Drosophila mutants with altered pyrimidine metabolism have been described ... uridine, but not to 0.07m~J-fluorouridine, and since pyrimidine bases may be.
900
BIOCHEMICAL SOCIETY TRANSACTIONS
Altered Pyrimidinesalvage Metabolism in a 5-Fluorouracil-Resistant Mutant of Drosophila melanogaster MARTIN M. CLYNES and EDWARD J. DUKE Department of Zoology, University College, Belfield, Dublin 4, Ireland Drosophila melanogaster is an ideal organism for the study of biochemical genetics in an intact animals system, because of its low chromosome number, shott life-cycle, and small, relatively non-repetitive genome (Lengyel et al., 1975) and because of the availability of genetically-definedmarker stocks (Lindsley & Grell, 1968). A number of Drosophila mutants with altered pyrimidine metabolism have been described (Nerrby, 1973; Okada et al., 1974; Strerman, 1974). Methods have been described for the isolation of mutants resistant to 5-fluorouracil(Duke &Glassman, 1968). Drosophilamelanogaster (Pac) are sensitive to 5-fluorouracil, but spontaneous resistant mutants occur. The work described here concerns a strain resistant to 0.07 mM-5-flUOrO~raCil.This strain has been shown to contain higher thymidylate synthetase activity than a sensitive strain selected also from the Pac stock (N. O’Byme & E. J. Duke, unpublished work). However, since the mutant was found to be cross-resistant to 0.07m~-5-fluorodeoxyuridine, but not to 0.07m~J-fluorouridine, and since pyrimidine bases may be converted into nucleosides or deoxynucleosides in Drosophila (Clynes & Duke, 1975), it seemed possible that alterations in pyrimidine-salvage metabolism might be involved in resistance. Enzyme activities were measured, under conditions of linearity with respect to time and enzyme concentration,as previously described (Clynes & Duke, 1975) except that concentrations of radioactive substrates were doubled. In order to avoid possible age differences in enzyme activities, extracts were always made
Table 1. SpeciJc activities of pyrimidine-salvage enzymes in Drosophila strains resistant and sensitive to 5-pUorouraci1 The specific activity of the sensitive extract was arbitrarily given the value of 100 for each enzyme in each experiment, and other activities expressed in terms of this. Typical values for the Pac strain (expressed in units of nmollh per mg of protein) were: uridine kinase, 4.1 ; thymidine kinase, 0.8 ; uracil phosphoribosyltransferase, 3.9. Specific activity (%) Expt. 1: Uridine kinase Thymidine kinase Uracil phosphoribosyltransferase
Expts. 2,3 and 4: Uridine kinase Thymidine kinase Uracil phosphoribosyltransferase Uridine kinase Thymidine kinase Uracil phosphoribosyltransferase Uridine kinase Thymidine kinase Uracil phosphoribosyltransferase
Resistant
Sensitive
Pac
302 116 251
100 100 100
102 111 117
Resistant
Sensitive
Adults from eggs of resistant flies grown on normal food
161 158 174 161 240 176 138 139 157
100 100 100 100 100 100 100 100 100
109 112 128 130 174 142 116 106 113 1976
564th MEETING, DUBLIN
901
from adults 1-4 days after emergence. The first experiment showed that the activities of uridine kinase (EC 2.7.1.48), thymidine kinase (EC 2.7.1.75) and uracil phosphoribosyltransferase (EC 2.4.2.9) were all increased in the resistant strain, and that the activities of these enzymes were similar in the sensitive and parent Pac strains (Table 1). To determine whether this was a genetically-determined increase in activity or a secondary adaptation or induction in the presence of 5-fluorouracil, enzyme activities were measured in the progeny of resistant flies, that had been allowed to lay eggs on normal food. These progeny had never been in contact with 5-fluorouracil, but the results show that they always had enzyme activities intermediate between resistant and sensitive values. Thus, the results do not definitely support either a genetic or inductive mechanism for the observed alterations in enzyme activities. It is possible that an adaptive alteration in enzyme activity might take several generations to re-stabilize at normal values. For instance, Baskin et al. (1975) have shown that, in a S-fluorodeoxyuridine-resistant variant of cultured mouse neuroblastoma with increased thymidylate synthetase activity, both resistance and increased activity are unstable during subsequent passage in the absence of the selective agent. Alternatively, it is possible that the ability of the resistant mutant to increase activities of these enzymes in the presence of 5-fluorouracil may be the biochemical basis for the mutation to resistance. However, increased activity of pyrimidine-salvage enzymes would be expected to increase rather than decrease 5-fluorouracil toxicity, supporting the suggestion that this increase is not the biochemical determinant of resistance. Considering the resistant population as a whole, one possibility is that lower activities of the three enzymes is a phenotypic expression of a recessive gene, which is lethal to homozygous larvae in the presence of 5-fluorouracil, but is retained in the resistant stock since it is non-lethal to heterozygotes. The reappearance of homozygotes in the resistant flies grown on normal food would result in lower overall specific activity in the adult population. Finally, the results in Table 1 indicate the possiblity that the enzymes of pyrimidine salvage in Drosophila melanogaster may be subject to some form of co-ordinate regulation. Baskin, F., Carlin, S. C., Kraus, P., Friedkin, M. & Rosenberg, R. N. (1975) Mol. Phurmacol. 11, 105-117
Clynes, M. M. & Duke, E. J. (1975) Biochem. Soc. Trans. 3, 1256-1257 Duke, E.J. & Glassman, E. (1968) Nature (London)220, 588-589 Lengyel, J., Spradling, A. & Penman, S. (1975) Methods Cell Biol. 10, 195-208 Lindsley, D. L. & Grell, E. H. (1968) Curnegie Ins?. Washington Publ. 627 Nsrby, S . (1973) Hereditus 73, 11-19 Okada, M., Kleinman, I. A. & Schneiderman, H. A. (1974) Dev. B i d . 37, 55-62 Strsman, P.(1974) Hereditus 78, 157-168
Turkey Liver Xanthine Dehydrogenase: Effects of Methanol on the Enzyme-Catalysed Oxidation of Reduced NicotinamideAdenine Dinucleotide fDE Nf FHAOLAIN,* MICHAEL J. HYNEST and MICHAEL P. COUGHLAN* *Department of Biochemistry and T Department of Chemistry, University College, Galway, Ireland
Methanol inactivation of substrate hydroxylation by xanthine dehydrogenase and related enzymes is progressive and turnover-dependent. It is widely accepted that the effects of methanol result from it forming (possibly after oxidation to formaldehyde) a complex with the modybdenum centres (Rajagopalan & Handler, 1964, 1967, 1968; Aleman et al., 1965; Rajagopalan et al., 1968; Coughlan et al., 1969; Pick et al., 1971). In contrast with its effects on xanthine oxidation, methanol treatment of chicken liver xanthine dehydrogenase results in a greatly enhanced ability to catalyse
Vol. 4
