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首页> 外文期刊>Molecular and Cellular Biology >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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Molecular cloning and characterization of a transcription factor for the copia retrotransposon with homology to the BTB-containing lola neurogenic factor.

机译：与含BTB的lola神经源因子同源的Copia逆转录转座子转录因子的分子克隆和表征。

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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-à-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.
机译：通过转染实验，我们先前在果蝇果蝇逆转录转座子中鉴定了一个72 bp的增强子序列，该序列与培养细胞中该移动元件的转录水平的控制有关。用得自果蝇的DH-33细胞核提取物的凝胶移位试验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文库的探针，我们分离出了一个编码110 kDa蛋白的cDNA克隆，该蛋白具有已知转录因子的共同特征;其中包括在C末端有两个锌指基序，在蛋白质的假定激活结构域中的三个富含谷氨酰胺的结构域，以及与Bric-à-brac，Tramtrack和Broad-复杂的BTB盒子。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中制备的重组蛋白的印迹实验确定。在培养的细胞中使用克隆因子的表达载体进行共转染测定时，克隆的报告基因以及与识别的靶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方法鉴定为涉及轴突的生长和引导。最终用洛拉基因表达载体对细胞进行转染测定，并与洛拉基因突变体进行原位杂交实验，最终提供证据表明，黑斑病鼠中的神经原性基因调节了锥孔逆转座子，在胚胎的中枢神经系统中具有阻遏作用。 。

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