Seasonal variation of spectral sensitivity in crayfish retinula cells.

螯虾复眼中含有两种视紫红质发色团：视黄醛（AI）和3-脱氢视黄醛（A2）。夏季的A2含量远低于一年中的大部分时间（Suzuki et al.，1984年，1985年）。已经有许多论文涉及小龙虾眼睛的光谱灵敏度，在最大响应波长的变化很大，在这里，我们重新研究了主要小网膜细胞（R1 -7）的光谱灵敏度曲线之间的差异，以及发色团在夏季和冬季条件下的变化。夏型克氏原螯虾的眼睛只含铝，只呈现一类光谱敏感曲线。冬季型的小龙虾的眼睛包含Al和A2，和选择性光适应的粗横纹肌制剂表明，视觉色素含有Al最大吸收在较短的波长比含有A2。然而，光谱灵敏度曲线的四种模式得到。由于给定的发色团是 ...更多信息 预计只产生一种类型的光谱敏感度曲线，已经提出在冬季可能存在两种不同的视蛋白。为了测试这一假设，我们已经产生了单克隆抗体，-视蛋白的小龙虾的眼睛的内容。获得了四种抗视紫红质的抗体。Western blotting检测结果表明，它们与35 kdd的条带结合。用免疫组化法观察四种抗体的抗原位点。在所有的横纹肌（R1 -8），包括顶端小网膜细胞中发现了两个。另一个仅定位于细胞R1 -7，第四个仅限于少数小网膜细胞。这些不同的分布模式使我们认为在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只眼睛中提取约200 μ g总RNA。然后用纯化的poly（A）^+ RNA合成单链和双链cDNA，并通过PCR方法进行扩增。使用简并视蛋白引物的PCR。我们从一种单一的视蛋白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的视蛋白相似，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）。用上述cDNA片段之一作为探针，我们用Southern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