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Drosophila FMR1 mutants are models of human fragile X syndrome. They show a loss of locomotor activity rhythm and severe degradation of eclosion timing. We analyzed the circadian behavior of FMR1 mutants (dfmr1B55) in two genetic backgrounds, yellow white (yw) and Canton S (CS). The arrhythmic phenotype of circadian locomotor activity in constant darkness (DD) did not significantly change in either genetic background. Surprisingly, eclosion timing was completely restored by backcrossing dfmr1B55 with yw or CS flies. Morphological analysis of the small ventrally located lateral neurons of FMR1 mutants revealed that the dorsal-projection area was significantly larger in arrhythmic than rhythmic flies. In addition, dfmr1B55 mutants in both genetic backgrounds had a significantly lower evening peak in the light-dark (LD) cycle. These results indicate that lack of FMR1 does not affect eclosion timing, but alters locomotor activity patterns in both LD and DD conditions by affecting the arborization of small ventrally located lateral neurons. Thus, the FMR1 gene may regulate the circadian-related locomotor activity of Drosophila.

Double-time (dbt) is a casein kinase gene involved in cell survival, proliferation, and circadian rhythms in the fruit fly, Drosophila melanogaster. Genetic and biochemical studies have shown that dbt and its mammalian ortholog casein kinase I epsilon (hckI epsilon) regulate the circadian phosphorylation of period (per), thus controlling per subcellular localization and stability. Mutations in these kinases can shorten the circadian period in both mammals and Drosophila. Since similar activities in circadian clock have been described for these kinases, we investigated whether the expression of mammalian casein kinase I can replace the activity of dbt in flies. Global expression of the full-length dbt rescued lethality of the null mutant dbt revVIII and rescued flies showed normal locomotor activity rhythms. Global expression of dbt also restored the locomotor activity rhythm of the arrhythmic genotype, dbt ar/dbt revVIII. In contrast, global expression of hckI epsilon or hckI alpha did not rescue lethality or locomotor activity of dbt mutants. Furthermore dbt overexpression in wild-type clock cells had only a small effect on period length, whereas hckI epsilon expression in clock cells greatly lengthened period to ~30.5 hours and increased the number of arrhythmic flies. These results indicate that hckI epsilon cannot replace the activity of dbt in flies despite the high degree of similarity in primary sequence and kinase function. Moreover, expression of hck Iepsilon in flies appears to interfere with dbt activity. Thus, caution should be used in interpreting assays that measure activity of mammalian casein kinase mutants in Drosophila, or that employ vertebrate CKI in studies of dPER phosphorylations.

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A new melanin biosynthesis inhibitor, melanoxazal, was isolated from the fermentation broth of Trichoderma sp. ATF-45 1 by successive purification procedures of carbon adsorption, ethyl acetate extraction and silica gel column chromatography. The inhibitor possesses a novel oxazole-containing structure with molecular formula, C8H9NO3. The structure was determined by means of NMR analyses to be (£)-4-(2'-formyl-3'-hydroxybuten-r-yl) oxazole, which is related to melanoxadin. Melanoxazal inhibited melanin formation in the larval haemolymph of the silkworm, Bombyx mori; [C50 value= 30.1 /ig/ml. Melanoxazal also showed a strong inhibitory activity against mushroom tyrosinase with IC50 value=4.2 wg/ml.Tyrosinase mediated melanin biosynthesis in insects has been considered to involve three key reactions: the monophenoloxidase (tyrosinase) catalyzed oxidation of tyrosine to 3,4-dihydroxyphenylalanine (dopa) in the first step, the diphenoloxidase catalyzed conversion of dopa to dopachrome via dopaquinone in second step, and formation of melanin by spontaneous polymerization of dopachrome in third step.1} Several investigations on melanin biosynthesis inhibitors of microbial origin, feldamycin,2) melanostatin,3'4) albocycline, OH-3984 Kl and K2,5'6) MR304A7) have been reported with screening methods using mushroomtyrosinase, melanin formation in Streptomyces bikiniensis and B16 melanoma cells. We have previously reported our screening program for melanin biosynthesis inhibitors from microorganisms, using the larval haemolymph of the silkworm, Bombyx rnori, in which we have isolated melanoxadin8) (2) and trichoviridin9) (3) from the cultured broth of fungal strains ATF-606and ATF-287,respectively. This method is advantageous in that it can screen manysamples in a shorter time than other methods. Furthermore, this screening method provides a useful approach in that it can find novel enzymatic inhibitors, because it contains a wider variety of enzymatic systems in the melanin metabolic pathway than the other single enzymatic systems. During the course of searching for melanin biosynthesis inhibitors, a novel oxazole compounddesignated as melanoxazal (1) (Fig. 1) was isolated from fungi. Taxonomical study indicated that the producing organism was Trichoderma sp. ATF-451.Structural studies of 1 showed that it possesses an oxazole skeleton with a molecular formula of C8H9NO3 which is struc- Fig. 1. The structures of melanoxazal (1), melanoxadin (2) and trichoviridin (3).