Abstract
Silk gland factor-1 (SGF-1) regulates transcription of the Bombyx sericin-1 gene via interaction with the SA site. In this study, two related SGF-1 polypeptides of apparent molecular masses of 40 and 41 kDa were purified. Specific interaction of these proteins with the SA site was demonstrated by electrophoretic mobility shift and dimethyl sulfate methylation interference assays. The SGF-1 40-kDa protein was partially sequenced and characterized as a new member of the fork head/HNF-3 family. Several full-length cDNAs encoding the SGF-1 40-kDa and possibly also the 41-kDa proteins were cloned and sequenced. SGF-1 mRNA is expressed consistently with the presumed role of the SGF-1 protein product in regulating the sericin-1 gene. The SGF-1 protein contains putative transactivation domains. We conclude that the 40- and 41-kDa SGF-1 proteins affect transcription of the sericin-1 gene via binding to the SA site.
Highlights
From the National Institute for Basic Biology, 38 Nishigonaka, Myodaiji-cho, Okazaki 444, Japan and the 'IlFaculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 227, Japan
A major tissue and sequencespecific complex was detected by means of electrophoretic mobility shift assay (EMSA) with a crude middle silk gland (MSG) extract using an oligonucleotide containing the SA element as a probe [4]
The same protein probably binds to two regions upstream of the H-chain fibroin gene [2], In the present report, we describe purification of the Silk gland factor-l (SGF-l) protein and molecular cloning of its corresponding cDNAs, Bombyx SGF-l is a homologue to the Drosophila fork head protein [6] and a new member of the fork head! HNF-3 family of transcriptional regulators
Summary
Vol 270, No 16, Issue of April 21, pp. 9340-9346, 1995 Printed in U.S.A. Silk Gland Factor-! Involved in the Regulation of Bombyx Sericin-! Gene Contains Fork Head Motif*. Silk gland factor-l (SGF-l) regulates transcription of the Bombyx sericin-l gene via interaction with the SA site. Silk glands and shown to be identical with SGF-3 [5] Another silk gland-enriched factor, named SGF-l, interacts with the SA site located in the proximal upstream region of the sericin-I gene promoter. A silk gland-enriched factor SGF-3 1 is known to bind to the SC site upstream of the sericin-l gene promoter [3]. Removal of this SC element curbs down in vitro transcription from the sericin-l gene promoter in crude middle silk gland (MSG) nuclear extract [3, 4]. 7560; Fax: 81-564-55-7566. 1 The abbreviations used are: SGF-3, silk gland factor-S; SGF-1, silk gland factor-1; MSG, middle silk glands; EMSA, electrophoretic mobility shift assay; ACN, acetonitrile; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s); HPLC, high pressure liquid chromatography
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