Yokoyama S
Department of Biology, Syracuse University, New York 13244, USA.
Methods Enzymol. 2000;315:312-25. doi: 10.1016/s0076-6879(00)15851-3.
To elucidate the molecular mechanisms of vertebrate color vision, it is essential to establish associations between amino acid substitutions and the directions of lambda max shifts of visual pigments. In this way, we can identify critical amino acid changes that may be responsible for lambda max shifts of visual pigments. In this process, we may consider only highly conserved residues, simply because the evolutionary conservation often implies functional importance. Using such an "evolutionary model" as a convenient tool in designing mutagenesis experiments, we can test specific hypotheses on the molecular mechanisms that are responsible for color vision in vertebrates. Virtually any vertebrate opsin cDNA can be expressed in COS cells, reconstituted with 11-cis-retinal, and the lambda max values of the regenerated pigments can be measured rather easily. By constructing mutant pigments with desired amino acid changes and conducting the in vitro assay and comparing their lambda max values with those of corresponding wild-type pigments, we can elucidate the molecular mechanisms of lambda max shifts--and color vision--of vertebrates rigorously.
为阐明脊椎动物色觉的分子机制，在视觉色素的氨基酸取代与最大吸收波长（λmax）移动方向之间建立联系至关重要。通过这种方式，我们可以识别可能导致视觉色素λmax移动的关键氨基酸变化。在此过程中，我们可能仅考虑高度保守的残基，这仅仅是因为进化保守性通常意味着功能重要性。将这样一个“进化模型”作为设计诱变实验的便捷工具，我们可以检验关于脊椎动物色觉分子机制的特定假设。实际上，几乎任何脊椎动物视蛋白cDNA都可以在COS细胞中表达，与11-顺式视黄醛重组，并且再生色素的λmax值可以相当容易地测量。通过构建具有所需氨基酸变化的突变色素，进行体外测定，并将它们的λmax值与相应野生型色素的λmax值进行比较，我们可以严格阐明脊椎动物λmax移动以及色觉的分子机制。
