The Drosophila melanogaster ade5 gene encodes a bifunctional enzyme for two steps in the de novo purine synthesis pathway

Steps 6 and 7 of de novo purine synthesis are performed by 5-aminoimidazole ribonucleotide carboxylase (AIRc) and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide synthetase (SAICARs), respectively. In vertebrates, a single gene encodes AIRc-SAICARs with domains homologous to Escherichia coli PurE and PurC. We have isolated an AIRc-SAICARs cDNA from Drosophila melanogaster via functional complementation with an E. coli purC purine auxotroph. This cDNA encodes AIRc yet is unable to complement an E. coli purE mutant, suggesting functional differences between Drosophila and E. coli AIRc. In vertebrates, the AIRc-SAICARs gene shares a promoter region with the gene encoding phosphoribosylamidotransferase, which performs the first step in de novo purine synthesis. In Drosophila, the AIRc-SAICARs gene maps to section 11B4-14 of the X chromosome, while the phosphoribosylamidotransferase gene (Prat) maps to chromosome 3; thus, the close linkage of these two genes is not conserved in flies. Three EMS-induced X-linked adenine auxotrophic mutations, ade4(1), ade5(1), and ade5(2), were isolated. Two gamma-radiation-induced (ade5(3) and ade5(4)) and three hybrid dysgenesis-induced (ade5(5), ade5(6), and ade5(8)) alleles were also isolated. Characterization of the auxotrophy and the finding that the hybrid dysgenesis-induced mutations all harbor P transposon sequences within the AIRc-SAICARs gene show that ade5 encodes AIRc-SAICARs.     

The purine synthesis gene Prat2 is required for Drosophila metamorphosis, as revealed by inverted-repeat-mediated RNA interference

PRAT (phosphoribosylamidotransferase; E.C. 2.4.2.14) catalyzes the first reaction in de novo purine nucleotide biosynthesis. In Drosophila melanogaster, the Prat and Prat2 genes are both highly conserved with PRAT sequences from prokaryotes and eukaryotes. However, Prat2 organization and expression during development is different from Prat. We used RNA interference (RNAi) to knock down expression of both Prat and Prat2 to investigate their functions. Using the GAL4-UAS system, Prat RNAi driven by Act5c-GAL4 or tubP-GAL4 causes variable pupal lethality (48-100%) and approximately 50% female sterility, depending on the transgenic strains and drivers used. This observation agrees with the phenotype previously observed for Prat EMS-induced mutations. Prat2 RNAi driven by Act5C-GAL4 or tubP-GAL4 also results in variable pupal lethality (61-93%) with the different transgenic strains, showing that Prat2 is essential for fly development. However, Prat2 RNAi-induced arrested pupae have a head eversion defect reminiscent of the "cryptocephal" phenotype, whereas Prat RNAi-induced arrested pupae die later as pharate adults. We conclude that Prat2 is required during the prepupal stage while Prat is more important for the pupal stage. In addition, Prat and Prat2 double RNAi results in more severe pupal lethal phenotypes, suggesting that Prat and Prat2 have partially additive functions during Drosophila metamorphosis.     

Multifunctional polypeptides for purine de novo synthesis

The pathway leading to the synthesis of purines for ATP, RNA, DNA and other cellular molecules involves the same enzymatic steps for all groups of organisms. However, the organization of the polypeptides catalyzing some of these steps differs strikingly from organism to organism.     

A putative de novo evolved gene required for spermatid chromatin condensation in Drosophila melanogaster

Comparative genomics has enabled the identification of genes that potentially evolved de novo from non-coding sequences. Many such genes are expressed in male reproductive tissues, but their functions remain poorly understood. To address this, we conducted a functional genetic screen of over 40 putative de novo genes with testis-enriched expression in Drosophila melanogaster and identified one gene, atlas, required for male fertility. Detailed genetic and cytological analyses showed that atlas is required for proper chromatin condensation during the final stages of spermatogenesis. Atlas protein is expressed in spermatid nuclei and facilitates the transition from histone- to protamine-based chromatin packaging. Complementary evolutionary analyses revealed the complex evolutionary history of atlas. The protein-coding portion of the gene likely arose at the base of the Drosophila genus on the X chromosome but was unlikely to be essential, as it was then lost in several independent lineages. Within the last ~15 million years, however, the gene moved to an autosome, where it fused with a conserved non-coding RNA and evolved a non-redundant role in male fertility. Altogether, this study provides insight into the integration of novel genes into biological processes, the links between genomic innovation and functional evolution, and the genetic control of a fundamental developmental process, gametogenesis.     

Sexual phenotype and vitellogenin synthesis in Drosophila melanogaster

An ovary transplanted from a Drosophila melanogaster female into a male will mature and form morphologically normal yolk-filled oocytes. Since it has been supposed that the yolk polypeptides come only from the female fat body, it was hypothesized that the implanted ovary induces the fat body of the male host to synthesize and secrete yolk polypeptides (YPs). To test this hypothesis, fat body preparations from females, untreated males, and males containing transplanted ovaries were cultured in vitro with ³⁵S-methionine and the medium was examined for the presence of newly labeled YPs. Female fat body secreted newly labeled YPs, but no freshly synthesized YPs were secreted by fat bodies from untreated males or from males containing transplanted ovaries. In vitro cultured ovaries, however, both from females and from male hosts did secrete newly synthesized YPs. Therefore, the YPs in an ovary that matured in a male come mainly from endogenous synthesis by the implanted ovary. To find whether males were responsive to the hormones that stimulate YP production in isolated female abdomens, we treated males with the juvenile hormone analogue ZR-515 and with 20-hydroxyecdysone. The latter, but not the former, was able to cause synthesis and secretion of three bands migrating precisely as YPs in SDS gels. Partial peptide digests of the 20-hydroxyecdysone-stimulated polypeptides in males showed them to be identical with those stimulated by 20-hydroxyecdysone or ZR-515 in isolated female abdomens and with the three YPs found in normal female hemolymph. Finally, YP synthesis was assayed in mutants that affect the phenotypic sex of a fly. It was found that flies bearing two X chromosomes and the mutations dsx, dsx^D, ix or three sets of autosomes continued to make YPs, but tra-3-pseudomales did not. These results suggest that the process of sex determination involves steps leading to synthesis of an ecdysteroid in females, which then activates synthesis of the YPs by the fat body. A hypothesis is suggested to explain the fact that two different hormones can stimulate YP synthesis and two different organs can synthesize YPs.     

Regulation of purine synthesis de novo in human fibroblasts by purine nucleotides and phosphoribosylpyrophosphate

Previous studies of purine nucleotide synthesis de novo have suggested that major regulation of the rate of the pathway is affected at either the phosphoribosylpyrophosphate (PP-Rib-P) synthetase reaction or the amidophosphoribosyltransferase (amido PRT) reaction, or both. We studied control of purine synthesis de novo in cultured normal, hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient, and PP-Rib-P synthetase-superactive human fibroblasts by measuring concentrations and rates of synthesis of PP-Rib-P and purine nucleotide end products, proposed effectors of regulation, during inhibition of the pathway. Incubation of cells for 90 min with 0.1 mM azaserine, a glutamine antagonist which specifically blocked the pathway at the level of conversion of formylglycinamide ribotide, resulted in a 5-16% decrease in purine nucleoside triphosphate concentrations but no consistent alteration in generation of PP-Rib-P. During this treatment, however, rates of the early steps of the pathway were increased slightly (9-15%) in normal and HGPRT-deficient strains, more markedly (32-60%) in cells with catalytically superactive PP-Rib-P synthetases, and not at all in fibroblasts with purine nucleotide feedback-resistant PP-Rib-P synthetases. In contrast, glutamine deprivation, which inhibited the pathway at the amido PRT reaction, resulted in time-dependent nucleoside triphosphate pool depletion (26-43% decrease at 24 h) accompanied by increased rates of PP-Rib-P generation and, upon readdition of glutamine, substantial increments in rates of purine synthesis de novo. Enhanced PP-Rib-P generation during glutamine deprivation was greatest in cells with regulatory defects in PP-Rib-P synthetase (2-fold), but purine synthesis in these cells was stimulated only 1.4-fold control rates by glutamine readdition. Stimulation of these processes in normal and HGPRT-deficient cells and in cells with PP-Rib-P synthetase catalytic defects was, respectively: 1.5 and 2.0-fold; 1.5 and 1.7-fold; and 1.6 and 4.1-fold. These studies support the following concepts. 1) Rates of purine synthesis de novo are regulated at both the PP-Rib-P synthetase and amido PRT reactions by end products, with the latter reaction more sensitive to small changes in purine nucleotide inhibitor concentrations. 2) PP-Rib-P exerts its role as a major regulator of purine synthetic rate by virtue of its interaction with nucleotide inhibitors to determine the activity of amido PRT. 3) Activation of amido PRT by PP-Rib-P is nearly maximal at base line in fibroblasts with regulatory defects in PP-Rib-P synthetase.     

Molecular nature of 11 spontaneous de novo mutations in Drosophila melanogaster

To investigate the molecular nature and rate of spontaneous mutation in Drosophila melanogaster, we screened 887,000 individuals for de novo recessive loss-of-function mutations at eight loci that affect eye color. In total, 28 mutants were found in 16 independent events (13 singletons and three clusters). The molecular nature of the 13 events was analyzed. Coding exons of the locus were affected by insertions or deletions >100 nucleotides long (6 events), short frameshift insertions or deletions (4 events), and replacement nucleotide substitutions (1 event). In the case of 2 mutant alleles, coding regions were not affected. Because approximately 70% of spontaneous de novo loss-of-function mutations in Homo sapiens are due to nucleotide substitutions within coding regions, insertions and deletions appear to play a much larger role in spontaneous mutation in D. melanogaster than in H. sapiens. If so, the per nucleotide mutation rate in D. melanogaster may be lower than in H. sapiens, even if their per locus mutation rates are similar.     

Developmental analysis of the torso-like phenotype in Drosophila produced by a maternal-effect locus

The segmental plan of the Drosophila embryo is already established at the blastoderm stage through the action of maternal effect genes which determine the polarity of the embryo and zygotically active genes involved in segmentation. We have analyzed the first example of a group of maternally acting genes which are necessary for establishing the developmental potential of the posterior 25% of the blastoderm. Females, homozygous for the X-linked maternal-effect mutation female sterile(1)Nasrat211 [fs(1)N211], produce embryos, characterized as torso-like, which lack all posterior endodermal derivatives as well as structures characteristic of abdominal segments 8 to 10. In addition, anterior endodermal derivatives are deficient and the absence of pharyngeal musculature causes a collapse of the cephalopharyngeal apparatus. The columnar blastoderm cell layer is defective at the posterior tip below the pole cells in these embryos. This defect, however, is presumably secondary to some abnormal feature of pole cell formation since in double mutants of fs(1)Nasrat211; tudor3 the blastoderm is normal but the embryos still show the torso-like phenotype. In situ hybridization with RNA probes derived from the fushi tarazu gene establishes that the cellular determination of the posterior blastoderm of embryos produced by fs(1)N211 is changed. This represents the first direct demonstration that a maternal-effect mutation alters the spatial distribution of a zygotic gene product involved in the segmental patterning of the embryo.     

Adenosine blocks hormone-induced meiotic maturation by suppressing purine de novo synthesis

We have examined adenosine (Ado) suppression of FSH-induced germinal vesicle breakdown (GVB) and its relationship to purine de novo synthesis. Oocyte-cumulus cell complexes (OCC) from PMSG-primed, immature mice were cultured 17-18 hr in medium containing 4 mM hypoxanthine (HX) or 300 microM dibutyryl cAMP (dbcAMP) to maintain meiotic arrest, and FSH was added to stimulate meiotic maturation. In the absence of FSH, Ado (1-250 microM) had no effect in dbcAMP-arrested oocytes but dose-dependently suppressed maturation in HX-treated oocytes. FSH-induced maturation was prevented by Ado, though more effectively in dbcAMP-supplemented cultures. Ado affected the magnitude, but not the kinetics pattern, of the response to FSH. Inosine also blocked meiotic induction, but only in dbcAMP-arrested oocytes. Purine de novo synthesis was nearly doubled in OCC by FSH treatment, and this response was completely prevented by Ado. FSH had no effect on HX salvage, although Ado reduced this activity by 98%. Inosine effects on metabolism were intermediate between the control and Ado groups. Experiments with radiolabeled energy substrates showed that Ado suppressed FSH activation of the pentose phosphate pathway but did not prevent significant activation of glycolysis or oxidation of pyruvate. Finally, in cultured follicles from primed mice, hCG-induced maturation was blocked by Ado as effectively as by the purine de novo synthesis inhibitor, azaserine. It is concluded that Ado has an inhibitory action on hormone-induced maturation that is due, at least in part, to suppression of glucose metabolism, leading to compromised purine de novo synthesis.     

Experimental phenocopy of a minute maternal-effect mutation alters blastoderm determination in embryos of Drosophila melanogaster

Maternal haploinsufficiency for a third chromosome Minute, M(3)i55, lowers rates of protein synthesis by approximately 30% during the syncytial nuclear cycles of early embryogenesis. The maternal effect of Mi55 also produces segmentation defects (denticle belt fusions) in the posterior abdomen of larvae. Furthermore, embryos from Minute mothers show abnormal expression patterns of the segmentation gene fushi tarazu (ftz) at the cellular blastoderm stage of embryogenesis. We developed a computer-aided analysis to describe the deviations in ftz expression which demonstrates that abnormally narrow ftz stripes occur in segment primordia that become fused in the larva. Unexpectedly, an abnormally wide ftz stripe occurs in segment primordia which do not develop abnormally. In addition, Mi55 produces a general narrowing of all ftz- interstripes. We phenocopied the Minute mutation by injecting wild-type embryos with cycloheximide concentrations which decreased protein synthesis rates to levels comparable with those of Minute embryos. Thus, a general decrease in protein synthesis during early embryogenesis leads to abnormal determination of posterior abdominal segment primordia.     

A new folate antimetabolite, 5,10-dideaza-5,6,7,8-tetrahydrofolate is a potent inhibitor of de novo purine synthesis

5,10-Dideazatetrahydrofolate (DDATHF) is a new antimetabolite designed as an inhibitor of folate metabolism at sites other than dihydrofolate reductase. DDATHF was found to inhibit the growth of L1210 and CCRF-CEM cells in culture at concentrations in the range of 10-30 nM. The inhibitory effect of DDATHF on the growth of L1210 and CCRF-CEM cells was reversed by either hypoxanthine or aminoimidazole carboxamide. Growth inhibition by DDATHF was prevented by addition of both thymidine and hypoxanthine, but not by thymidine alone. 5-Formyltetrahydrofolate reversed the effects of DDATHF in a dose-dependent manner. DDATHF had no appreciable inhibitory activity against either dihydrofolate reductase or thymidylate synthase in vitro, but was found to be an excellent substrate for folylpolyglutamate synthetase. DDATHF had little or no effect on incorporation of either deoxyuridine or thymidine into DNA, in distinct contrast to the effects of the classical dihydrofolate reductase inhibitor, methotrexate. DDATHF was found to deplete cellular ATP and GTP over the same concentrations as those inhibitory to leukemic cell growth, suggesting that the locus of DDATHF action was in the de novo purine biosynthesis pathway. The synthesis of formylglycinamide ribonucleotide in intact L1210 cells was inhibited by DDATHF with the same concentration dependence as inhibition of growth. This suggested that DDATHF inhibited glycinamide ribonucleotide transformylase, the first folate-dependent enzyme of de novo purine synthesis. DDATHF is a potent folate analog which suppresses purine synthesis through direct or indirect inhibition of glycinamide ribonucleotide transformylase.     

Healing of Euchromatic Chromosome Breaks by Efficient de novo Telomere Addition in Drosophila melanogaster

Many arthropod species are infected with maternally inherited endosymbionts that induce a shift in the sex ratio of their hosts by feminizing or killing males (cytoplasmic sex-ratio distorters, or SRDs). These endosymbionts can have profound impacts on evolutionary processes of their hosts. Here, I derive analytical expressions for the coalescent effective size N_e of populations that are infected with SRDs. Irrespective of the type of SRD, N_e for mitochondrial genes is given by the number of infected females. For nuclear genes, the effective population size generally decreases with increasing prevalence of the SRD and can be considerably lower than the actual size of the population. For example, with male-killing bacteria that have near perfect maternal transmission, N_e is reduced by a factor that is given to a good approximation by the proportion of uninfected individuals in the population. The formulae derived here also yield the effective size of populations infected with mutualistic endosymbionts or maternally inherited bacteria that induce cytoplasmic incompatibility, although in these cases, the reduction in N_e is expected to be less severe than for cytoplasmic SRDs.SIMPLE null models are essential in science. In population genetics, this role is filled by the Wright–Fisher model and its retrospective counterpart, the Kingman coalescent. Both of these models have proven to be immensely useful in spite of the fact that natural populations usually violate the assumptions made in these models. The reason for this is that often, the Wright–Fisher model can be rescaled so that it behaves in many important respects like a more complex population model. This rescaling is achieved through the concept of the effective population size, N_e. Roughly speaking, a complex population model is said to have a certain N_e if the haploid Wright–Fisher model with population size N_e experiences the same amount of random genetic drift as the complex model. Reflecting the different ways in which drift can be measured, N_e can be defined in different ways, e.g., as the inbreeding, the variance, or the coalescent effective population size. Different definitions often produce the same value for N_e, but may also yield drastically different numbers (Kimura and Crow 1963).The coalescent effective population size is defined through the factor by which time needs to be rescaled in a complex population model to produce the standard coalescent with time scale given by the population size N (Nordborg and Krone 2002). It has been argued that this is the most useful definition for N_e because “the coalescent essentially embodies all of the information that can be found in sampled genetic data” (Sjödin et al. 2005). More recently, Wakeley and Sargsyan (2009) have proposed two extensions of the coalescent effective population size in which they advocate including a mutation parameter in the definition and also allowing for a nonlinear relationship between N_e and N.One frequently encountered feature in natural populations that complicates population genetics is infection with maternally inherited endosymbionts. In particular, many arthropod species harbor a great number of phylogenetically diverse microorganisms that influence their hosts'' biology in different ways (Bourtzis and Miller 2003; Bourtzis and Miller 2006; Bourtzis and Miller 2009). Because of their maternal transmission, many of these microorganisms—for example, the bacteria Wolbachia pipientis and Cardinium hertigii—have evolved intricate manipulations of their hosts'' reproductive system that allows them to spread in a host population through exploitation of male hosts (reproductive parasitism, reviewed in Engelstädter and Hurst 2009a). Most manipulations involve a shift in the sex ratio of their hosts (both primary and at the population level), and the inducing endosymbionts are consequently referred to as cytoplasmic sex-ratio distorters (SRDs). In some species, genetic males develop into females if they are infected (“feminization”: Martin et al. 1973; Rigaud 1997; Bouchon et al. 1998; Hiroki et al. 2002). More commonly, infected males are killed by the endosymbionts early in their development (male killing: reviewed in Hurst et al. 2003). The adaptive advantage of this strategy is seen in an early fitness boost in the surviving females in a brood, for example, through reduced sibling competition or cannibalism of the dead brothers (Hurst 1991; Hurst and Majerus 1993; Jaenike et al. 2003). Some examples for species infected with male-killing or feminizing SRDs are given in Huigens and Stouthamer 2003).

TABLE 1

Empirical examples for cytoplasmic SRDs with parameter estimates

Effect of testosterone on purine synthesis de novo in rat perineal muscle in vivo

We examined in vivo the influence of testosterone on purine synthetis de nov, in the levator ani and gastrocnemius muscles of the rat. The hypoxanthine, adenine and guanine contents and the rate of incorporation of [¹⁴C]formate into these purine bases were determined in castrated adult and prepubertal rats (groups 1 and 2) both before and after orchiectomy and, in the second case, at different times after testosterone treatment. Substantially similar behavior was found in both groups, with some specific differences. The results showed an increase in the basal levels after castration (except for a dramatic decrease in adenine and a rise in the Gua/Ade molar ratio in prepubertal rats) and a return to basal levels after hormone administration, which was also accompanied by variations in the Gua/Ade molar ratio. The kinetics of purine nucleotide synthesis de novo and, spefically, of the overall reactions: IMP formation from PRib-PP, IMP → AMP and IMP → GMP, were followed by evaluating the incorporation curves of [¹⁴C]formate into hypoxanthine, adenine and guanine. Our results show that testosterone administration enhanced the incorporation rate and gave characteristic patterns: a diphasic cyclic oscillation of the Ade values in adult castrated rats, and single peaks having a specific shape in the other cases. The Gua/Ade labeling ratio was unchaned in castrated rats and increased in both groups during ther first 5 days after testosterone treatment, after which values even fell below normal; in most cases, values overlapped the pattern of the Gua/Ade molar ratio. The specific profile of the curves indicated that testosterone initially accelerated the turnover of guanylic acid and in the second phase re-established the normal behavior and ratio of AMP and GMP formation. These results indicate that the ‘inosinic branch point’ was subject to regulation by testosterone. The profiles of the incorporation curves and of the Gua/Ade ratio were indicative of a primary and secondary response to hormone action.     

Insertional inactivation of the gene for collagen-binding protein has a pleiotropic effect on the phenotype of Staphylococcus aureus.

This report describes phenotypical changes caused by the insertional inactivation of the gene for the collagen-binding protein in Staphylococcus aureus PH100. Insertional inactivation resulted in reductions in the amount of fibronectin-binding protein in PH100 and the ability of intact cells to aggregate in the presence of fibronectin. However, the capacity of PH100 to adhere to immobilized fibronectin remained the same.