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Nucleic Acids Research, 1995, Vol. 23, No. 14 2648-2653
© 1995

MOLECULAR BIOLOGY

The fat body cell-free system for tissue-specific transcription of plasma protien gene of Bombyx mori

Eriko Mine, Hiroshi Sakurai1, Susumu Izumi and Shiro Tomino*

Department of Biology, Tokyo Metropolitan University Minamiohsawa, hachioji-shi, Tokyo 192–03, Japan 1Laboratory of Molecular Genetics, Keio University School of Medicine Shinanomachi, Shinjuku-ku, Tokyo 160, Japan

*To whom correspondence should be addressed

Received April 13, 1995. Accepted June 12, 1995.

A nuclear extract was prepared from the larval fat body of the silkworm, Bombyx mori, and a homologous In vitro system was developed for the transcription of major plasma protein gene of B.mori. The gene for SP1, a storage protein of B.mori, and adenovlrus 2 major late (AdML) gene were faithfully transcribed under relatively high template concentrations in the nuclear extract prepared from the fat body of female fifth instar larvae. Complete inhibition of gene transcription by a low concentration of {alpha}-amanitin Indicated that the reaction is catalyzed by RNA polymerase II. At low template concentration (0.6 nM) the fat body nuclear extract transcribed the homologous SP1 gene with high efficiency, while AdML gene and larval cuticle protein gene were only barely transcribed in the same extract. The SP1 gene deleted upstream of the TATA box sequence showed little effect on transcription, whereas mutations that destroy TATA sequence totally abolished the gene transcription. These results suggested that the core promoter region of SP1 gene spanning between positions –44 and +16 is essential for the fat body specific transcription In vitro.


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