Characterization of the activity of β-galactosidase from Escherichia coli and Drosophila melanogaster in fixed and non-fixed Drosophila tissues

Abstractβ-Galactosidase encoded by the Escherichia coli lacZ gene, is widely used as a reporter molecule in molecular biology in a wide variety of animals. β-Galactosidase retains its enzymatic activity in cells or tissues even after fixation and can degrade X-Gal, a frequently used colormetric substrate, producing a blue color. Therefore, it can be used for the activity staining of fixed tissues. However, the enzymatic activity of the β-galactosidase that is ectopically expressed in the non-fixed tissues of animals has not been extensively studied. Here, we report the characterization of β-galactosidase activity in Drosophila tissues with and without fixation in various experimental conditions comparing the activity of two evolutionarily orthologous β-galactosidases derived from the E. coli lacZ and Drosophila melanogaster DmelGal genes. We performed quantitative analysis of the activity staining of larval imaginal discs and an in vitro assay using larval lysates. Our data showed that both E. coli and Drosophila β-galactosidase can be used for cell-type-specific activity staining, but they have their own preferences in regard to conditions. E. coli β-galactosidase showed a preference for neutral pH but not for acidic pH compared with Drosophila β-galactosidase. Our data suggested that both E. coli and Drosophila β-galactosidase show enzymatic activity in the physiological conditions of living animals when they are ectopically expressed in a desired specific spatial and temporal pattern. This may enable their future application to studies of chemical biology using model animals

Analysis of a cellular structure observed in the compound eyes of Drosophila white; yata mutants and white mutants

We previously identified the Drosophila yata mutant, which showed phenotypes including progressive vacuolization of the white-coloured compound eye, progressive shrinkage of the brain and a shortened lifespan. The yata gene was shown to be involved in controlling intracellular trafficking of the Amyloid precursor protein-like protein, which is an orthologue of Amyloid precursor protein, which is a causative molecule of Alzheimer's disease. In this study, we examined the phenotype of the compound eye of the yata mutant using electron microscopy and confocal microscopy. We found that abnormal cellular structures that seemed to originate from bleb-like structures and contained vesicles and organelles, such as multivesicular bodies and autophagosomes, were observed in aged white; yata mutants and aged white mutants. These structures were not observed in newly eclosed flies and the presence of the structures was suppressed in flies grown under constant dark conditions after eclosion. The structures were not observed in newly eclosed red-eyed yata mutants or wild-type flies, but were observed in very aged red-eyed wild-type flies. Thus, our data suggest that the observed structures are formed as a result of changes associated with exposure to light after eclosion in white mutants, white; yata mutants and aged flies