Metabolism of dihydrouracil in Rhodosporidium toruloides.

Previous studies, including those done with a similar species, have indicated that dihydrouracil is formed by the breakdown of uracil and is degraded into N-carbamyl-beta-alanine. (Fink et al., J. Biol. Chem. 201:349-355, 1953; S. R. Vilks and M. Y. Vitols, Mikrobiologiya 42:567-583, 1973; O. A. Milstein and M. L. Bekker, J. Bacteriol. 127:1-6, 1976). In the present work the conversion of dihydrouracil to uracil is studied in Rhodosporidium toruloides, and the growth characteristics of mutants that have lost the ability to use dihydrouracil as a source of nitrogen are examined. It is concluded that dihydrouracil must be converted to uracil before catabolism of the pyrimidine ring can take place.

Urea: obligate intermediate of pyrimidine-ring catabolism in Rhodosporidium toruloides.

Urea has been shown to be an obligate intermediate in and the penultimate product of the catabolism of pyrimidine-ring nitrogen in Rhodosporidium toruloides (Rhodotorula). One of a series of mutants selected for its inability to utilize uracil as a sole source of nitrogen was unable to utilize urea also. The mutant accumulated urea and failed to form 14CO2 during supplementation with [2-14C]uracil. Radioautograms from the resulting cell extracts and media failed to reveal expected intermediates. Cell-free extracts of the mutant were shown to lack urease activity. Revertants of the mutant were essentially wild type in all tested attributes. Elements of the reductive pathway for pyrimidine catabolism are present in Rhodosporidium (O. A. Milstein and M. L. Bekker, J. Bacteriol. 127: 1-6, 1976), but is has not been determined whether this pathway is involved with production of urea.

Complementation Analysis of Metabolite-Resistant Mutations with Forced Heterokaryons of NEUROSPORA CRASSA.

The double mutant strain pyr-3 arg-12(s) is a prototroph because a common precursor of arginine and pyrimidine is supplied by the arginine pathway. Growth of this strain is inhibited by exogenous citrulline or arginine. Citrulline-resistant mutants of this strain were selected, and they resulted from modifier mutations at other loci. Forced heterokaryons were used to study complementation among these modifiers. Since the complementation test requires the scoring of non-growth as the positive result, there was concern that variations in nuclear ratios could give erroneous results. This possibility does not seem significant, since groups of mutants established by complementation correspond with groups established by physiological, enzymatic, and recombinational measurements.-The technique has revealed that the most frequently mutated loci are arg-1 and what is probably un-3. Arg-1 mutations affect the conversion of citrulline to argininosuccinate, while un-3 mutations reduce the citrulline uptake rate. Since most of these mutations are of the intracistronic complementing type, a complementation map was constructed for most of the affected loci. The high proportion of complementors in each map can be explained by assuming that partially functioning gene products are more likely to complement with each other than are those which are nonfunctional.