Holometabolous insects drastically change their body structures during post-embryonic development and metamorphosis. Their developmental processes are mainly controlled by two insect hormones, juvenile hormone (JH) and ecdysteroid. By contrast with the study for the mode of ecdysteroid action, little is known about the mechanism how JH exerts its function. For the purpose of investigating the JH functions, I focused on the regulatory mechanisms of expression of the major plasma protein gene in the silkworm, Bombyx mori. Plasma proteins termed "SP1" and "30K proteins" are synthesized by the fat body cells of the silkworm, B. mori , in a sex- and stage-specific manner during the larval development. I have successfully established a primary culture of the fat body cells to investigate the regulatory mechanisms of plasma protein gene expression.The primary cultures of fat body cells contained at least two cell types: small, oval cells, and large, spherical cells. The cells adhered and migrated on the culture dish. By the seventh day of cultivation, the cells have clustered to form the fat body-like structures that were maintained for at least three months. Plasma proteins were actively synthesized in the primary cultured cells isolated from the fat body of the final instar larvae only when the cells tightly adhered to the culture dish. Immunocytochemical analysis revealed that only 10-15% of the clustered cells synthesized plasma proteins in our culture system. These results indicate that the primary culture comprises heterogeneous cells which are morphologically and functionally distinct. The patterns of SP1 syntheses in primary cultures faithfully reproduced its sex-dependency in vivo. Subsequently, I established a method for introduction of DNAs into the fat body cells to analyze the regulatory element of 30K protein gene. Chimeric genes containing the 5\u27 sequences (-1668 to +14) of 30K protein 6G1 gene fused to the firefly luciferase gene were introduced into fat body cells using a method of electroporation. Optimum introduction of DNA was obtained at 90 V, 1075 μF, 1 pmol of DNA and 4 x 10^6 cells in 0.5 ml of serum-free Grace\u27s insect medium as an electroporation buffer using a 0.4 cm cuvette. When the constructs were transfected into fat body cells, the luciferase gene was accurately transcribed under the control of the 30K protein 6G1 gene promoter. By contrast, the fusion gene consisting of the 5\u27 upstream region (-1480 to +16) of B. mori LCP30 gene and the luciferase gene did not express in the fat body cells . These results show that the promoter constructs introduced into the fat body cells exhibit their promoter-specific expression. The analysis of 5\u27 upstream sequence of 30K protein 6G1 gene revealed that the enhancer elements are located in the sequence between nucleotide (nt) position -176 and -48 of the 30K protein gene. It was considered that the synthesis of 30K protein might be negatively regulated by JH in vivo. In this thesis, I clearly showed that JH suppressed the 30K protein synthesis by use of the primary culture system of fat body cells . Next, I analyzed the JH-responsive element of 30K protein 6G1 genes using the electroporation method. Analysis using a series of deletion mutants revealed that the JH-responsive elements of 30K protein 6G1 gene are located downstream of nt position -176 with its enhancer elements.東京都立大学, 2000-03-25, 博士(理学), 甲第568号東京都立大
