Ralph Hillman

Effects of tyrosine hydroxylase mutants on locomotor activity in Drosophila: a study in functional genomics.

The brain of the adult fruit fly, Drosophila melanogaster, contains tyrosine hydroxylase, the rate-limiting enzyme required for catecholamine biosynthesis, as well as dopa decarboxylase. Catecholamines, principally dopamine, are also present. We have previously shown that pharmacological inhibition of tyrosine hydroxylase with α-methyl-p-tyrosine results in a dose-related inhibition of locomotor activity in adult organisms. Similar results were found with reserpine, a well-known inhibitor of catecholamine uptake into storage granules. The drug-induced inhibition could be prevented in each case by the concomitant administration of l-dopa. The single-copy gene coding for tyrosine hydroxylase in Drosophila is pale (ple). Both null and temperature-sensitive loss of function mutant alleles of ple are recessive embryonic lethals. Heterozygous null mutant flies have normal locomotor activity demonstrating that only a single dose of the wild type form of ple is required to support normal function. Both hemizygous and homozygous temperature-sensitive ple mutants (plets1) also show normal locomotor activity at the permissive temperature for this mutant allele (18°C), which progressively declines as the temperature is increased to its restrictive level (29°C). These abnormal locomotor effects are reversible by l-dopa. Thus the effects on locomotor activity resulting from the pharmacological inhibition of catecholamine synthesis or storage are the same as those resulting from lack of tyrosine hydroxylase expression. These findings indicate that brain catecholamine loss decreases locomotor activity in the fly, as it does in mammals, and demonstrate the ability of functional genomic studies to mimic that of pharmacological inhibition of enzyme function or other similar processes.

Reproduction and development in Drosophila are dependent upon catecholamines

Drosophila melanogaster, maintained on culture media containing alpha-methyl-p-tyrosine (alpha-MT) at millimolar levels for 7 days, fail to produce viable progeny. Lesser concentrations delay development. This effect of alpha-MT, an inhibitor of tyrosine hydroxylase (TH), is partially reversible by co-administration of L-dihydroxyphenylalanine, the product of TH. Potent inhibitors of other steps in the pathway for catecholamine biosynthesis are inactive except for a partial effect with dopamine B-hydroxylase inhibition. the effect of alpha-MT is due to a combination of ovulation suppression coupled with decreases in embryonic and larval viability. Effects similar to those of alpha-MT are found with millimolar levels of reserpine, prazosin and to a lesser extent, rauwolscine. No significant effects are found with propranolol, chlorpromazine, sulpiride and SK&F 83566. Mutant alleles of the gene coding for TH are known to be lethal at the embryonic stage when homozygous in both Drosophila and mice. Taken together, these results indicate that in addition to their established roles in the nervous system catecholamines function in animal development via an action mediated through alpha-adrenoceptors.

In, vitro studies of protein synthesis in Drosophila

Abstract Comparative in , vitro studies of protein synthesis in Oregon-R wild-type and A 53g mutant Drosophila , melanogaster indicate that the A 53g genotype has an effect at the translation level. The A 53g post-microsomal supernatant fluid stimulates the incorporation of C14 amino acids by cell-free systems using ribosomes prepared from either A 53g or Oregon-R wild-type flies. The level of in , vitro charging of Drosophila and calf liver tRNA with C14 amino acids by the A 53g post-microsomal supernatant fraction is also greater than the level of charging by the Oregon-R supernatant fluid.

Effects of adrenergic agents on locomotor behavior and reproductive development in Drosophila

Inhibition of tyrosine hydroxylase, the rate‐limiting enzyme in catecholamine biosynthesis, by α‐methyl‐p‐tyrosine (αMT) at media concentrations of 0.3–3.0 mM, markedly inhibited (>90%) fly reproduction and development as evidenced by progeny count. Under these conditions adult spontaneous locomotor activity (SMA) was also dose‐dependently reduced. However, no significant effects on behavior were observed at 0.3 mM. The behavioral effects of αMT were prevented by coadministration of L‐DOPA. Similar effects on behavior and development were produced by reserpine at doses of 0.1–1.0 mM. The higher doses affected behavior, but no behavioral effects occurred at 0.1 mM. Rescue from the effects of reserpine was also achieved with L‐DOPA. Treatment at 1 mM with the mammalian catecholamine receptor blockers propranolol (β1β2), raclopride (D2), SK&F 83566 (D1), prazosin (α1), and rauwolscine (α2) did not affect behavior. The two α adrenoceptor inhibitors, however, markedly decreased fly development. These results suggest that the receptor mechanisms mediating the effects of catecholamines on behavior and development are different. The locomotor behavior of adult progeny of parents treated with the lowest doses of αMT and reserpine described above was markedly suppressed by these drug treatments. This result indicates an increased sensitivity of the progeny to compounds which share the ability to deplete tissue catecholamines. Thus, alterations in catecholamine function during development may have behavioral consequences in surviving organisms. Drug Dev. Res. 50:142–146, 2000.

A developmental role for catecholamines in Drosophila behavior.

Tyrosine hydroxylase (TH), the enzyme which catalyzes the conversion of tyrosine to L-DOPA and is the rate limiting step in catecholamine biosynthesis, is genetically expressed during development in Drosophila. Null mutant alleles of the single copy gene which codes for this enzyme are developmentally lethal as is a conditional TH mutant at its restrictive temperature. In adult flies, inhibition of TH by alpha-methyl-p-tyrosine (alphaMT) decreases locomotor activity in a dose-dependent manner. This behavioral effect is accompanied by reductions in brain levels of dopamine, the primary CNS catecholamine in Drosophila, and can be prevented by the coadministration of L-DOPA. Similar effects are found with reserpine and at the restrictive temperature in flies with a temperature conditional mutation for TH. In agreement with published studies in mammals, inhibition of TH by alphaMT during Drosophila development results in enhanced expression of this enzyme in the progeny of surviving adults. This biochemical outcome is accompanied behaviorally by increased sensitivity to the locomotor effects of both alphaMT and reserpine, drugs which act via depletion of brain catecholamines. Since TH is the rate limiting enzyme responsible for the conversion of tyrosine to L-DOPA and L-DOPA is converted to dopamine by aromatic amino acid decarboxylase (AAAD), the results indicate that depletion of catecholamine levels in the fly embryo results in increased dopamine biosynthesis in the next generation accompanied by alterations in behavior.

Macromolecular complexes of aminoacyl-tRNA synthetases in Drosophila

Abstract Aminoacyl-tRNA synthetases in Drosophila are present in large macromolecular aggregates having mol. wt in excess of 10 6 Daltons. After cellular fractionation these enzymes are found in the post-microsomal hard pellet, the post-microsomal soft pellet, and the post-microsomal supernatant fractions. The distribution of specific enzyme activities within these fractions varies and is dependent upon the specific tRNA synthetase being studied.

The role of the GABAB receptor and calcium channels in a Drosophila model of Parkinson's Disease

Transgenic Drosophila melanogaster carrying the human gene for alpha synuclein is an animal model for the study of Parkinsons Disease. Climbing activity in these flies is reduced as a result of the effect of this protein on the locomotor activity of the transgenic fly. L-DOPA and gamma amino butyric acid (GABA) reverse the loss of this activity when placed in the food fed to these flies. While muscimol, a GABA(A) receptor agonist has no effect in this system, baclofen and the allosteric agonists CG 7930 and GS 39783 which affect the GABA(B) receptor reverse this activity. This latter effect is eliminated when these compounds are fed in conjunction with the GABA(B) receptor antagonist 2-hydroxysaclofen. In addition, fendiline which is a Ca(++) receptor blocker also reverses the loss of climbing ability. Because there is a calcium channel close to the GABA(B) receptor on the cell surface, these data are indicative of a relationship between the roles of the GABA(B) receptor, the calcium channel and the effect of alpha-synuclein on the motor activity of the transgenic fly.

Multiple gene control of the tRNA aminoacylating system in Drosophila melanogaster

The stimulation of in vitro tRNA aminoacylation by post-microsomal supernatant enzymes isolated from Abnormal Abdomen ( A 53 g ) adult flies is a function of the residual genome of these flies rather than of the major mutant gene, A 53 g . Genes controlling this stimulation are located on the X chromosome as well as on the autosomes. These observations are discussed in terms of the phenotypic effect of the major mutant gene in response to changes in protein synthesis which are under the control of modifier genes responsible for aberrant genetic translation mechanisms.

Competent Chick Ectoderm: Nonspecific Response to RNA

Presumptive chick neuroectoderm responds to RNA from brain and heart by forming neural tubes, but it does not respond to liver RNA. This dif ferential response can be correlated with the presence of Folin-positive material in those RNA preparations which elicit the formation of neural structures.

Gene control of soluble protein levels during development of abnormal abdomen in Drosophila.

Abstract The mutant genotype Abnormal abdomen ( A 53 g ) of Drosophila melanogaster causes an increase in the amount of soluble protein per fly when compared to a wild-type strain ( Ore-R ). This increase is first detected at 50 hr after puparium formation and is preceded 2 hr earlier by an increase in total RNA. A direct correlation is found between the expressivity of the A 53 g mutation in the adult fly and the total soluble protein per mg body weight of that fly. Quantitative analyses of supernatant protein from mutant and wild-type flies on polyacrylamide disk gel electrophoresis reveal that the increases observed in the mutant are due to increases in specific electrophoretic classes of proteins and not to a general stimulation of all proteins. Reciprocal crosses between mutant and wild-type flies indicate that the penetrance and expressivity of the A 53 g phenotype is under maternal control and that an increased soluble protein content in F1 flies is found only when the mutant genotype is contributed to the F1 through the maternal gamete.