Mine E, Sakurai H, Izumi S, Tomino S
Department of Biology, Tokyo Metropolitian University, Japan.
Nucleic Acids Res. 1995 Jul 25;23(14):2648-53. doi: 10.1093/nar/23.14.2648.
A nuclear extract was prepared for 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 adenovirus 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.
制备了家蚕幼虫脂肪体的核提取物，并开发了一种同源体外系统用于家蚕主要血浆蛋白基因的转录。家蚕的一种储存蛋白SP1的基因以及腺病毒2主要晚期（AdML）基因，在从雌性五龄幼虫脂肪体制备的核提取物中，在相对较高的模板浓度下被如实地转录。低浓度的α-鹅膏蕈碱对基因转录的完全抑制表明该反应是由RNA聚合酶II催化的。在低模板浓度（0.6 nM）下，脂肪体核提取物高效转录同源的SP1基因，而AdML基因和幼虫表皮蛋白基因在同一提取物中几乎不被转录。TATA框序列上游缺失的SP1基因对转录影响不大，而破坏TATA序列的突变则完全消除了基因转录。这些结果表明，SP1基因位于-44至+16位之间的核心启动子区域对于体外脂肪体特异性转录至关重要。
