Spatial and biological characterisation of the complete quinic acid utilisation gene cluster in Aspergillus nidulans

Abstract

Heterologous probing of restriction digests of chromosomal DNA from Aspergillus nidulans with radioactively labelled probes encoding dehydroshikimate dehydratase (QA-4) and a repressor gene (QAI-S) from Neurospora crassa revealed a pattern of hybridisation inconsistent with an equivalent single copy of each gene in A. nidulans. Screening of size-selected and total genome A. nidulans DNA libraries allowed the isolation of four unique classes of sequence, two of which hybridised to the QA-4 probe, and two of which hybridised to the QA1-S probe. In each case, one of each pair of unique sequences was able to complement the equivalent mutations qutC (=QA-4) and qutR (=QA1-S) in A. nidulans, whereas the second of each pair was unable to complement the same mutation. The complementing sequences were physically mapped relative to the previously cloned A. nidulans QUT gene cluster, demonstrating that QUTR is distal and divergently transcribed from QUTA with approximately 3.6 kb between the ATG translational start codons, and that QUTC is transcribed in the same direction as QUTD on the other side of the cluster, approximately 1.65 kb downstream of the QUTD TAA translational stop signal. The physical and genetic maps of the QUT gene cluster correlate precisely. The non-complementing A. nidulans DNA sequences that hybridise to the N. crassa QA-4 (=QUTC) and QA1-S (=QUTR) fulfill many of the criteria characteristic of pseudogenes. The derived protein sequence of the QUTG gene shows a striking similarity to the protein sequence of bovine myo-inositol monophosphatase, indicating that they evolved from a common ancestor, and suggests a role for the QUTG gene, for which no function has previously been discovered, in expression of the QUT gene cluster.