An analysis of the embryonic defects in Punch mutants of Drosophila melanogaster

Punch (Pu), a complex genetic locus, encodes GTP cyclohydrolase, the first enzyme in the pteridine biosynthetic pathway. In the larval and adult stages of the Drosophila life cycle, the function of the locus can be monitored by enzyme assays. Although enzyme activity cannot be detected prior to larval stages, the locus must also have earlier functions since most homozygous Pu mutants die during embryogenesis. In order to assess the role of the locus during this stage of development, morphological examinations of embryos from different classes of Pu mutants were performed. An exact correspondence has been found between genetic and morphological classes of Pu mutations. The locus is required during two periods of embryogenesis. These requirements are genetically separable as shown by mutants with defects specific to each period. An early function utilizes both maternal and zygotic components. Mutants defective for these components have abnormal segment patterns. Late in embryogenesis, a Pu product is necessary for the proper pigmentation of larval cuticle and proper orientation and differentiation of other larval structures, particularly in the head region. A cold-sensitive period corresponds to this later function as determined by temperature-shift experiments. Some of the phenotypes observed correspond to known physiological roles of pteridines; others are unexpected and unexplained.     

Characterization of New Punch Mutations: Identification of Two Additional Mutant Classes

The Punch locus of Drosophila melanogaster which encodes the pteridine biosynthetic enzyme, GTP cyclohydrolase, is genetically complex. Lethal alleles of the locus resolve into an array of interallelic complementation groups, and at least one class of mutations is developmentally specific, affecting GTP cyclohydrolase activity only in the heads of adults. All previously isolated Punch alleles were identified on the basis of a mutant eye color phenotype. By screening mutagenized chromosomes over Punch region deficiencies, we have now isolated new alleles on the basis of lethal and visible phenotypes. Most of these alleles fall into previously identified genetic classes, but two new classes of mutations were also found. One class contains two alleles that behave as dominant lethal mutations in some genetic backgrounds. The other class represents a second developmentally specific set of alleles that affect the function of the Punch locus only during embryogenesis.     

A Genetic Analysis of the Pteridine Biosynthetic Enzyme, Guanosine Triphosphate Cyclohydrolase, in DROSOPHILA MELANOGASTER

Strains with mutant eye color were surveyed for levels of GTP cyclohydrolase (GTP CH), the first enzyme acting in the biosynthesis of pteridines, the pigments causing red eye color in Drosophila. Six strains were found to have reduced GTP CH activity. In five of the six strains, the reduction of activity is apparent only in the adult head of homozygous mutants. We show that mutations in Punch (2-97, Pu) have severe effects on GTP CH activity. In most cases, the reduction of activity is apparent in all tissues and stages that express the enzyme. The activity of GTP CH is shown to be closely correlated with the number of Pu+ genes in the genome. One ethyl methanesulfonate (EMS)-induced Pu mutant has a GTP CH enzyme that is unstable when compared with the wild-type enzyme. Mutations in Pu fall into three general classes. The largest class has a recessive lethal and eye color phenotype, 50% or higher GTP CH activity in heterozygotes, and equivalent defects in all tissues. A second class is dominant in eye color phenotype and recessive lethal, with less than 50% GTP CH activity in heterozygotes. The third class is homozygous viable and has severe reduction of activity in the adult head, but no or less severe loss in other tissues.     

Correlation of guanosine triphosphate cyclohydrolase activity and the synthesis of pterins in Drosophila melanogaster

The enzyme guanosine triphosphate cyclohydrolase (GTP cyclohydrolase), which in bacteria is known to be the first enzyme in the biosynthetic pathway for the synthesis of pteridines, has been discovered in extracts of Drosophila melanogaster. Most of the enzyme (80%) is located in the head of the adult fly. An analysis of enzyme activity during development in Drosophila has revealed the presence of a relatively small peak of activity at pupariation and a much larger peak that appears at about the time of eclosion. Enzyme activity declines rapidly as the fly ages. Analyses for the production of the typical pteridine pigments of Drosophila have indicated that the small peak of GTP cyclohydrolase activity evident at pupariation coincides with the appearance of isoxanthopterin, sepiapterin, and pterin, and the larger peak at eclosion roughly corresponds to the accumulation of drosopterin as well as to the appearance in larger amounts of pterin and sepiapterin. These observations strongly suggest that in Drosophila, like bacteria, GTP cyclohydrolase is involved in the biosynthesis of pteridines. Analyses of a variety of zeste mutants of Drosophila melanogaster have shown that these mutants all contain GTP cyclohydrolase equal approximately to the amount found in the wild-type fly. These observations do not support the suggestions made by Rasmusson et al. (1973) that zeste is the structural locus for GTP cyclohydrolase.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (GB33929).     

Tissue-Specific and Complex Complementation Patterns in the Punch Locus of DROSOPHILA MELANOGASTER

Mutations in the Punch locus result in loss of GTP cyclohydrolase activity, but all mutations do not affect the enzyme in the same way. There are at least three classes of Punch mutations. One class results in a dominant eye color, recessive lethal phenotype. A second class of mutations also causes a recessive lethal phenotype, but heterozygous mutants have normal eye color. They show loss of GTP cyclohydrolase function in all tissues where activity can be measured. Alleles comprising a third class are recessive eye color mutations that are homozygous viable. Individuals with this third type of mutation show loss of enzyme activity in the eye, but show normal or near-normal activity elsewhere. In order to examine the organization and function of this locus further, we have performed interallelic complementation tests on 25 Punch mutations, monitoring viability and enzyme activity in prepupae and adults. Most allele combinations are lethal. Those that complement do so in ways that are tissue-or stage-specific and unpredictable. Tests of mutants with tissue-specific phenotypes and of individuals mutant for complementing Punch lethal alleles lead us to conclude that Punch is a complex locus, both with respect to its organization and to its products.     

Control of drosopterin synthesis in Drosophila melanogaster: Mutants showing an altered pattern of GTP cyclohydrolase activity during development

The reaction catalyzed by GTP cyclohydrolase is the first unique step of pteridine biosynthesis in Drosophila melanogaster and is therefore likely to be an important control point. GTP cyclohydrolase activity varies during development, showing two distinct peaks of activity—one at pupariation and a much larger peak at emergence. Most of the early pupal enzyme is located in the body region, whereas in late pupal and early adult life most of the activity is found in the head. Mixing experiments indicate that developmental changes in activity are not due to changes in the level of a direct effector of GTP cyclohydrolase. The mutants raspberry and prune show an increased GTP cyclohydrolase activity at pupariation relative to wild type, but a decreased enzyme activity at emergence. The changes in GTP cyclohydrolase activity are reflected in changes in pteridine levels in these mutants. Several lines of evidence suggest that neither locus is the structural gene for GTP cyclohydrolase. The raspberry and prune gene products may play a specific role in regulating GTP cyclohydrolase activity during development.This work was supported by a grant from the Australian Research Grants Committee D2 75/15248.     

Regulation of GTP cyclohydrolase I and dihydropteridine reductase in rat pheochromocytoma PC 12 cells

The addition of 8-bromo cyclic AMP, forskolin, theophylline, and 3-isobutyl-1-methylxanthine to the medium of PC 12 cells resulted in an increase in GTP cyclohydrolase I activity, but had no effect on dihydropteridine reductase activity, except theophylline which caused a decrease in dihydropteridine reductase activity at 96 h. GTP cyclohydrolase I activity peaked at 24 h and returned to normal 96 h after drug treatment. Cycloheximide decreased GTP cyclohydrolase I activity at 48 and 96 h, but had little effect on dihydropteridine reductase activity. The addition of reserpine selectively increased only GTP cyclohydrolase I activity. The addition of tetrahydrobiopterin and sepiapterin, however, coordinately inhibited both GTP cyclohydrolase I and dihydropteridine reductase activities. It appears that GTP cyclohydrolase I activity in PC 12 cells is regulated by cyclic AMP stimulation and by end-product inhibition, whereas dihydropteridine reductase activity is only subject to pterin inhibition.     

Selection and mutant properties of Pichia guilliermondii yeasts with derepressed GTP cyclohydrolase, the enzyme of the 1st step in flavinogenesis

A positive method is proposed for selecting Pichia guilliermondii mutants with derepressed GTP cyclohydrolase. Mutants with the incompletely blocked gene RIB2 were used as parent strains; these can grow in a medium without riboflavin (RF) only if the enzyme is derepressed as the result of iron deficiency in cells. Strains growing in a medium without RF at the optimal supply of cells with iron were selected as regulatory mutants. The mutants accumulated 6,7-dimethylpterin in high concentrations and a small amount of RF in the medium and in the cells. The activity of GTP cyclohydrolase rather than that of RF synthase increased in the mutants; the activity of RF kinase and FAD pyrophosphorylase was not elevated. Hybrids produced by crossing the regulatory mutants with wild type strains did not accumulate 6,7-dimethylpterin in the medium and the activity of the GTP cyclohydrolase did not increase; this is indicative of the negative regulation for the expression of the structural gene for GTP cyclohydrolase. The authors propose a model for the regulation of GTP cyclohydrolase and RF synthase at the gene level involving iron ions as a corepressor.     

Leafy Cotyledon Mutants of Arabidopsis

We have previously described a homeotic leafy cotyledon (lec) mutant of Arabidopsis that exhibits striking defects in embryonic maturation and produces viviparous embryos with cotyledons that are partially transformed into leaves. In this study, we present further details on the developmental anatomy of mutant embryos, characterize their response to abscisic acid (ABA) in culture, describe other mutants with related phenotypes, and summarize studies with double mutants. Our results indicate that immature embryos precociously enter a germination pathway after the torpedo stage of development and then acquire characteristics normally restricted to vegetative parts of the plant. In contrast to other viviparous mutants of maize (vp1) and Arabidopsis (abi3) that produce ABA-insensitive embryos, immature lec embryos are sensitive to ABA in culture. ABA is therefore necessary but not sufficient for embryonic maturation in Arabidopsis. Three other mutants that produce trichomes on cotyledons following precocious germination in culture are described. One mutant is allelic to lec1, another is a fusca mutant (fus3), and the third defines a new locus (lec2). Mutant embryos differ in morphology, desiccation tolerance, pattern of anthocyanin accumulation, presence of storage materials, size and frequency of trichomes on cotyledons, and timing of precocious germination in culture. The leafy cotyledon phenotype has therefore allowed the identification of an important network of regulatory genes with overlapping functions during embryonic maturation in Arabidopsis.     

Pterin and folate salvage. Plants and Escherichia coli lack capacity to reduce oxidized pterins

Dihydropterins are intermediates of folate synthesis and products of folate breakdown that are readily oxidized to their aromatic forms. In trypanosomatid parasites, reduction of such oxidized pterins is crucial for pterin and folate salvage. We therefore sought evidence for this reaction in plants. Three lines of evidence indicated its absence. First, when pterin-6-aldehyde or 6-hydroxymethylpterin was supplied to Arabidopsis (Arabidopsis thaliana), pea (Pisum sativum), or tomato (Lycopersicon esculentum) tissues, no reduction of the pterin ring was seen after 15 h, although reduction and oxidation of the side chain of pterin-6-aldehyde were readily detected. Second, no label was incorporated into folates when 6-[(3)H]hydroxymethylpterin was fed to cultured Arabidopsis plantlets for 7 d, whereas [(3)H]folate synthesis from p-[(3)H]aminobenzoate was extensive. Third, no NAD(P)H-dependent pterin ring reduction was found in tissue extracts. Genetic evidence showed a similar situation in Escherichia coli: a GTP cyclohydrolase I (folE) mutant, deficient in pterin synthesis, was rescued by dihydropterins but not by the corresponding oxidized forms. Expression of a trypanosomatid pterin reductase (PTR1) enabled rescue of the mutant by oxidized pterins, establishing that E. coli can take up oxidized pterins but cannot reduce them. Similarly, a GTP cyclohydrolase I (fol2) mutant of yeast (Saccharomyces cerevisiae) was rescued by dihydropterins but not by most oxidized pterins, 6-hydroxymethylpterin being an exception. These results show that the capacity to reduce oxidized pterins is not ubiquitous in folate-synthesizing organisms. If it is lacking, folate precursors or breakdown products that become oxidized will permanently exit the metabolically active pterin pool.     

Mechanism of suppression in Drosophila. V. Localization of the purple mutant of Drosophila melanogaster in the pteridine biosynthetic pathway

The suppressible eye color mutant purple (pr) of Drosophila melanogaster is known to be unable to synthesize a wild-type complement of pteridine eye pigments. This study measures the reduced levels of drosopterins, sepiapterin, and an unidentified presumed pteridine in pr and pr ^bw. Pteridine analyses in double mutants combining pr with one of three other eye color mutants sepia, Henna-recessive³, and prune², suggest that the metabolic block in pr occurs prior to sepiapterin biosynthesis. Measurements of GTP and GTP cyclohydrolase in pr showed wild-type levels and indicate the metabolic block in pr to be at one of the steps converting dihydroneopterin triphosphate to sepiapterin. Quantitation of pteridines in suppressed purple [su(s) ²; pr and pr; su(pr) ^e3] shows restoration of pteridines to wild-type or nearly wild-type levels.T. G. W. is a predoctoral trainee supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.The Oak Ridge National Laboratory is operated by Union Carbide Corporation for the U.S. Energy Research and Development Administration.