Molecular cloning and characterization of a transcription factor for the copia retrotransposon with homology to the BTB-containing lola neurogenic factor
Molecular cloning and characterization of a transcription factor for the copia retrotransposon with homology to the BTB-containing lola neurogenic factor
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DOI：
10.1128/mcb.17.1.482
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发表时间：
1997-01-01
影响因子：
5.3
通讯作者：
Heidmann, T
中科院分区：
文献类型：
--
作者：
Cavarec, L;Jensen, S;Heidmann, T
通过转染实验，我们之前在果蝇copia反转录转座子中鉴定了一个72-bp的增强子序列，该序列参与培养细胞中该移动元件的转录水平的控制，用来自果蝇hydei的DH-33细胞的核提取物进行的凝胶转移分析进一步证明了至少两个核因子与该增强子序列的特异性相互作用，使用该序列作为筛选 我们从 DH-33 细胞 RNA 构建的表达 cDNA 文库，我们分离了编码 110-kDa 蛋白质的 cDNA 克隆，其具有与已知转录因子相同的特征；这些结构域包括 C 端的两个锌指基序、推定的蛋白质激活结构域中的三个富含谷氨酰胺的结构域，以及与 Bric-a-brac、Tramtrack 和 Broad-Complex BTB 盒具有同源性的 N 端结构域。该转录因子的精确 DNA 识别序列已通过凝胶位移测定和细菌重组蛋白足迹实验确定。在具有克隆因子表达载体的培养细胞中的共转染测定中，通过 copia 报告基因以及与鉴定的靶 DNA 序列偶联的最小启动子的转录激活，证明了克隆元件的功能。 Southern印迹和核苷酸序列分析揭示了果蝇中的一个相关基因（lola基因），该基因先前通过遗传学方法鉴定为参与轴突生长和引导，在用lola基因表达载体培养的细胞中进行的转染测定以及与lola基因突变体的原位杂交实验最终提供了证据，证明copia逆转录转座子是由黑腹果蝇中的这种神经源性基因调节的。 黑腹果蝇，对胚胎的中枢神经系统具有抑制作用。
By transfection experiments, we previously identified a 72-bp enhancer sequence within the Drosophila copia retrotransposon which is involved in the control of the transcription level of this mobile element in cells in culture, Gel shift assays with nuclear extracts from Drosophila hydei-derived DH-33 cells further demonstrated specific interactions of at least two nuclear factors with this enhancer sequence, Using this sequence as a probe for the screening of an expression cDNA library that we constructed from DH-33 cells RNA we have isolated a cDNA clone encoding a 110-kDa protein with features common to those of known transcription factors; these include a two zinc-finger motif at the C terminus, three glutamine-rich domains in the presumptive activation domain of the protein, and an N-terminal domain which shares homology with the Bric-a-brac, Tramtrack, and Broad-Complex BTB boxes. The precise DNA recognition sequence for this transcription factor has been determined by both gel shift assays and footprinting experiments with a recombinant protein made in bacteria. The functionality of the cloned element was demonstrated upon transcriptional activation of copia reporter genes, as well as of a minimal promoter coupled with the identified target DNA sequence, in cotransfection assays in cells in culture with an expression vector for the cloned factor. Southern blot and nucleotide sequence analyses revealed a related gene in Drosophila melanogaster (the lola gene) previously identified by a genetic approach as involved in axon growth and guidance, Transfection assays in cells in culture with lola gene expression vectors and in situ hybridization experiments with lola gene mutants finally provided evidence that the copia retrotransposon is regulated by this neurogenic gene in D. melanogaster, with a repressor effect in the central nervous systems of the embryos.