| Budget Amount *help |
¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1991: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1990: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
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
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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
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