Hiramatsu, C., Radlwimmer, F. B., Yokoyama, S. and Kawamura S. (2004). Mutagenesis and reconstitution of middle-to-long-wave-sensitive visual pigments of New World monkeys for testing the tuning effect of residues at sites 229 and 233. Vision Research, 44 (19):2225-2231.

On the basis of sequence comparison, spectral differences among the alleles of red-green opsin gene in New World monkeys had been ascribed to amino acid residues at sites 180, 229, 233, 277, and 285. While the significant spectral effects had been demonstrated for sites 180, 277 and 285 by site-directed mutagenesis (the "three-site rule"), effects at sites 229 and 233 had remained untested. We measured absorption spectra of the reconstituted red-green opsins from the tri-allelic squirrel monkey and the mono-allelic owl monkey. The peak absorption spectra (lambda max) of squirrel monkey opsins were 532, 545, and 558 nm and that of owl monkey pigment 539 nm, being consistent with the prediction from the three-site rule. Our site-directed mutagenesis for sites 229 and 233 showed that their mutational effects for lambda max values were negligible. These results preclude the necessity of examining exon 4, encoding the residues at sites 229 and 233, of red-green opsin genes for colour-vision typing of New World monkeys.