EVOLUTION OF SHORTER AND MORE HYDROPHILIC TRANSTHYRETIN N-TERMINI BY STEPWISE CONVERSION OF EXON-2 INTO INTRON-1 SEQUENCES (SHIFTING THE 3'-SPLICE-SITE OF INTRON-1)

Transthyretin cDNA was cloned from Eastern Grey Kangaroo liver and its nucleotide sequence determined. Analysis of the derived amino acid se quence of kangaroo transthyretin, together with data obtained previous ly for transthyretins from other vertebrate species [Duan, W., Richard son, S. J., Babon, J. J., Heyes, R. J., Southwell, B. R., Harms, P. J. , Wettenhall, R. E. H., Dziegielewska, K. M., Selwood, L., Bradley, A. J., Brack, C. M. & Schreiber, G. (1995) Eur. J. Biochem. 227, 396-406 ], showed that the N-terminus is the region which changes most distinc tly during evolution. It has been shown for human, mouse and rat trans thyretins, that this region is encoded by DNA at the border of exon 1 and exon 2. Therefore, this section of transthyretin genomic DNA was a mplified by PCR and directly sequenced for the Buffalo Rat, Tammar Wal laby, Eastern Grey Kangaroo, Stripe-faced Dunnart, Short-tailed Grey O possum and White Leghorn Chicken. The splice sites at both ends of int ron 1 were identified by comparison with the cDNA sequences. The obtai ned data suggest that the N-termini of transthyretin evolved by succes sive shifts of the 3' splice site of intron 1 in the 3' direction, res ulting in successive shortening of the 5' end of exon 2. At the protei n level, this resulted in a shorter and more hydrophilic N-terminal re gion of transthyretin. Successive shifts in splice sites may be an evo lutionary mechanism of general importance, since they can lead to step wise changes in the properties of proteins. This could be a molecular mechanism for positive Darwinian selection.