The GC content of exons and intron sequences was taken into account to evaluate the over-representation of some residues around splice sites in each yeast species. Nucleotides were considered to be preferentially conserved at a given position when their individual proportion represented at least 40% of the nucleotides registered at this location.
The analysis of the degree of conservation of the exonic and intronic nucleotides adjacent to the splice motifs shows that non-random sequence patterns are conserved at sites flanking the core splicing sites in S. cerevisiae introns [Spingola et al., 1999; Lopez & Seraphin, 1999] as in all other hemiascomycetous yeast introns [Bon et al., 2003].
Thus, a G generally precedes the 5' splice site in most yeast species. This nucleotide preference can be enlarged to AAG in the yeast species most closely related to S. cerevisiae. The intronic context of the splicing sites also appeared to be somewhat conserved. Except for a few exceptions, an A and a T were always found in the downstream region of the 5' site, around the branch site and in the upstream region of the 3' site [Bon et al., 2003].
The nature of this preferential nucleotide usage appears to be species-dependent although some general rules exist. The significance of such conservation is not clarified. These non-random sequences closely resemble the ?proto-splice site? (MAG-intron-R) postulated for intron insertion events [Csank et al., 1990; Horowitz & Krainer, 1994; Bhattacharya et al., 2000; Lorkovic et al., 2000], suggesting that they may be remnants of preferential sites for the insertion of introns in pre-mRNA genes. An other possibility is that they might be required for efficient splicing [Long et al., 1998].
All of these observations helped us to define an extension of the core splice motifs in each yeast species [Bon et al., 2003].