Abstract
Abstract: :
Purpose:IRBP is very unsual for a retinoid–binding protein in that it consists of four 300 aa "modules" or "repeats". Interestingly, there are only 2 modules in the osteichthyes studied to date. Ohno (1970) considered that gene duplication events occured before the emergence of vertebrates, prior to the radiation of the ray finned fish (actinopterygii) and lobe–finned fish (sarcopterygii), and again later during the evolution of the actinopterygii. Here we seek to determine when during the evolution of vertebrates did the duplication events occur that lead to the modern modular structure of IRBP. Methods: Living vertebrate representatives at key points in evolution were selected to study the structure of IRBP: Gar fish (early actinopterygii), Lungfish (sarcopterygii), shark (predate osteichthyes), lamprey (earliest vertebrate), and amphioxus (vertebrate ancestor). Western blot analysis utilized antibodies generated against bovine IRBP, or recombinant zebrafish or Xenopus IRBPs. Control reactions were adsorbed with the corresponding protein. LC tandem mass spectrometry was used to establish the identity of IRBP. Results: For each species, Western blot analysis revealed a single high molecular weight protein from the IPM that crossreacted with the antibody. The relative molecular ratio for these proteins were as follows: Gar fish (127 kD), shark (140 kD) and lamprey (145 kD). Conclusions: The fact that IRBP in all the species studied is similar to the size of mammalian and amphibian IRBPs suggest that it has a similar gene structure also consisting of four repeats. This will be formally shown by ongoing cloning studies. At this point we can conclude the following: 1) The half–size of IRBP in most osteichthyes occured as a consequence of gene loss early during the emergence of the ray–finned fish after the divergence of the gar fish. 2) The gene quadruplication that lead to the four modular structure of mammalian IRBP appears to have been a very early event in the evolution of the vertebrates even predating the divergence of lampreys.
Keywords: gene/expression • protein structure/function • retinoids/retinoid binding proteins