Prophenol oxidase A3 in Drosophila melanogaster: activation and the PCR-based cDNA sequence

Phenol oxidase exists in Drosophila hemolymph as a prophenol oxidase, A₁ and A₃, that is activated in vivo with a native activating system, AMM-1, by limited proteolysis with time. The polypeptide in purified prophenol oxidase A₃ has a molecular weight of approximately 77,000 Da. A PCR-based cDNA sequence coding A₃ has 2501 bp encoding an open reading frame of 682 amino acid residues. The potential copper-binding sites, from Trp-196 to Tyr-245, and from Asn-366 to Phe-421, are highly homologous to the corresponding sites in other invertebrates. The availability of prophenol oxidase cDNA should be useful in revealing the biochemical differences between A₁ and A₃ isoforms in Drosophila melanogaster that are refractory or unable to activate prophenol oxidase.     

Nucleotide sequence of a Drosophila melanogaster cDNA encoding a calnexin homologue

A cDNA which encodes a calnexin (Cnx)-like protein from Drosophila melanogaster has been characterized. The deduced amino acid sequence shares several regions of homology with Cnx from other sources with two conserved motifs each repeated four times. The gene was found to be transcribed in various tissues and at all developmental stages. We have mapped the gene at chromosomal position 99A and we have also mapped the related gene coding for Drosophila calreticulin at 85E.     

Molecular characterization of the Drosophila melanogaster urate oxidase gene, an ecdysone-repressible gene expressed only in the malpighian tubules.

The urate oxidase (UO) gene of Drosophila melanogaster is expressed during the third-instar larval and adult stages, exclusively within a subset of cells of the Malpighian tubules. The UO gene contains a 69-base-pair intron and encodes mature mRNAs of 1,224, 1,227, and 1,244 nucleotides, depending on the site of 3' endonucleolytic cleavage prior to polyadenylation. A direct repeat, 5'-AAGTGAGAGTGAT-3', is the proposed cis-regulatory element involved in 20-hydroxyecdysone repression of the UO gene. The deduced amino acid sequences of UO of D. melanogaster, rat, mouse, and pig and uricase II of soybean show 32 to 38% identity, with 22% of amino acid residues identical in all species. With use of P-element-mediated germ line transformation, 826 base pairs 5' and approximately 1,200 base pairs 3' of the D. melanogaster UO transcribed region contain all of the cis elements allowing for appropriate temporal regulation and Malpighian tubule-specific expression of the UO gene.     

The nucleotide sequence of a full length cDNA clone encoding rat liver urate oxidase

Recently we reported the sequence of a cDNA clone (pUOX-1), isolated from a lambda gt11 cDNA library, which encoded for rat liver urate oxidase (EC 1.7.3.3), but this clone lacked the nucleotide sequences encoding the N-terminal region for this enzyme. Using the cDNA insert from the pUOX-1 clone as a probe, we have now isolated a full length cDNA clone, pUOX-2, from a lambda gt10 library by plaque hybridization. Nucleotide sequence analysis of the pUOX-2 clone showed that it has 1379 base pairs with an open reading frame coding for 303 amino acid residues corresponding to a molecular mass of 34,931 daltons. In addition to the open reading frame the pUOX-2 contains 439 bp of 3'-untranslated and 41 bp of 5'-untranslated sequences. The consensus polyadenylation signal AATAAA precedes a stretch of poly(A)+ residues at the 3' end.     

Purine-induced expression of urate oxidase and enzyme activity in Atlantic salmon (Salmo salar). Cloning of urate oxidase liver cDNA from three teleost species and the African lungfish Protopterus annectens

The peroxisomal enzyme urate oxidase plays a pivotal role in the degradation of purines in both prokaryotes and eukaryotes. However, knowledge about the purine-induced expression of the encoding gene is lacking in vertebrates. These are the first published sequences of fish urate oxidase, which were predicted from PCR amplified liver cDNAs of Atlantic salmon (Salmo salar), Atlantic cod (Gadus morhua), Atlantic halibut (Hippoglossus hippoglossus) and African lungfish (Protopterus annectens). Sequence alignment of different vertebrate urate oxidases revealed amino acid substitutions of putative functional importance in the enzyme of chicken and lungfish. In the adult salmon, expression of urate oxidase mRNA predominated in liver, but was also identified in several nonhepatic organs including brain, but not in skeletal muscle and kidney. Juvenile salmon fed diets containing bacterial protein meal (BPM) rich in nucleic acids showed a significant increase in liver urate oxidase enzyme activity, and urea concentrations in plasma, muscle and liver were elevated. Whereas salmon fed the 18% BPM diet showed a nonsignificant increase in liver mRNA levels of urate oxidase compared with the 0% BPM-fed fish, no further increase in mRNA levels was found in fish receiving 36% BPM. The discrepancy between urate oxidase mRNA and enzyme activity was explained by rapid mRNA degradation or alternatively, post-translational control of the activity. Although variable plasma and liver levels of urate were detected, the substrate increased only slightly in 36% BPM-fed fish, indicating that the uricolytic pathway of Atlantic salmon is intimately regulated to handle high dietary purine levels.     

Purification and properties of dihydropterin oxidase from Drosophila melanogaster

The enzyme dihydropterin oxidase has been purified to apparent homogeneity from the fruit fly Drosophila melanogaster. This enzyme uses a variety of 2-amino-4-oxo-7,8-dihydropteridine compounds as substrates, including 2-amino-4-oxo-7,8-dihydropteridine (called dihydropterin), Km = 0.11 microM; 6-lactoyl-7,8-dihydropterin, Km = 1.80 microM; and 7,8-dihydrobiopterin, Km = 1.25 microM. The products in each case are the corresponding fully oxidized compounds 2-amino-4-oxopteridine, oxidized 6-lactoyl-7,8-dihydropterin, and 6-L-erythro-dihydroxypropylpterin, respectively. During the reaction, 1 mol of molecular oxygen is consumed per mole of substrate oxidized, and hydrogen peroxide is produced. The molecular weight of the enzyme is approximately 51,500. The enzyme apparently contains two polypeptide chains of identical molecular weight. The prosthetic group of the enzyme has been identified as FAD. From the determination of the occurrence of the enzyme in the various stages of the life cycle of D. melanogaster and from other considerations, the tentative conclusion is reached that the physiological role of dihydropterin oxidase is to convert dihydropterin to 2-amino-4-oxopteridine, a reaction that is believed to be essential in the formation of 2-amino-4-oxo-7-hydroxypterin in D. melanogaster.     

Regulator gene of phenyl oxidase activity in Drosophila melanogaster

A recessive lethal mutation l(2)hemo causing the occurrence of melanotic tumors in homozygous Drosophila larvae was found. The study of phenoloxidase (PO) activity revealed that the number of hemocytes with PO activity in homozygous larvae was significantly reduced (0.4 +/- 0.24%), compared to wild-type larvae (6.3 +/- 0.5%). On injury followed by injection with bacterial cells, the formation of melanotic thrombus did not occurred and hemocytes with PO activity were not recorded in homozygotes of line P103. Suppression of the activity of PO isozymes A1 and A3 was detected by means of electrophoretic analysis of homozygotes. According to gene mapping data, the localization of this mutation did not match any structural gene for known PO forms and is therefore related to a regulatory gene controlling the activity of the immune system of Drosophila.     

Cloning and characterization of a complex satellite DNA from Drosophila melanogaster.

The sequence organization of the 1.688 satellite DNA (density 1.688 g/cm³ in CsCl) has been investigated, and this satellite has been found to differ from the other D. melanogaster satellite DNAs in having a much greater sequence complexity. Purification of 1.688 satellite DNA by successive equilibrium density centrifugations yielded a fraction 77% pure. Segments of satellite DNA were isolated by molecular cloning in the plasmid vector pSC101. One recombinant plasmid contained a segment of 1.688 satellite DNA 5.8 kilobase pairs in size and was stable during propagation in E. coli. Recognition sites for restriction enzymes from Haemophilus aegyptius (Hae III), Haemophilus influenzae f (Hinf) and Arthrobacter luteus (Alu I) were mapped in the satellite DNA of this hybrid plasmid. The spacing of Hae III, Hinf and two Alu I sites at regular intervals of about 365 base pairs is strong evidence that the sequence complexity of this satellite DNA is 365 base pairs. Further evidence comes from the finding that both gradient-purified and cloned 1.688 satellite DNA renature with their Hae III sites in register. The Hae III and Hinf sites in gradient-purified satellite DNA have been shown by Manteuil, Hamer and Thomas (1975) and Shen, Wiesehahn and Hearst (1976) to be distributed at intervals of 365 base pairs and integral multiples thereof. These investigators proposed that some of the sites in an otherwise regular array have been randomly inactivated. Cloned satellite DNA provided a hybridization probe for sensitive studies of the arrangement of these recognition sites in gradient-purified satellite DNA. Some regions of satellite DNA were found to contain many fewer recognition sites than expected from the proposed models. These findings suggest that different regions of 1.688 satellite DNA may exhibit different arrangements of Hae III and Hinf recognition sites.     

Aldehyde oxidase activity in the tumorous-head strain of Drosophila melanogaster

Gross aldehyde oxidase activity from the egg-stage through 10-day-old adults and distribution of the enzyme in eye-antennal imaginal discs in third instar larvae were determined for the tumorous-head strain of Drosophila melanogaster. Aldehyde oxidase activity of several laboratory strains was measured for comparative purposes. Aldehyde oxidase activity was 100% higher during embryogenesis in tuh(ASU) eggs than in Oregon-R-C eggs. A second period of elevated aldehyde oxidase activity was observed during metamorphosis where tuh(ASU) pupae averaged 65% more enzyme activity than Oregon-R-C. Therefore, during determination and differentiation of the eye-antennal imaginal disc, the tuh(ASU) strain possesses a high aldehyde oxidase activity. Wild-type Drosophila melanogaster antennal imaginal discs are aldehyde oxidase positive, whereas attached eye imaginal discs are apparently aldehyde oxidase negative. A sample of eye-antennal imaginal discs from tuh(ASU) third instar larvae revealed that either one or both eye discs of 64% of the larvae were aldehyde oxidase positive. Aldehyde oxidase activity may be correlated with the homoeotic transformation in parts of the eye disc.     

Phenol oxidase activity and the Lozenge locus of Drosophila melanogaster

We have found that the phenol oxidase activity in 50-hr Drosophila melanogaster pupae is much greater than that of adult flies. The mutants lz and lz ^g have all of the phenol oxidase components present in wild type, whereas the mutant tyr-1 has all of the wild-type components but the activity of each component is greatly reduced in comparison with wild-type activity. The newly discovered lozenge allele, lz ^rfg, lacks all phenol oxidase activity.Predoctoral fellow supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.The Oak Ridge National Laboratory is operated for the U.S. Atomic Energy Commission by Union Carbide Corporation.     

Identification of a cDNA clone encoding DIP1-binding protein in Drosophila melanogaster

The Drosophila melanogaster L27a gene encodes a ribosomal protein which is a member of the L15 family of ribosomal proteins. D.m. L27a is closely related to the mammalian protein that has been found differentially expressed in lung cancer tissues and therefore could be involved in the control of cell proliferation such as the ribosomal protein S6. Our work elucidates the role of DIP1 which is a novel protein that we found in Drosophila. We performed a two-hybrid system assay and identified the L27a protein as an interactor of DIP1. The interaction was then validated by in vitro binding assays. DIP1, similar to other nuclear proteins in eukaryotes, is localized to the nuclear periphery and chromatin domain in all nuclei, but disappears at the metaphase. It is possible that in D.m. L27a protein, via interaction with DIP1, could be involved in protein synthesis as well as in cell cycle regulation.     

Cloning and expression of a cDNA encoding mouse kidney -amino acid oxidase

A cDNA encoding -amino acid oxidase (DAO;EC 1.4.3.3) has been isolated from a BALB/c mouse kidney cDNA library by hybridization with the cDNA for the porcine enzyme. Analysis of the nucleotide (nt) sequence of the clone revealed that it has a 1647-nt sequence with a 5′-terminal untranslated region of 68 nt that encodes 345 amino acids (aa), and a 3′-terminal untranslated region of 544 nt that contains the polyadenylation signal sequence ATTAAA. The deduced aa sequence showed 77 and 78% aa identity with the porcine and human enzymes, respectively. Two catalytically important aa residues, Tyr²²⁸ and His³⁰⁷, of the porcine enzyme, were both conserved in these three species. RNA blot hybridization analysis indicated that a DAO mRNA, of 2 kb, exists in mouse kidney and brain, but not liver. Synthesis of a functional mouse enzyme in Escherichia coli was achieved through the use of a vector constructed to insert the coding sequence of the mouse DAO cDNA downstream from the tac promoter of plasmid pKK223-3, which was designed so as to contain the lac repressor gene inducible by isopropyl-β- -thiogalactopyranoside. Immunoblot analysis confirmed the synthesis and induction of the mouse DAO protein, and the molecular size of the recombinant mouse DAO was found to be identical to that of the mouse kidney enzyme. Moreover, the maximum activity of the mouse recombinant DAO was estimated to be comparable with that of the porcine DAO synthesized in E. coli cells.