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Seasonal variation of spectral sensitivity in crayfish retinula cells.

小龙虾视网膜细胞光谱敏感性的季节性变化。

基本信息

批准号：
02640546
负责人：
HARIYAMA Takahiko
金额：
$ 0.96万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

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

小龙虾的复眼拥有两种视紫红质发色团：视黄醛（AI）和3-脱氢视黄醛（A2）。夏季A2含量远低于一年中的含量(Suzuki et al., 1984, 1985)。已经有很多论文讨论了小龙虾眼睛的光谱敏感性，最大响应波长变化很大，在这里，我们重新研究夏季和冬季条件下主要视网膜细胞（Rl-7）光谱敏感性曲线的差异以及生色团的变化。夏季型小龙虾的眼睛只含有铝，并且只表现出一类光谱灵敏度曲线。冬季型小龙虾的眼睛同时含有Al和A2，粗横纹组织制剂的选择性光适应表明，含有Al的视觉色素在比含有A2的视觉色素更短的波长处吸收最大。然而，获得了四种光谱灵敏度曲线。由于预计给定的发色团仅产生一种类型的光谱灵敏度曲线，因此有人提出冬季可能存在两种不同的视蛋白。为了检验这一假设，我们针对小龙虾眼睛的视蛋白含量制备了单克隆抗体。获得了四种抗视紫红质的抗体。通过蛋白质印迹，它们以 35 kdd 与条带结合。通过免疫组织化学观察所有四种抗体的抗原位点。在所有横纹细胞(R1-8)中都发现了两个，包括顶端网膜细胞。另一个仅局限于R1-7细胞，第四个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 只眼睛中提取了约 200 杯总 RNA。然后从纯化的poly(A)^+ RNA合成单链和双链cDNA，并通过PCR方法进行扩增。使用带有简并视蛋白引物的 PCR。我们从单一种类的视蛋白 mRNA 中克隆并测序了一些 EDNA 片段。推导的氨基酸序列表明，小龙虾视蛋白与其他已知视蛋白相似，因为存在保守的关键残基（K用于视网膜结合，两个C用于二硫键S和T用于磷酸化等）。使用上述 cDNA 片段之一作为探针，我们通过 Southern 杂交检查了 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。较少的