Cloning and partial characterization of the xanthine dehydrogenase gene of Calliphora vicina, a distant relative of Drosophila melanogaster

In vitro enzymatic assays have shown that an enzyme with typical xanthine dehydrogenase (XDH) activities and electrophoretic mobility slightly different from that of Drosophila XDH is present in Calliphora tissues. A Calliphora genomic sequence has been isolated by low-stringency hybridization to the Drosophila rosy gene (XDH), and partially sequenced. This sequence has been shown to be unique, polymorphic, and it maps on chromosome I. Sequence comparisons provide compelling evidence that it belongs to the XDH gene of Calliphora. Interspecies transformation experiments, aimed at investigating functional as well as structural divergence of the XDH genes of Calliphora and Drosophila, are now possible.     

Genetic Limits of the Xanthine Dehydrogenase Structural Element within the Rosy Locus in DROSOPHILA MELANOGASTER

Experiments are described that provide an opportunity to estimate the genetic limits of the structural (amino acid coding) portion of the rosy locus (3:52.0) in Drosophila melanogaster, which controls the enzyme, xanthine dehydrogenase (XDH). This is accomplished by mapping experiments which localize sites responsible for electrophoretic variation in the enzyme on the known genetic map of null-XDH rosy mutants. Electrophoretic sites are distributed along a large portion of the null mutant map. A cis-trans test involving electrophoretic variants in the left- and right-hand portions of the map leads to the conclusion that the entire region between these variants is also structural. Hence most, if not all, of the null mutant map of the rosy locus contains structural information for the amino acid sequence of the XDH polypeptide. Consideration is given to the significance of the present results for the general problem of gene organization in higher eukaryotes.     

Extension of the Limits of the Xdh Structural Element in DROSOPHILA MELANOGASTER

Experiments expanding the array of mutants affecting the xanthine dehydrogenase (XDH) structural element in Drosophila melanogaster are described. These include rosy eye color mutants which exhibit interallelic complementation, and mutants with normal eye color but lowered levels of XDH. Evidence is presented which argues that these are structural alterations in the enzyme. Recombination experiments were performed using these mutants as well as some electrophoretic variants. The two ends of the rosy locus are marked with mutant sites which are clearly structural in nature; the XDH structural element and the rosy null mutant map are completely concordant. A possible procedure to recover control element mutants is described.     

Organization of the Rosy Locus in DROSOPHILA MELANOGASTER: Further Evidence in Support of a CIS-Acting Control Element Adjacent to the Xanthine Dehydrogenase Structural Element

The present report summarizes our recent progress in the genetic dissection of an elementary genetic unit in a higher organism, the rosy locus (ry:3--52.0) in Drosophila melanogaster. Pursuing the hypothesis that the rosy locus includes a noncoding control region, as well as a structural element coding for the xanthine dehydrogenase (XDH) peptide, experiments are described that characterize and map a rosy locus variant associated with much lower than normal levels of XDH activity. Experiments are described that fail to relate this phenotype to alteration in the structure of the XDH peptide, but clearly associate this character with variation in number of molecules of XDH per fly. Large-scale fine-structure recombination experiments locate the genetic basis for this variation in the number of molecules of XDH per fly to a site immediately to the left of the XDH structural element within a region previously designated as the XDH control element. Moreover, experiments clearly separate this "underproducer" variant site from a previously described "overproducer" site within the control region. Examination of enzyme activity in electrophoretic gels of appropriate heterozygous genotypes demonstrates the cis-acting nature of this variation in the number of molecules of XDH. A revision of the map of the rosy locus, structural and control elements is presented in the light of the additional mapping data now available.     

The Rosy Locus in Drosophila Melanogaster: Xanthine Dehydrogenase and Eye Pigments

The rosy gene in Drosophila melanogaster codes for the enzyme xanthine dehydrogenase (XDH). Mutants that have no enzyme activity are characterized by a brownish eye color phenotype reflecting a deficiency in the red eye pigment. Xanthine dehydrogenase is not synthesized in the eye, but rather is transported there. The present report describes the ultrastructural localization of XDH in the Drosophila eye. Three lines of evidence are presented demonstrating that XDH is sequestered within specific vacuoles, the type II pigment granules. Histochemical and antibody staining of frozen sections, as well as thin layer chromatography studies of several adult genotypes serve to examine some of the factors and genic interactions that may be involved in transport of XDH, and in eye pigment formation. While a specific function for XDH in the synthesis of the red, pteridine eye pigments remains unknown, these studies present evidence that: (1) the incorporation of XDH into the pigment granules requires specific interaction between a normal XDH molecule and one or more transport proteins; (2) the structural integrity of the pigment granule itself is dependent upon the presence of a normal balance of eye pigments, a notion advanced earlier.     

Identification of an amber nonsense mutation in the rosy516 gene by germline transformation of an amber suppressor tRNA gene.

Seven xanthine dehydrogenase and cross-reacting material negative Drosophila melanogaster rosy stocks were screened for amber and ochre nonsense mutations. Amber and ochre nonsense suppressors were created by site-directed mutagenesis starting from a wild-type tRNA(Tyr) gene. The suppressor tRNA genes were subcloned into a pUChsneo transformation vector providing heat-shock controlled neomycin resistance. The seven rosy stocks were germline transformed with amber and ochre tDNA(Tyr), and the G1 generation was screened for Geneticin resistance. Surviving rosy516 flies transformed with the amber suppressor showed an eye colour intermediate between the original ry516 stock and the wild-type, suggesting that ry516 is an amber nonsense mutant. This was confirmed by sequencing the relevant part of the ry516 gene; the analysis revealed a C-to-T transition in a CAG glutamine codon at nucleotide 1522 of the wild-type rosy gene.     

Cloning and dosage compensation of the 6-phosphogluconate dehydrogenase gene (Pgd+) of Drosophila melanogaster

Using a heterologous rat cDNA probe, we have identified a 14.7 kbp Drosophila melanogaster genomic clone containing the X-linked gene Pgd+, which encodes the enzyme 6-phosphogluconate dehydrogenase (6PGD). We used in situ hybridization to larval polytene chromosomes, a somatic transient expression assay for enzyme activity, and the rescue of the lethal Pgd- phenotype by germline transformation to verify the identity of the gene. A 7.4 kbp fragment including the gene and approximately 1.2 kbp of upstream and 1.8 kbp of downstream sequences was relocated to autosomal ectopic sites by germline transformation; this transduced gene exhibits levels of enhanced activity in males comparable to those of the indigenous gene at its normal X chromosome locus. We conclude that the sequences responsible for dosage compensation of Pgd+ are included in this fragment.     

A deleted portion of one of the two xanthine dehydrogenase genes causes translucent larval skin in the oq mutant of the silkworm (Bombyx mori)

A silkworm mutant, oq, has translucent larval skin because it is deficient in xanthine dehydrogenase (XDH) activity and is unable to synthesize uric acid, which is normally accumulated in the larval epidermis and makes the skin white and opaque. Two XDH bands were found in zymograms of the silkworm fat body: an intense band (XDHalpha) and a faint one (XDHbeta). The oq mutant lacks only XDHalpha, which seemed to be the major source of XDH activity in the fat body. An 8-bp deletion found in BmXDH1, a silkworm XDH gene, generates a premature stop codon. The resulting truncated BmXDH1 protein lacks three molybdenum cofactor-binding domains necessary for enzyme activity. BmXDH2, the other XDH gene, does not show any apparent deficiencies. BmXDH1 expressed in yeast cells yielded an activity band with the same mobility as that of XDHalpha in zymograms. BmXDH1 of the oq mutant did not yield active XDH in yeast, while the activity was restored by filling in the deleted sequence. These results showed that BmXDH1 deletion in the oq mutant is responsible for the absence of significant XDH activity, resulting in the translucent larval skin of the mutant phenotype.     

The transformer gene in Ceratitis capitata provides a genetic basis for selecting and remembering the sexual fate

The medfly Ceratitis capitata contains a gene (Cctra) with structural and functional homology to the Drosophila melanogaster sex-determining gene transformer (tra). Similar to tra in Drosophila, Cctra is regulated by alternative splicing such that only females can encode a full-length protein. In contrast to Drosophila, however, where tra is a subordinate target of Sex-lethal (Sxl), Cctra seems to initiate an autoregulatory mechanism in XX embryos that provides continuous tra female-specific function and act as a cellular memory maintaining the female pathway. Indeed, a transient interference with Cctra expression in XX embryos by RNAi treatment can cause complete sexual transformation of both germline and soma in adult flies, resulting in a fertile male XX phenotype. The male pathway seems to result when Cctra autoregulation is prevented and instead splice variants with truncated open reading frames are produced. We propose that this repression is achieved by the Y-linked male-determining factor (M).     

An analysis of xanthine dehydrogenase negative mutants of the rosy locus in Drosophila melanogaster.

Eighteen alleles of the rosy locus in Drosophila melanogaster were characterized to identify putative nonsense mutants. Seven alleles exhibited no evidence of intragenic complementation, no evidence of immunological complementation, no evidence of immunological cross-reactivity to antibodies elicited by wild type xanthine dehydrogenase (XDH), and of course were completely deficient in measurable XDH activity. It is possible that one or more of these highly negative ry alleles are nonsense mutants. The remaining eleven ry alleles code for XDH molecules that retain some antigenic similarities to the wild type enzyme as assessed by immunoelectrophoresis and six of these eleven were capable of intragenic complementation.     

Cytogenetic Analysis of the Chromosomal Region Immediately Adjacent to the Rosy Locus in DROSOPHILA MELANOGASTER

This report describes the genetic analysis of a region of the third chromosome of Drosophila melanogaster extending from 87D2–4 to 87E12–F1, an interval of 23 or 24 polytene chromosome bands. This region includes the rosy (ry, 3–52.0) locus, carrying the structural information for xanthine dehydrogenase (XDH). We have, in recent years, focused attention on the genetic regulation of the rosy locus and, therefore, wished to ascertain in detail the immediate genetic environment of this locus. Specifically, we question if rosy is a solitary genetic unit or part of a larger complex genetic unit encompassing adjacent genes. Our data also provide opportunity to examine further the relationship between euchromatic gene distribution and polytene chromosome structure.——The results of our genetic dissection of the rosy microregion substantiate the conclusion drawn earlier (Schalet, Kernaghan and Chovnick 1964) that the rosy locus is the only gene in this region concerned with XDH activity and that all adjacent genetic units are functionally, as well as spatially, distinct from the rosy gene. Within the rosy micro-region, we observed a close correspondence between the number of complementation groups (21) and the number of polytene chromosome bands (23 or 24). Consideration of this latter observation in conjunction with those of similar studies of other chhromosomal regions supports the hypothesis that each polytene chromosome band corresponds to a single genetic unit.     

Cloning and characterization of the Tribolium castaneum eye-color genes encoding tryptophan oxygenase and kynurenine 3-monooxygenase.

The use of eye-color mutants and their corresponding genes as scorable marker systems has facilitated the development of transformation technology in Drosophila and other insects. In the red flour beetle, Tribolium castaneum, the only currently available system for germline transformation employs the exogenous marker gene, EGFP, driven by an eye-specific promoter. To exploit the advantages offered by eye-pigmentation markers, we decided to develop a transformant selection system for Tribolium on the basis of mutant rescue. The Tribolium orthologs of the Drosophila eye-color genes vermilion (tryptophan oxygenase) and cinnabar (kynurenine 3-monooxygenase) were cloned and characterized. Conceptual translations of Tc vermilion (Tcv) and Tc cinnabar (Tccn) are 71 and 51% identical to their respective Drosophila orthologs. We used RNA interference (RNAi) to show that T. castaneum larvae lacking functional Tcv or Tccn gene products also lack the pigmented eyespots observed in wild-type larvae. Five available eye-color mutations were tested for linkage to Tcv or Tccn via recombinational mapping. No linkage was found between candidate mutations and Tccn. However, tight linkage was found between Tcv and the white-eye mutation white, here renamed vermilion(white) (v(w)). Molecular analysis indicates that 80% of the Tcv coding region is deleted in v(w) beetles. These observations suggest that the Tribolium eye is pigmented only by ommochromes, not pteridines, and indicate that Tcv is potentially useful as a germline transformation marker.     

Germline autonomy of maternal-effect mutations altering the embryonic body pattern of Drosophila

Nine maternal-effect loci Drosophila melanogaster were tested in germline mosaics to determine whether the wildtype gene activity is required in somatic or germline components of the maternal ovary. Mutations in these loci affect the anterior-posterior or dorso-ventral body pattern. In all nine loci (torso, trunk, exuperantia, vasa, valois, staufen, tudor, dorsal, Toll) a mutant genotype in the germ cells is sufficient to produce all aspects of the mutant embryonic phenotype, even when those germ cells are surrounded by wildtype somatic tissues.     

Two functional but noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility

The trace biogenic amine tyramine is present in the nervous systems of animals ranging in complexity from nematodes to mammals. Tyramine is synthesized from tyrosine by the enzyme tyrosine decarboxylase (TDC), a member of the aromatic amino acid family, but this enzyme has not been identified in Drosophila or in higher animals. To further clarify the roles of tyramine and its metabolite octopamine, we have cloned two TDC genes from Drosophila melanogaster, dTdc1 and dTdc2. Although both gene products have TDC activity in vivo, dTdc1 is expressed nonneurally, whereas dTdc2 is expressed neurally. Flies with a mutation in dTdc2 lack neural tyramine and octopamine and are female sterile due to egg retention. Although other Drosophila mutants that lack octopamine retain eggs completely within the ovaries, dTdc2 mutants release eggs into the oviducts but are unable to deposit them. This specific sterility phenotype can be partially rescued by driving the expression of dTdc2 in a dTdc2-specific pattern, whereas driving the expression of dTdc1 in the same pattern results in a complete rescue. The disparity in rescue efficiencies between the ectopically expressed Tdc genes may reflect the differential activities of these gene products. The egg retention phenotype of the dTdc2 mutant and the phenotypes associated with ectopic dTdc expression contribute to a model in which octopamine and tyramine have distinct and separable neural activities.     

Domain mapping of tube, a protein essential for dorsoventral patterning of the Drosophila embryo.

The tube protein plays an essential role in the signal transduction pathway that establishes dorsoventral polarity in the Drosophila melanogaster embryo. Characterization of each of four tube mutants revealed a substitution or insertion in the amino-terminal half of the protein. This portion of the tube protein is also evolutionarily conserved, as demonstrated by isolation and sequencing of the Drosophila virilis tube gene. Moreover, RNA microinjection assays and germline transformation experiments demonstrated that the amino-terminal domain alone provides substantial levels of gene function: constructs encoding only the amino-terminal domain restore dorsoventral polarity to embryos lacking any maternal tube function. In the carboxyterminal domain, sequence conservation is concentrated in the five octapeptide repeats. Although the repeat-containing domain by itself provides no rescue of the tube maternal effect phenotype, it is necessary for wild-type levels of tube activity. This domain is thus likely to play an ancillary role in axis formation.