Analysis of pyrimidine catabolism in Drosophila melanogaster using epistatic interactions with mutations of pyrimidine biosynthesis and beta-alanine metabolism

Abstract

The biochemical pathway for pyrimidine catabolism links the pathways for pyrimidine biosynthesis and salvage with beta-alanine metabolism, providing an array of epistatic interactions with which to analyze mutations of these pathways. Loss-of-function mutations have been identified and characterized for each of the enzymes for pyrimidine catabolism: dihydropyrimidine dehydrogenase (DPD), su(r) mutants; dihydropyrimidinase (DHP), CRMP mutants; beta-alanine synthase (betaAS), pyd3 mutants. For all three genes, mutants are viable and fertile and manifest no obvious phenotypes, aside from a variety of epistatic interactions. Mutations of all three genes disrupt suppression by the rudimentary gain-of-function mutation (r(Su(b))) of the dark cuticle phenotype of black mutants in which beta-alanine pools are diminished; these results confirm that pyrimidines are the major source of beta-alanine in cuticle pigmentation. The truncated wing phenotype of rudimentary mutants is suppressed completely by su(r) mutations and partially by CRMP mutations; however, no suppression is exhibited by pyd3 mutations. Similarly, su(r) mutants are hypersensitive to dietary 5-fluorouracil, CRMP mutants are less sensitive, and pyd3 mutants exhibit wild-type sensitivity. These results are discussed in the context of similar consequences of 5-fluoropyrimidine toxicity and pyrimidine catabolism mutations in humans.

Figure 1.

Interrelationships of uracil and β-alanine metabolic pathways in Drosophila. Loss-of-function mutations of de novo pyrimidine biosynthesis genes (e.g., rudimentary) produce a truncated wing phenotype that is normalized by enhanced pyrimidine salvage or by blocked pyrimidine catabolism. Thus, mutations in pyrimidine catabolism are recessive suppressors of rudimentary phenotypes. Also, the black mutant cuticle phenotype results from diminished levels of β-alanine, a compound that is primarily derived by catabolism of uracil. The semidominant, feedback-insensitive r^Su(b) mutation results in enhanced uridine-5′-monophosphate (UMP) and uracil availability, thereby suppressing the black mutant phenotype. Thus, mutations in pyrimidine catabolism are epistatic to (i.e., block) the suppression of black by r^Su(b). Details and references are in the text.

Figure 2.

The su(r) gene region. Above the double horizontal line are locations of exons for the su(r) (FlyBase Reg-3–RC transcript) and CG12118 (FlyBase CG12118–RA transcript) genes, as well as the insertion site of the P{EPgy2}04394 transposon (Celniker et al. 2002; Bellen et al. 2004). Below the double horizontal line are the extents and locations of su(r) mutations [su(r)¹, DQ027826; su(r)^C4, DQ257368; su(r)^C5, DQ257369; su(r)^C43, DQ257371; su(r)^C11, DQ257370] as well as the genomic DNA insert within the P{PYD1+} transposon.

Figure 3.

Suppression of the rudimentary wing phenotype by su(r) and CRMP^sup mutations. (A) Typical severe rudimentary wing of a r^C animal. (B) Entirely wild-type wing of a su(r)¹ r^C animal. (C) Moderate rudimentary wing of a r^C; CRMP^supB3 animal.

Figure 4.

su(r)¹ DNA contains a nonconservative substitution of an invariant amino acid residue. A segment of the DPD protein from a variety of species is aligned: Dictyostelium discoideum (AAQ11981), Caenorhabditis elegans (Q18164), pig (B54718), mouse (AAH39699), human (AAA57474), and the D. melanogaster CG2194 gene (AAN64918), as well as the su(r)¹ protein (AAY44826). Numbers in parentheses refer to the position within each complete protein of the first amino acid in the protein fragment shown. The distinctive glutamate substitution in su(r)¹ DPD is underlined.

Figure 5.

The CRMP gene region. Above the double horizontal line are locations of exons for the rheb (FlyBase Rheb–RA transcript) and CRMP (FlyBase CRMP–RA transcript) genes and the insertion site of the P{EP}3238 transposon (Rørth 1996; Celniker et al. 2002). Below the double horizontal line are the extents and locations of CRMP mutations (CRMP^supB3, DQ257367; CRMP^supI3, DQ257373; CRMP^supI2, DQ257372; CRMP^supA4, DQ257377) and the genomic DNA insert within the P{PYD2+} transposon.

Figure 6.

The CRMP^supA4 mutation encodes a nonconservative substitution of an invariant amino acid residue. A segment of the DHP and CRMP proteins from a variety of species is aligned: Pseudomonas aeruginosa (AAG03830), Saccharomyces kluyveri (AAF69237), Arabidopsis thaliana (AAO33381), D. discoideum (AAO33383), C. elegans (DHP-1, Q21773; DHP-2, Q18677), mouse (DHP, AAK00644), human (DHP, AAH34395; CRMP-1, AAH00252; CRMP-2, AAA93202; CRMP-4, Q14195; CRMP-5, AAF80348), D. melanogaster (AAO33382), as well as the CRMP^supA4 protein (ABB73022). Numbers in parentheses refer to the position within each complete protein of the first amino acid shown in the protein fragment. The distinctive valine substitution in CRMP^supA4 DHP/CRMP is underlined.

Figure 7.

CRMP^sup mutations block suppression of the black cuticle phenotype by r^Su(b). (A) Typical black body exhibited by a w; b; CRMP^supA4/TM3 fly. (B) Suppressed black phenotype of a w; b; CRMP^supA4 P{r^Su(b)}/TM3 fly. (C) The genotype w; b; CRMP^supA4 P{r^Su(b)}/CRMP^supA4 produces a black cuticle phenotype. Eye coloration of these flies is due to markers on the transposon and chromosomes used in the crosses and is otherwise unrelated to the black phenotype.

Figure 8.

The pyd3 gene region. Above the double horizontal line are locations of exons for the pyd3 gene (FlyBase pyd3–RA tanscript) and the insertion site of the P{GawB}2343 transposon (Celniker et al. 2002; Hayashi et al. 2002; FlyBase). Below the double horizontal line are the extents and locations of pyd3 mutations (pyd3^Lb5, DQ257375; pyd3^La2, DQ257374; pyd3^Lb10, DQ257376) and the genomic DNA insert within the P{PYD3+} transposon.

Figure 9.

Sensitivities of pyrimidine catabolism mutants to dietary 5-fluorouracil. Animals were reared on varying concentrations of 5-FU as described in materials and methods, and emerging adult progeny were counted. The mean number of surviving experimental (hatched bars) and control siblings (solid bars) are presented (± SEM of triplicate vials). (A) Parents were su(r)/Y or P{EPgy2}04394/Y [non-su(r)] males and C(1)DX, y f/Y females, producing progeny of the same genotypes: experimental su(r) males and control attached-X females [su(r)⁺]. (B) Parents were CRMP/TM3 or CRMP⁺/TM3 males and Df(3R)noi-B/TM3 females, producing experimental hemizygotes [CRMP/Df(3R)noi-B] and control heterozygous CRMP⁺ siblings [CRMP/TM3 and Df(3R)noi-B/TM3]. (C) Parents were pyd3/TM3 or P{GawB}2343/TM3 (non-pyd3) males and Df(3R)dsx10M/TM3 females, producing experimental hemizygotes [pyd3/Df(3R)dsx10M] and heterozygous control siblings [pyd3/TM3 and Df(3R)dsx10M/TM3].