ABSTRACT
Using the developmental mutant ecd1, the biological activity of 20-hydroxyecdysone (20E) and 20E metabolites 3-dehydro-20-hydroxyecdysone (3D20E), 3-epi-20-hydroxyecdysone (3D20E), 3-epi-20-hydroxyecdysone-3-phosphate (20E'3P), 20,26-dihydroxyecdysone (20,26E), and 20-hydroxyecdysonoic acid (20Eoic) was tested for their ability to induce the transcription of the steroid-inducible gene P1 in the Drosophila larval fat body. 3D20E was the most efficient ecdysteroid in the initiation of P1 gene transcription therefore the formation of 3D20E and the 3-epimer could not be regarded as an inactivation pathway in Drosophila larvae. Formation of 20,26E and 20Eoic may be an inactivation pathway in this biological model.
Subject(s)
Adipose Tissue/physiology , Drosophila/genetics , Invertebrate Hormones/pharmacology , RNA, Messenger/genetics , Adipose Tissue/drug effects , Animals , Blotting, Northern , Drosophila/growth & development , Ecdysteroids , Gene Expression/drug effects , Larva , Molecular Weight , Mutation , RNA, Messenger/drug effects , TemperatureABSTRACT
Cultured Kc cells of Drosophila melanogaster are sensitive to the insect moulting hormone, 20-hydroxy-ecdysone (20-OH-E). Morphological changes of Kc-treated cells were observed and electron microscopic analysis of pseudopodia shows a large increase in the number of microtubules, all arranged in the same orientation. The 60 C beta tubulin gene which is expressed only in 20-OH-E-treated cells encodes a 2.6-kb mRNA which is essentially cytoplasmic and polyadenylated. The corresponding premessenger is 7 kb in length and is absent in untreated cells. Two peaks of expression of the 60 C beta tubulin gene are observed during Drosophila development: at midembryogenesis (stage 8-13 h) and at the late third instar larvae-early pupae stage. By use of the Ecdysone 1 mutant, 60 C beta tubulin gene expression was demonstrated to be regulated in part by 20-OH-E during Drosophila development. Through these two complementary biological models of study, the mode and role of beta tubulin gene regulation are discussed.
Subject(s)
Drosophila melanogaster/genetics , Ecdysterone/pharmacology , Gene Expression Regulation/drug effects , Tubulin/genetics , Animals , Blotting, Northern , Cell Compartmentation , Cell Line , Cell Nucleus/metabolism , Cytoplasm/metabolism , Drosophila melanogaster/growth & development , Microtubules/ultrastructure , Multigene Family , Mutation , RNA, Messenger/genetics , Transcription, GeneticABSTRACT
During third-instar larval development of Drosophila melanogaster, the fat body tissue synthesizes six major methionine-containing polypeptides, three of which are the alpha, beta, and gamma subunits of the hexameric larval serum protein LSP-1, a fourth is the single subunit of the hexameric larval serum protein LSP-2, and the other two are polypeptides P6 and P1. Genomic DNA clones of the six structural genes for the polypeptides were isolated and characterized. Each gene maps by in situ hybridization at a single chromosomal site and appears to be present as a single copy in the genome. The LSP-1 and LSP-2 genes show striking regulatory similarities: The LSP-1 beta and gamma transcripts are first detected in fat bodies within an hour after the second molt, and the LSP-1 alpha and LSP-2 transcripts a few hours later; the four transcripts are subsequently maintained at high levels during most of the third instar and rapidly decrease shortly before pupariation. Ecdysterone increases the levels of at least three of the four LSP transcripts in the fat bodies when ecdysterone-deficient larvae from the temperature-sensitive mutant ecd1 are supplemented with the hormone. The regulatory characteristics of the P6 and P1 genes differ in several ways from those of the LSP genes. Expression of the P6 and P1 genes begins later than the LSP genes, and the levels of the transcripts remain high at the end of the third instar after the LSP transcripts have markedly decreased. Ecdysterone increases the level of the P1 transcript, but not of the P6 transcript, in ecdysterone-deficient ecd1 larvae.
Subject(s)
Blood Proteins/genetics , Drosophila melanogaster/genetics , Gene Expression Regulation , Genes , Adipose Tissue/metabolism , Animals , Blood Proteins/metabolism , Chromosome Mapping , Drosophila melanogaster/physiology , Larva , Protein BiosynthesisABSTRACT
HPLC allowed separation of twelve major labeled compounds after injection of 3H-ecdysone into Pieris pharate pupae. These compounds were identified as six pairs of metabolites (3 alpha and 3 beta epimers), comprising ecdysone, 20-hydroxyecdysone, 26-hydroxyecdysone, 20,26-dihydroxy-ecdysone and the polar metabolites P and 20-hydroxy-P. These last two products could not be enzymatically split by any hydrolase tested and are weak acids arising respectively from 26-hydroxyecdysone and 20,26-dihydroxyecdysone. They might be 26-oic compounds. Epimerization appears as a fundamental inactivation process in Pieris and could well be a general characteristic of closed systems (eggs and pupae). No significant amounts of hydrolyzable conjugates were detected in our biological system (pharate pupae and pupae).
