Specificity of chromosome damage caused by the Rex element of Drosophila melanogaster

Alkylresorcinols, natural amphiphilic compounds commonly found in cereal grains, markedly decreased mutagenic activity of four standard mutagens examined in the Ames test. The effect was the strongest in the case of indirect-acting mutagens, benzo[a]pyrene and 2-aminofluorene. In the case of direct-acting mutagens, daunorubicin and methyl methanesulfonate, the diminution of the mutagenic activity by the alkylresorcinols was smaller but still noticeable. In the Sister Chromatid Exchanges test (SCEs) with cultured in vitro human blood-derived lymphocytes, a significant decrease of SCEs frequency induced by benzo[a]pyrene was observed in the presence of alkylresorcinols. These preliminary results seem to be important in the aspect of possible antimutagenic and anticarcinogenic potency of alkylresorcinols found in cereal grains.     

Purification of choline acetyltransferase from Drosophila melanogaster

Choline acetyltransferase (EC 2.3.1.6) from Drosophila melanogaster (Canton S, wild type) was purified 12,500-fold to a final specific activity of 500 mumol min-1 mg protein-1. The purification used homogenized fly heads and consisted of polyethylene glycol precipitation, DEAE-Bio-Gel A chromatography, Octyl-Sepharose chromatography, and affinity chromatography using solid phase Green A-agarose. The molecular weight of the native enzyme, as determined by molecular exclusion chromatography, was approximately 67,000 daltons. The final enzyme preparation showed two major protein bands at 67,000 and 54,000 daltons on polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS). After molecular exclusion chromatography, both SDS gel bands were present in the single symmetrical peak that contained the enzyme activity. Two-dimensional tryptic peptide maps prepared from the individual SDS gel bands indicated that they have very similar primary structures. Both SDS gel bands were precipitated by two different monoclonal antibodies derived against Drosophila choline acetyltransferase activity. The structural and immunological relatedness of the two SDS gel bands indicates that the enzyme is essentially homogeneous and that, in the native state, it may consist of more than one polypeptide chain.     

Characterization and cloning of tripeptidyl peptidase II from the fruit fly, Drosophila melanogaster

We describe the characterization, cloning, and genetic analysis of tripeptidyl peptidase II (TPP II) from Drosophila melanogaster. Mammalian TPP II removes N-terminal tripeptides, has wide distribution, and has been identified as the cholecystokinin-degrading peptidase in rat brain. Size exclusion and ion exchange chromatography produced a 70-fold purification of dTPP II activity from Drosophila tissue extracts. The substrate specificity and the inhibitor sensitivity of dTPP II is comparable to that of the human enzyme. In particular, dTPP II is sensitive to butabindide, a specific inhibitor of the rat cholecystokinin-inactivating activity. We isolated a 4309-base pair dTPP II cDNA which predicts a 1354-amino acid protein. The deduced human and Drosophila TPP II proteins display 38% overall identity. The catalytic triad, its spacing, and the sequences that surround it are highly conserved; the C-terminal end of dTPP II contains a 100-amino acid insert not found in the mammalian proteins. Recombinant dTPP II displays the predicted activity following expression in HEK cells. TPP II maps to cytological position 49F4-7; animals deficient for this interval show reduced TPP II activity.     

Disruption of mesectodermal lineages by temporal misexpression of the Drosophila POU-domain transcription factor, drifter

The horizontal and vertical considerations that must be taken into account during the diagnosis and subsequent treatment of the dentally and skeletally compromised patient are discussed. The treatment planning and treatment of a difficult malocclusion that required comprehensive orthodontics with surgical intervention are presented.     

Evolutionary history of the sex-peptide (Acp70A) gene region in Drosophila melanogaster

A phosphorothioate oligonucleotide (PS-ODN) with sequence identical to the repeat sequence of the mammalian telomere, 5'-d(TTAGGG)-3', was incubated with a Burkitt's lymphoma-derived (OMA-BL1) cell line. This hexanucleotide inhibits telomerase activity in cell lysates, lengthens cell doubling time, and induces apoptosis. Concatenated repeats (12-, 18-, and 24-mers) of the 5'-d(TTAGGG)-3' motif induce similar cellular responses. Scrambled sequences do not efficiently inhibit telomerase activity or significantly alter cell growth and viability. The in vivo efficacy of this PS-ODN was evaluated in a xenograft human-nude mouse model. Once tumors were established these animals were administered the telomere mimic, 5'-d(TTAGGG)-3', a control scrambled sequence 5'-d(TGTGAG)-3', or saline for 14 days via a subcutaneous osmotic pumps in a blinded study monitoring tumor size with dose and time. A significant decrease in tumor size was observed in animals given 50 micrograms/mouse/day 5'-d(TTAGGG)-3', but not following 5'-d(TGTGAG)-3', relative to the saline-treated animals. The antitumor activity of the 6-mer telomere mimic demonstrated a dose dependency including a reduction in metastatic nodules in the spleen. No activity was observed with the scrambled controls. In addition to antitumor activity we observed an increase in the mouse hematopoietic progenitor cell populations, BFU-e and CFU-GM. These results demonstrated the effects of a short hexameric oligonucleotide telomere mimic in vitro and in vivo and the potential utility of short oligonucleotides as telomerase inhibitors.     

DNA base sequence changes induced by diethyl sulfate in postmeiotic male germ cells of Drosophila melanogaster

The DNA base sequence changes induced by diethyl sulfate (DES) were analyzed in postmeiotic male germ cells of Drosophila melanogaster. 31 transmissible vermilion mutants were recovered in F1 and F2 generations, with a frequency of 2.6 x 10(-4) for the F1, and of 1.8-13 x 10(-4) for the F2. The results show that DES induces both base pair substitutions (93%) and deletions (7%). In accord with its relatively high ability to alkylate oxygens in DNA, the most frequent type of sequence alteration among the basepair changes are GC-AT transitions, accounting for 73% of mutations, followed by transversions AT-TA (10%). DES also induced AT-GC transitions and AT-CG transversions. Both induced deletions were intralocus deletions, not occurring between basepair repeats. No influence of neighboring bases on the mutation position was found.     

Genetic controls of meiotic recombination and somatic DNA metabolism in Drosophila melanogaster

Recombination-defective meiotic mutants and mutagen-sensitive mutants of D. melanogaster have been examined for their effects on meiotic chromosome behavior, sensitivity to killing by mutagens, somatic chromosome integrity, and DNA repair processes. Several loci have been identified that specify functions that are necessary for both meiotic recombination and DNA repair processes, whereas mutants at combination and DNA repair processes, whereas mutants at other loci appear to be defective in only one pathway of DNA processing.     

Purification and characterization of prophenoloxidases from pupae of Drosophila melanogaster

The effects of heparin and other polyanions on the myotoxicity of Bothrops jararacussu venom and purified bothropstoxin (BthTX) were investigated. The release rate of creatine kinase (CK) from isolated extensor digitorum longus muscle and the plasma CK activity of mice were used to quantify the results. The myotoxic effects of B. jararacussu venom or BthTX were inhibited by preincubation of these agents with one of the following: a heterogeneous heparin preparation (designated 'heparin'), low mol. wt heparin (H-4500) or dextran sulfates (DS-8000 and DS-500,000). Non-sulfated dextran (D-40,000) and two chondroitin sulfates were ineffective. The antimyotoxic effects of the polyanions are ascribed to their forming inactive acid-base complexes with the basic myotoxins of Bothrops venoms. Gel-filtration experiments in Sephadex provided direct evidence for complex formation between heparin and BthTX. Intravenous (i.v.) administration of H-4500 or DS-8000 opposed the increase in plasma CK activity induced by a subsequent i.m. injection of venom or BthTX. In contrast, pretreatment with i.v. heparin or DS-500,000 enhanced the venom-induced increase in plasma CK activity. This effect was not observed (1) when the animals were treated with a polyvalent antivenom, which inhibits the coagulation and local stasis induced by Bothrops venoms, and (2) when BthTX, which has no thrombotic or hemorrhagic properties, was the myotoxic agent. The potentiation of the venom-induced increase in plasma CK activity by heparin and DS-500,000 is ascribed to improved washout of the CK released from damaged fibers, because of the anticoagulant properties of the drugs.     

Molecular screening for P-element insertions in a large genomic region of Drosophila melanogaster using polymerase chain reaction mediated by the vectorette

As an alternative to existing methods for the detection of new insertions during a transposon mutagenesis, we adapted the method of vectorette ligation to genomic restriction fragments followed by PCR to obtain genomic sequences flanking the transposon. By combining flies containing a defined genomic transposon with an excess of flies containing unrelated insertion sites, we demonstrate the specificity and sensitivity of the procedure in the detection of integration events. This method was applied in a transposon-tagging screen for BJ1, the Drosophila homolog of the vertebrate gene Regulator of Chromosome Condensation (RCCI). Genetic mobilization of a single genomic P element was used to generate preferentially new local insertions from which integrations into a genomic region surrounding the BJ1 gene were screened. Flies harboring new insertions were phenotypically selected on the basis of the zeste1-dependent transvection of white. We detected a single transposition to a 13-kb region close to the BJ1 gene among 6650 progeny that were analyzed. Southern analysis of the homozygous line confirmed the integration 3 kb downstream of BJ1.     

Phenotypic enhancement of longevity by environmental urea in Drosophila melanogaster

The phenotypic enhancement of longevity through a variety of environmental treatments, including dietary manipulations, has been observed in various species of animals, both vertebrate and invertebrate. Elucidating the mechanisms underlying such effects has provided insights into the physiological processes contributing to the determination of lifespan. Here, we report the enhancement of longevity in adult Drosophila melanogaster maintained on food supplemented with urea, a metabolic waste product occurring naturally in Drosophila cultures, especially at high larval densities. The impact of urea on longevity is shown to be through a decrease in the age-independent parameter (A) of the Gompertz equation, rather than the age-dependent parameter (alpha), which reflects the "rate of aging." We also present evidence suggesting that the urea-induced increase in longevity is mediated exclusively through a reduction in some aspect(s) of reproduction in adult flies maintained on urea-supplemented food.