Isolation and expression of the acetate-inducible isocitrate lyase gene (acu-3) from Neurospora crassa: evidence for a second constitutive isozyme

Abstract

Heterologous hybridisation of the Aspergillus nidulans structural gene for isocitrate lyase (acuD) to a lambda genomic library of Neurospora crassa identified a recombinant phage containing the hybridising sequence on an internal 9 kb EcoRI fragment. A restriction fragment length polymorphism (RFLP) enabled the fragment to be assigned to linkage group V (LG V), the location of the acetate-inducible isocitrate lyase, acu-3 of Neurospora. Functional ectopic complementation by co-transformation of an am-, acu- double mutant using independent plasmid clones, carrying the entire 9 kb EcoRI fragment (pICLG1) and the selectable marker am+ (NADP-glutamate dehydrogenase), demonstrated that the clone contains the entire acetate-inducible transcription unit. However, Northern analysis revealed two species of mRNA, only one of which was inducible on acetate. Native polyacrylamide gel electrophoresis separated two iso-enzymic activities, again only one of which was acetate-inducible and deficient in acu-3- mutants. Further hybridisation of the acu-3 gene probe to an electrophoretic karyotype of Neurospora crassa identified sequences in an additional linkage group as well as in LG V, as anticipated. The isozymes are therefore sequence-related.

The acetate regulatory gene facB of Aspergillus nidulans encodes a Zn(II)2Cys6 transcriptional activator

“…Structural genes of acetate metabolism have been cloned from several fungi, e.g. N. crassa (Thomas et al 1988;Gainey et al 1991), A. nidulans (Ballance and Turner 1986;Sandeman and Hynes 1989), Saccharomyces cerevisiae (Fernandez et al 1992; SchoÈ ler and SchuÈ ller 1993) and Coprinus cinereus (Mellon et al 1987).…”

A regulator gene for acetate utilisation from Neurospora crassa

“…The protein fraction was purified further using DNA affinity chromatography. The affinity column was constructed with a 671-bp BamHI-ApaI fragment from the promoter region of the acu-3 gene (encoding isocitrate lyase; Gainey et al 1991Gainey et al , 1992Mizote et al 1996). The promoter fragment was selected because it produces a reliable gel shift with the DEAEpurified protein, and it is required in vivo for induction of the acu-3 gene in response to acetate (Bibbins et al 1998).…”

“…Genetic loci encoding these enzymes have been defined in N. crassa through the isolation of acetate nonutilising mutants (acu) with single enzyme deficiencies: acu-3 codes for isocitrate lyase, acu-5 for acetyl-CoA synthetase, acu-6 for phosphoenolpyruvate carboxykinase (EC 4.1.1.32), acu-8 for acetyl-CoA hydrolase (EC 3.1.2.1), acu-9 for malate synthase, and acu-13 for NAD + -specific malate dehydrogenase (EC 1.1.1.37) Fincham 1968a, 1968b;Connerton 1990;Connerton et al 1992;Owen et al 1992). The genes encoding isocitrate lyase, acetyl-CoA synthetase, acetylCoA hydrolase and malate synthase have been isolated and their nucleotide sequences determined Marathe et al 1990;Gainey et al 1991Gainey et al , 1992Sandeman et al 1991).…”

Molecular Characterization of Mutants of the Acetate Regulatory GenefacBofAspergillus nidulans

“…One of the primers was end-labeled using T4 polynucleotide kinase (Promega Corp.) and [␥-32 P]dATP (4000 Ci/mmol; Bresatec) as recommended by the supplier (Promega Corp.). The acuD Ϫ414-Ϫ202 probe was generated from pICL1, which contains the entire acuD gene on a 4.9-kb EcoRI fragment in pUC7 (kindly provided by Dr. G. Turner; Gainey et al, 1992;J. Brown and G. Turner, unpublished).…”