Figure 3.—

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.