant background. Mycelium was grown for 20–24 hr in minimal medium with 10 mm ammonium tartrate as the nitrogen source and the indicated carbon sources at 1% except for acetate and proline (50 mm). (B) Expression in mycelium grown for 20 hr in the indicated glucose concentrations with 10 mm ammonium tartrate as the nitrogen source. (C) Effects of nonmetabolizable glucose analogs on expression in the wild-type and creA204 strains. 2-DOG and 3-MeGlc were present at the indicated concentrations. Mycelia were grown in 1% glucose–10 mm ammonium tartrate medium for 16 hr and then transferred to the indicated media with 10 mm ammonium chloride present as the nitrogen source for a further 4 hr before harvesting. (D) Expression in various mutant backgrounds. Mycelia of strains carrying the acuG∷lacZ fusion gene in the indicated genetic background were grown in 0.1% glucose-minimal media for 16 hr with 10 mm ammonium chloride as the nitrogen source. The indicated carbon sources were added and growth was continued for 8 hr before harvesting. Harvested mycelium was extracted and assayed for β-galactosidase. The specific activity is expressed as for Figure 3.

Effects of the acuK248 and acuM301 mutations on gene expression. (A) Effects on AcuF-LacZ expression. Mycelia of acuF-lacZ-containing strains of the indicated relevant genotypes were grown for 16 hr in 1% glucose–10 mm ammonium tartrate and then transferred to the indicated media for 4 hr before harvesting, extraction, and assaying as described in the legends to Figure 3 and Figure 4. The nitrogen source was 10 mm ammonium chloride in all cases except for the glucose-plus-proline treatment where 10 mm proline was present as the sole nitrogen source. Carbon sources were glucose (1%), proline (50 mm), acetate (50 mm), and malate (10 mm). (B) Effect on AcuG-LacZ expression. Mycelia of strains carrying the acuG∷lacZ fusion gene of the indicated relevant genotypes were grown for 16 hr in 1% glucose–10 mm ammonium tartrate and then transferred to the indicated media for 4 hr. Carbon sources were glucose (1%), glycerol (1%), proline (50 mm), and acetate (50 mm). Ammonium chloride (10 mm) was present as a nitrogen source. (C) Effects on acuF and acuG transcripts. RNA was extracted from the strains of the indicated relevant genotypes grown under the same conditions as indicated in A. The same Northern blot was hybridized sequentially to an acuF probe (a 2.3-kb PstI–XhoI fragment with coordinates −961 to +1359), an acuG probe (a 1.7-kb EcoRV fragment beginning at coordinate +58 and containing the rest of the coding region), and the EcoRI fragment of the histone H3 gene. (D) Effects on acuN transcripts. RNA was as for C with hybridization to the same probes as for Figure 3A.

Growth of relevant mutants on carbon sources. The following carbon sources were added to minimal medium with 10 mm ammonium chloride as the nitrogen source: glucose (1%), glycerol (0.5%), acetate (50 mm), ethanol (0.5%), l-proline (50 mm), l-alanine (50 mm), malate (10 mm), quinate (0.2%), valerate (10 mm), butyrate (20 mm), and Tween 80 (0.2%). All acids were adjusted to a pH of ∼6.5 with sodium hydroxide where necessary. Tween 80 is a source of the long-chain fatty acid oleate. Growth was for 2–3 days at 37° except for malate (6 days).

Analysis of expression of acuN and the effects of the acuN356 mutation. (A) Northern blot analysis of expression in a wild-type strain. RNA was extracted from mycelium grown for 16 hr in 1% glucose–10 mm ammonium tartrate medium and then transferred to medium with or without glucose (1%) with ammonium chloride (10 mm) as the nitrogen source with the following additions as indicated: acetate (50 mm); l-glutamate (10 mm); and γ-aminobutyric acid–GABA (10 mm) for 4 hr. acuN RNA was detected by hybridization with a 3.8-kb EcoRI fragment (coordinates −461 to +3376). Hybridization with the EcoRI fragment of the histone H3 gene (Ehinger et al. 1990) was used as a loading control. (B) Effects of acuN 5′ sequences on expression of a lacZ reporter. Mycelium from strains with single copies of plasmids with the indicated 5′ sequences driving lacZ expression were grown for 20–24 hr in medium containing one of the carbon sources: glucose (1%), glycerol (0.5%), acetate (50 mm), proline (50 mm), and quinate (0.5%) together with ammonium chloride (10 mm) as the nitrogen source before harvesting, extraction, and determination of β-galactosidase activities. The specific activity is expressed as Miller units per minute per milligram of protein (Davis et al. 1988) and represents the average of three independent experiments. The bars show standard errors. (C) Effects of the acuN356 mutation on acuN expression. RNA was extracted from wild type and an acuN356 strain grown under the same conditions as described in A and the Northern blot hybridized as in A.

The acuK and acuM genes. (A) The structure of the genes showing intron positions and the sequences coding for the indicated domains. The sequence changes and the resulting generation of stop codons in the acuK248 and acuM301 mutations are shown. (B) Comparison of the predicted Zn(2) Cys(6) domains of AcuK and AcuM homologs in filamentous ascomycetes. Sequences were aligned using Clustal W (Thompson et al. 1994) and visualized with Macboxshade (http://www.ch.embnet.org/software/BOX_form.html). Identical residues are indicated by a solid background and similar residues by a shaded background. Sequences were obtained from NCBI (http://www.ncbi.nlm.nih.gov/) and from the Broad Institute Fungal Genome Initiative (http://www.broad.mit.edu/annotation/fungi/fgi/). Species and accession numbers were the following: AcuK—A. nidulans (AN7468.3), A. oryzae (BAE57193), A. fumigatus (XP_749712), Neurospora crassa (XP_957577), and Gibberella zeae (XP_388802); and AcuM—A. nidulans (AN6293.3), A. oryzae (BAE59871), A. fumigatus (XP_755565), M. grisea (XP_360718), N. crassa (CAD37032), and G. zeae (XP_381738).