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These two features are likely to be introduced by the subtraction technique [ 25, 26], which favours the cloning of relatively short fragments with lower degrees of conservation.
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We recently identified another calcium-responsive enhancer activity lying within a large restriction fragment of IG1 (VA, unpublished data), presumably arising from the possible co-operation between two or more CNS with no detectable activity when assayed individually, or from a subsequence that had not been listed as a CNS because of a lower degree of conservation.
A higher score indicates a higher rate and lower degree of conservation.
At least, the lower degree of conservation of the non-expressed genes supports the latter.
The second observation is the lower degree of conservation of chaperonin substrates among organisms, which suggests that chaperonin substrates emerged later on in evolutionary history.
Previous studies reported a lower degree of conservation of ASEs than CSEs and constructed models in which exons were generated as ASEs [ 21, 49, 50].
The extracellular loops (ELs), especially EL2 and EL3 in GPRC5A and GPRC5D (Additional file 4, 5), show a lower degree of conservation than the rest of the proteins.
Notably, the D6 subdomain shows a significantly lower degree of conservation in the Walker A motif and the Walker B motif deviates from the consensus sequence (DE > DN).
Therefore, we can explain the lower degree of conservation by the emergence of chaperonin-dependent substrates later on in evolutionary history.
Finally, these un-annotated sequences could represent partial transcripts with mostly UTRs which, in general, show lower degree of conservation among species.
The lower degree of conservation of truncated sequences compared to full-length ORF1 sequences (Table 3) could be due to diversifying selection concomitant with the expansive generation of effectors.
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