title: Rhodopsin from the fish, Astyanax: role of tyrosine 261 in the red shift.
abstract: OBJECTIVE
  To isolate and characterize the rhodopsin cDNA from the fish, Astyanax fasciatus, and to determine the effect of tyrosine 261 on its spectral tuning.
  
  
  METHODS
  The rhodopsin cDNA was cloned using reverse transcription-polymerase chain reaction amplification and then sequenced. A mutant, Y261F, was generated by site-directed mutagenesis. Both wild type and mutant were transiently expressed in COS-1 cells, regenerated with 11-cis retinal, and purified by immunoaffinity chromatography. Ultraviolet-visible spectrophotometry was used to determine wavelength of maximum absorption.
  
  
  RESULTS
  A fasciatus rhodopsin cDNA exhibits 80% amino acid identity with bovine rhodopsin. In contrast to all known rhodopsins, this rhodopsin contains a tyrosine instead of a phenylalanine at amino acid position 261. Indeed, this particular amino acid replacement has been implicated in the long wavelength absorption of the red cone pigment. Site-directed mutagenesis was used to change the Astyanax amino acid 261 to phenylalanine (Y261F). Expression of the Y261F mutant in COS-1 cells showed an absorbance maximum of 496 nm, compared to 504 nm for the wild type pigment.
  
  
  CONCLUSIONS
  A naturally occurring fish rhodopsin is red shifted about 8 nm due to one critical amino acid substitution.
authors.affiliation: Department of Biology, Syracuse University, NY 13244, USA.
authors.forename: R
authors.initials: R
authors.lastname: Yokoyama
authors.forename: B E
authors.initials: BE
authors.lastname: Knox
authors.forename: S
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D005399:Fishes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D014443:Tyrosine; D012243:Rhodopsin