Jones G, O'Mahony P, Chang S, Schachtschabel U
Molecular and Cellular Biology Section, School of Biological Sciences, University of Kentucky, Lexington, KY 40506, USA.
Gene. 1996 Sep 16;173(2):209-14. doi: 10.1016/0378-1119(96)00100-x.
The promoters of three juvenile hormone (JH)-sensitive, and one JH-insensitive hexamerin-encoding genes (Hex) were isolated from Trichoplusia ni, and sequences necessary for, or affecting, transcriptional activity were identified by biochemical and functional methods. The transcription start points (tsp) for each of the four Hex were determined biochemically, by both primer extension and the sequencing of multiple, independent full-length cDNA clones. The function of each inferred tsp, as an actual tsp, was confirmed by in vitro transcription assay. The transcription initiator sequence, GNACAGT, was identical for three of the Hex, while the fourth used a divergent motif. Using the in vitro transcription system, a minimal core promoter of 60 bp (bp -34 to +24) of the BJHSP1 (basic JH suppressible protein 1) gene, containing a single TATA box motif approximately 30 bp upstream of the tsp, was functionally sufficient to support alpha-amanitin-sensitive transcription. The same construct was also transcriptionally functional in a homologous cell line transfection assay. The corresponding region of the other Hex also contains a similarly positioned TATA box motif, and promoter constructs for each, that included that included the tsp, initiator and inferred basal transcription apparatus binding site, were all transcriptionally functional in a cell line transfection assay. The action of sequences 5' to the minimal promoter region in modulating the rate of transcription was shown by a cell line transfection assay of a nested deletion series of the promoter for the BJHSP1 gene, in which the results identified a strongly suppressive element between positions -160 and -109. This system of Hex genes, including those sensitive to JH and one not sensitive, should be useful in a comparative approach toward identifying those regulatory motifs that are functionally necessary to transduce the regulatory action of JH.
从粉纹夜蛾中分离出了三个对保幼激素（JH）敏感和一个对JH不敏感的六聚蛋白编码基因（Hex）的启动子，并通过生化和功能方法鉴定了转录活性所需的或影响转录活性的序列。通过引物延伸和多个独立全长cDNA克隆的测序，以生化方法确定了四个Hex基因各自的转录起始点（tsp）。通过体外转录试验证实了每个推断的tsp作为实际tsp的功能。三个Hex基因的转录起始序列GNACAGT相同，而第四个使用了不同的基序。利用体外转录系统，BJHSP1（碱性JH抑制蛋白1）基因的一个60 bp（-34至+24 bp）的最小核心启动子，在tsp上游约30 bp处含有一个单一的TATA盒基序，在功能上足以支持对α-鹅膏蕈碱敏感的转录。相同的构建体在同源细胞系转染试验中也具有转录功能。其他Hex基因的相应区域也含有位置相似的TATA盒基序，并且每个基因的启动子构建体（包括tsp、起始子和推断的基础转录装置结合位点）在细胞系转染试验中均具有转录功能。通过对BJHSP1基因启动子的嵌套缺失系列进行细胞系转染试验，显示了最小启动子区域5'端序列在调节转录速率方面的作用，结果确定了在-160和-109位之间有一个强抑制元件。这个Hex基因系统，包括对JH敏感的和一个不敏感的基因，在比较鉴定那些转导JH调节作用所需的功能调控基序的方法中应该是有用的。
