Fryxell K J, Meyerowitz E M
EMBO J. 1987 Feb;6(2):443-51. doi: 10.1002/j.1460-2075.1987.tb04774.x.
We have used two techniques to isolate and characterize eye-specific genes from Drosophila melanogaster. First, we identified genes whose expression is limited to eyes, photoreceptor cells, or R7 photoreceptor cells by differential screening with [32P]cDNAs derived from the heads of mutant flies that have reduced amounts of these tissues and cells (Microcephalus, glass3, and sevenless, respectively). Secondly, we identified opsin genes by hybridization with synthetic [32P]oligonucleotides that encode domains that have been conserved between some opsin genes. We found seven clones that contain genes expressed only in the eye or optic lobes of Drosophila; three are expressed only in photoreceptor cells. One is expressed only in R7 photoreceptor cells and hybridizes to some of the previously mentioned oligonucleotides. The complete DNA sequence of the R7-specific opsin gene and its 5' and 3' flanking regions was determined. It is quite different from other known Drosophila opsin genes, in that it is not interrupted by introns and shares only 37-38% amino acid identity with the proteins encoded by these genes. The predicted protein structure contains many characteristics that are common to all rhodopsins, and the sequence differences help to identify four domains of the rhodopsin molecule that have been conserved in evolution.
我们运用了两种技术从黑腹果蝇中分离并鉴定眼特异性基因。首先，我们通过用来自头部组织和细胞数量减少的突变果蝇（分别为小头症、玻璃眼3和无七受体）的[32P]cDNA进行差异筛选，鉴定出那些表达局限于眼睛、光感受器细胞或R7光感受器细胞的基因。其次，我们通过与合成的[32P]寡核苷酸杂交来鉴定视蛋白基因，这些寡核苷酸编码在一些视蛋白基因之间保守的结构域。我们发现了七个克隆，其包含的基因仅在果蝇的眼睛或视叶中表达；其中三个仅在光感受器细胞中表达。一个仅在R7光感受器细胞中表达，并与上述一些寡核苷酸杂交。我们确定了R7特异性视蛋白基因及其5'和3'侧翼区域的完整DNA序列。它与其他已知的果蝇视蛋白基因有很大不同，因为它没有被内含子打断，并且与这些基因编码的蛋白质仅共享37 - 38%的氨基酸同一性。预测的蛋白质结构包含所有视紫红质共有的许多特征，序列差异有助于识别视紫红质分子在进化中保守的四个结构域。
