Dopamine is an important neurotransmitter in the central nervous system of both Drosophila and mammals. Despite the evolutionary distance, functional parallels exist between the fly and mammalian dopaminergic systems, with both playing roles in modulating locomotor activity, sexual function, and the response to drugs of abuse. In mammals, dopamine exerts its effects through either dopamine 1-like (D1-like) or D2-like G protein-coupled receptors. Although pharmacologic data suggest the presence of both receptor subtypes in insects, only cDNAs encoding D1-like proteins have been isolated previously. Here we report the cloning and characterization of a newly discovered Drosophila dopamine receptor. Sequence analysis reveals that this putative protein shares highest homology with known mammalian dopamine 2-like receptors. Eight isoforms of the Drosophila D2-like receptor (DD2R) transcript have been identified, each the result of alternative splicing. The encoded heptahelical receptors range in size from 461 to 606 aa, with variability in the length and sequence of the third intracellular loop. Pharmacologic assessment of three DD2R isoforms, DD2R-606, DD2R-506, and DD2R-461, revealed that among the endogenous biogenic amines, dopamine is most potent at each receptor. As established for mammalian D2-like receptors, stimulation of the Drosophila homologs with dopamine triggers pertussis toxin-sensitive Gi͞o-mediated signaling. The D2-like receptor agonist, bromocriptine, has nanomolar potency at DD2R-606, -506, and -461, whereas multiple D2-like receptor antagonists (as established with mammalian receptors) have markedly reduced if any affinity when assessed at the fly receptor isoforms. The isolation of cDNAs encoding Drosophila D2-like receptors extends the range of apparent parallels between the dopaminergic system in flies and mammals. Pharmacologic and genetic manipulation of the DD2Rs will provide the opportunity to better define the physiologic role of these proteins in vivo and further explore the utility of invertebrates as a model system for understanding dopaminergic function in higher organisms. Dopamine is an essential catecholamine in the central nervous system of both mammals (1) and Drosophila (2). In mammals, dopamine modulates neurologic, cardiovascular, endocrine, and renal functions. In addition, this neurotransmitter regulates motor activity, sexual behavior, and the response to drugs of abuse (1, 3-6). In Drosophila, known dopamine-mediated functions overlap with those of mammals. Experimental evidence suggests that in flies dopamine modulates locomotor activity, sexual function and the response to cocaine, nicotine, and alcohol (7-11).Dopaminergic signaling is mediated through seven transmembrane domain (TM) G protein-coupled receptors that fall within the class A or rhodopsin family based on amino acid homology and conservation of amino acid signature motifs. The dopamine receptor family is divided into two major subfamilies: the D1-like receptors (D1 and D5) and D2-like receptors (D2, D3, and ...

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Cholera, the pandemic diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, continues to be a major public health challenge in the developing world. Cholera toxin, which is responsible for the voluminous stools of cholera, causes constitutive activation of adenylyl cyclase, resulting in the export of ions into the intestinal lumen. Environmental studies have demonstrated a close association between V. cholerae and many species of arthropods including insects. Here we report the susceptibility of the fruit fly, Drosophila melanogaster, to oral V. cholerae infection through a process that exhibits many of the hallmarks of human disease: (i) death of the fly is dependent on the presence of cholera toxin and is preceded by rapid weight loss; (ii) flies harboring mutant alleles of either adenylyl cyclase, Gsα, or the Gardos K+ channel homolog SK are resistant to V. cholerae infection; and (iii) ingestion of a K+ channel blocker along with V. cholerae protects wild-type flies against death. In mammals, ingestion of as little as 25 μg of cholera toxin results in massive diarrhea. In contrast, we found that ingestion of cholera toxin was not lethal to the fly. However, when cholera toxin was co-administered with a pathogenic strain of V. cholerae carrying a chromosomal deletion of the genes encoding cholera toxin, death of the fly ensued. These findings suggest that additional virulence factors are required for intoxication of the fly that may not be essential for intoxication of mammals. Furthermore, we demonstrate for the first time the mechanism of action of cholera toxin in a whole organism and the utility of D. melanogaster as an accurate, inexpensive model for elucidation of host susceptibility to cholera.

Superoxide dismutases (SOD) play a major role in the intracellular defense against oxygen radical damage to aerobic cells. In eucaryotes, the cytoplasmic form of the enzyme is a 32-kDa dimer containing two copper and two zinc atoms (CuZn SOD) that catalyzes the dismutation of the superoxide anion (02) to H202 and 02Superoxide-mediated damage has been implicated in a number of biological processes, including aging and cancer; however, it is not certain whether endogenously elevated levels of SOD will reduce the pathological events resulting from such damage. To understand the in vivo relationship between an efficient dismutation of 02 and oxidative injury to biological structures, we generated transgenic strains of Drosophila melanogaster overproducing CuZn SOD. This was achieved by microinjecting Drosophila embryos with P-elements containing bovine CuZn SOD cDNA under the control of the Drosophila actin Sc gene promoter. Adult flies of the resulting transformed lines which expressed both mammalian and Drosophila CuZn SOD were then used as a novel model for evaluating the role of oxygen radicals in aging. Our data show that expression of enzymatically active bovine SOD in Drosophila flies confers resistance to paraquat, an 02-generating compound. This is consistent with data on adult mortality, because there was a slight but significant increase in the mean lifespan of several of the transgenic lines. The highest level of expression of the active enzyme in adults was 1.60 times the normal value. Higher levels may have led to the formation of toxic levels of H202 during development, since flies that died during the process of eclosion showed an unusual accumulation of lipofuscin (age pigment) in some of their cells. In conclusion, our data show that free-radical detoxification has a minor but positive effect on mean longevity for several strains.

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When U1 and U2 small nuclear ribonucleoproteins (snRNPs) purified by a procedure which preserves their immunoprecipitability by autoimmune antibodies (Hinterberger et al., J. Biol. Chem. 258:2604-2613, were submitted to extensive digestion with micrococcal nuclease, we found that their degradation pattern was sharply dependent upon magnesium concentration, indicating that they undergo a profound structural modification. At low Mg2+ (s5 mM), both particles only exhibit a core-resistant structure previously identified as being common to all but U6 snRNAs (Liautard et al., J. Mol. Biol. 162:623-643, 1982). At high Mg2+ (.7 mM), U1 and U2 snRNPs behave differently from one another. In U1 snRNP, most U1 snRNA sequence is protected, except for the 10 5'-terminal nucleotides presumably involved in splicing and a short sequence between nucleotides 102 and 108. Another region spanning nucleotides 60 to 79 is only weakly protected. This structural modification was demonstrated to be reversible. In U2 snRNP, the U2 snRNA sequence remains exposed in its 5' part up to nucleotide 92, and the 3'-terminal hairpin located outside the core structure becomes protected.All but U3 small nuclear RNAs (snRNAs) assemble in the cell with specific proteins to form small nuclear ribonucleoproteins (snRNPs) which are precipitable by sera from patients with systemic lupus erythematosus or mixed connective tissue disease. These U snRNPs (for a review, see C. Brunel, J. Sri-Widada, and P. Jeanteur, Progress in Molecular and Subcellular Biology, in press) interact with heterogenous nuclear RNPs (containing the heterogenous population of premessengers), which are now recognized as the real splicing substrate (8). Recent reports have strengthened the initial suggestions by Lerner et al. (13) and Rogers and Wall (20) that U1 snRNP is involved in splicing. Among these are the crucial demonstration that an in vitro splicing system is inhibited by monoclonal anti-Sm, patient anti-Sm, or anti-RNP antibodies but not by scleroderma-polymyositis overlap syndrome serum-precipitating U2 snRNP (18) and the biochemical evidence that U1 snRNP binds to an in vitro transcript containing splice sites (16) or that U1 snRNP stimulates in vitro splicing of adenoviral sequences (8).In the course of isolating U snRNPs from HeLa cells, we first purified a mixture containing Ul, Ul-, U4, U5, and U6 particles (5) and then purified the individual U1 snRNP (21). These particles have a common and simple protein complement which constitutes only the low-molecular-weight polypeptides (9 to 14 kilodaltons) among those immunoprecipitated by anti-RNP or anti-Sm antibodies. As such, they retained antigenicity with respect to anti-Sm but not anti-RNP (1) and were therefore designated as core snRNPs. Although certainly far from being functional, these core particles allowed us to identify the binding site for these common core proteins as being a single-stranded region containing the sequence A(U)nG, with n -3, bordered by two double-stranded stems (14). This site corresponds t...

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Null mutants for Cu/Zn superoxide dismutase (CuZnSOD)