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Down-regulation of expression of a pupal cuticle protein gene (GmPCP52) was investigated during metamorphosis in Galleria during normal development and in response to 20-hydroxyecdysone (20 E) and a juvenile hormone analogue (epofenonane). The developmental profile of GmPCP52 transcription was traced by nuclear run-on transcription assays. Transcription of the GmPCP52 gene is highest shortly after pupal ecdysis. There is a rapid decline between a pupal age of 12 and 18 h. Transcription becomes undetectable at 24 h. 20 E accelerates cessation of transcription, but induces a short second period of GmPCP52 transcriptional activity. Epofenonane prolongs transcription and induces a second round of transcriptional activity in relation to the synthesis of a second pupal cuticle. Analysis of changes in poly(A) tail length of GmPCP52 mRNA demonstrated control of expression at the level of mRNA translatability and stability. At 6 to 9 h poly(A) tails of GmPCP52 mRNA have lengths of 70 to 170 A-residues. From 9 h on mRNA with about 50 As accumulates. This material is regarded as translationally inactive. From 18 h on, further poly(A) shortening and degradation of the transcript occurs. Again, 20 E has an accelerating effect. In accordance with the results of the run-on experiments, there is a second increase in GmPCP52 mRNA with poly(A) tail lengths greater than 50 A. Epofenonane causes delay but does not prevent the changes observed in untreated animals. The results demonstrate, that expression of the GmPCP52 gene is regulated at the level of transcription, translation, as well as transcript accumulation and degradation. Targets of hormonal action are discussed.

To trace the developmentally regulated expression of a gene coding for a pupal cuticle protein (GmPCP52), total RNA of carcasses was extracted at different times during metamorphosis and subjected to Northern blot analysis. Blots were probed with a dig-labelled antisense in vitro transcript of GmPCP52 cDNA.In untreated animals, GrnPCP52 mRNA is absent during the wandering stage in last instar larvae. The amount of mRNA rises after pupal ecdysis to a maximum at a pupal age of 18 h. At 42 h it again becomes undetectable. Injection of the JH analogue epofenonane immediately after pupal ecdysis delays the decline of GmPCP52 mRNA, without the transcript have completely disappeared at 60 h. Thereafter it comes up again from day 3 to day 7 in the course of synthesis of a secondary pupal cuticle. Injection of 20E causes precocious disappearance of CMPCP52 mRNA. It becomes undetectable at day 2. Comparison of the Northern blot data with results of in vitro translation experiments reveals participation of translational control. o 1996 Wiley-Liss. Inc.

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