Seasonal variation of spectral sensitivity in crayfish retinula cells.

小龙虾复眼具有两种视紫红质发色团：视网膜（AI）和3-脱氢视网膜（A2）。夏季A2含量远低于全年（Suzuki et al., 1984, 1985）。在此，我们重新研究了在夏季和冬季条件下，主要视网膜细胞（Rl-7）的光谱灵敏度曲线和发色团的变化之间的差异。夏季型小龙虾的眼睛只含有Al，只显示一类光谱灵敏度曲线。冬季型小龙虾的眼睛中同时含有Al和A2，粗横纹肌制剂的选择性光适应性表明，含有Al的视色素比含有A2的视色素在更短的波长处吸收最多。但得到了四种光谱灵敏度曲线。由于一个给定的发色团通常只产生一种光谱灵敏度曲线，因此有人提出在冬季可能存在两种不同的视蛋白。为了验证这一假设，我们生产了单克隆抗体，用于测定小龙虾眼睛中的视蛋白含量。获得4种视紫红质抗体。在35 kdd时，用免疫印迹法将它们捆绑在条带上。免疫组化观察4种抗体的抗原位点。在所有横纹肌（Rl-8）中均发现2个，包括视网膜顶端细胞。另一种仅局限于Rl-7细胞，第四种仅局限于少数视网膜细胞。这些不同的分布模式使我们认为小龙虾视网膜中确实存在不同的视蛋白。我们现在已经克隆了视蛋白CDNA。从1200只眼睛中提取了大约200杯的总RNA。然后从纯化的poly(A)^+ RNA合成单链和双链CDNA，并通过PCR方法扩增。用退化视蛋白引物进行PCR。我们从一种视蛋白mRNA中克隆并测序了一些EDNA片段。推导出的氨基酸序列表明，小龙虾视蛋白与其他已知的视蛋白相似，因为存在保守的关键残基（K为视网膜结合，两个C为二硫键，S和T为磷酸化等）。使用上述CDNA片段之一作为探针，我们用南杂交检测pcr扩增的基因组DNA。在主视蛋白带附近检测到第二个微弱信号，表明存在另一个视蛋白基因。少

The compound eye of the crayfish possesses two kinds of chromophore of rhodopsin, retinal(AI)and 3-dehydroretinal(A2). The A2 content in summer is much lower than during the reat of the year(Suzuki et al., 1984, 1985). There have been many papers dealing with the spectral sensitivity of the crayfish eye, with wide variation in the wavelength at maximum response,Here, we re-investigate the difference between the spectral sensitivity curves in the main retinula cells(Rl-7), and the changes in the chromophores, for summer and winter conditions. The eyes of the summer-type crayfish contained only Al and showed only one class of spectral sensitivity curve. The eyes of the winter-type crayfish contained both Al and A2, and selective light adaptation of the crude rhabdom preparations showed that the visual pigment containing Al absorbs maximally at a shorter wavelength than that containing A2. However, four patterns of spectral sensitivity curves were obtained. Since a given chromophore is ex … More pected to produce only one type of spectral sensitivity curve, it has been proposed that there may be two different opsin present in winter. In order to test this hypotheses, we have produced monoclonal antibodies, -to the opsin content of the eye of crayfish. Four antibodies to rhodopsin were obtained. They were bound to the band at 35 kdd by western blotting. The antigen sites of all four antibodies were observed by immunohistochemistry. Two were found in all rhabdomeres(Rl-8), including the apical retinula cell. Another was localized to cells Rl-7 only, and the fourth was confined to only a few retinula cells. These different patterns of distribution lead us to suggest that there are indeed different opsins present in the crayfish retina.We have now cloned the opsin CDNA. About 200 mug of total RNA was extracted from 1200 eyes. Single- and double-stranded CDNA were then synthesized from purified poly(A)^+ RNA, and amplified by the PCR method. Using PCR with degenerate opsin primers. We cloned and sequenced some EDNA fragments from a single kind of opsin mRNA. The deduced amino aced sequence showed that crayfish opsin is similar to other known opsins, in view of the presence of conserved key residues(K for retinalbinding, two C for disulfide bonds S and T for phosphorylation etc.). Using one of the above CDNA fragments as a probe, we examined the PCR-amplified genomic DNA with southern hybridization. A second, weak signal was detected near the main opsin band, suggesting the presence of another opsin gene. Less