Aspergillus nidulans can utilize carbon sources that result in the production of TCA cycle intermediates, thereby requiring gluconeogenesis. We have cloned the acuG gene encoding fructose-1,6 bisphosphatase and found that expression of this gene is regulated by carbon catabolite repression as well as by induction by a TCA cycle intermediate similar to the induction of the previously studied acuF gene encoding phosphoenolpyruvate carboxykinase. The acuN356 mutation results in loss of growth on gluconeogenic carbon sources. Cloning of acuN has shown that it encodes enolase, an enzyme involved in both glycolysis and gluconeogenesis. The acuN356 mutation is a translocation with a breakpoint in the 59 untranslated region resulting in loss of expression in response to gluconeogenic but not glycolytic carbon sources. Mutations in the acuK and acuM genes affect growth on carbon sources requiring gluconeogenesis and result in loss of induction of the acuF, acuN, and acuG genes by sources of TCA cycle intermediates. Isolation and sequencing of these genes has shown that they encode proteins with similar but distinct Zn(2) Cys(6) DNA-binding domains, suggesting a direct role in transcriptional control of gluconeogenic genes. These genes are conserved in other filamentous ascomycetes, indicating their significance for the regulation of carbon source utilization.

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The purine hydroxylases I and I1 of Aspergillus niduluns [previously called xanthine dehydrogenases I and 11: Scazzocchio, Holl and Foguelman, Eur. J. Biochem. 36, 428-445 (2973)l have been studied in crude extracts. The two enzymes differ in their substrate specificities, purine hydroxylase 11 being able to accept nicotinate as a substrate and unable to hydroxylate xanthine. The kinetics of inhibition with allopurinol and oxypurinol are also different, the two analogues being pseudo-irreversible inhibitors of purine hydroxylase I, while allopurinol is a competitive inhibitor of purine hydroxylase I1 and oxypurinol shows anti-competitive inhibition. Differences in electrophoretic mobility and molecular size are also shown. We have failed to show the formation of hybrid purine hydroxylase 1/11 molecules. While a common evolutionary origin of the purine hydroxylases could be postulated, the data reveal a considerable divergence.

In Aspergillus nidulans uric acid can be produced from xanthine via purine hydroxylase I (xanthine dehydrogenase) or via the xanthine alternative pathway (Darlington and Scazzocchio, Biochem. Biophys. Acta, 166, 569--571; 1968). A mutation defective in the xanthine alternative pathway of Aspergillus nidulans is described. By combining this mutation with hxB-20 which results in complete loss of purine hydroxylase I and II activities, but which conserves cross-reacting material, it is possible to block completely uric acid production and thus investigate which are the effective in vivo inducers of three enzymes under the control of the positive regulatory gene uaY: adenine deaminase, purine hydroxylase I (measured as cross-reacting material) and urate oxidase. It is concluded that uric acid is the only effective physiological inducer, while its 2 and 8 thio-analogues serve as gratuitous inducers.