© 2012 Landes Bioscience. Do not distribute. Figure 2. REP sequence orientation within GI doublets. (A) Alignment of 101 GI REP doublets from SBW25 (seven are shown) that are found at a distance of 71 bp to each other. REP sequences within the doublet are found in opposite orientations and are divided by a less conserved spacer sequence. Each REP sequence consists of a palindrome, a 5’ and a 3’ flanking sequence. The bases in the center of each palindrome indicate the orientation within the doublet. TT is found in the center of the first palindrome and AA in the center of the second, hence, the shown doublet is of type AA-TT. Three conserved As and Ts are found at the 5’ and 3’ end respectively, indicating the co-option of this REP doublet class as transcription terminator. (B) Alignment of the less commonly found AA-TT GI doublet conformation separated by 43 bp. Note that the conserved As and Ts at the 5’ and 3’ end of the alignment do not exist. However, As are found at the 5’ end of the bc sequence and at the 3’ end of the b sequence similar to GI doublets in TT-AA orientation. (C) A potential scenario for the evolution of AA-TT GI doublets from TT-AA GI doublets. An accidental transposition of the 3’ and 5’ end of two co-localized TT-AA GI doublet could have been sufficient to create the new AA-TT REP doublet type. act to maintain RAYT function—even if RAYT function is required for the move- ment of REPINs. This means that either RAYTs are the non-functional remains of once active transposons, or they are maintained by virtue of some complex relationship with the host cell, or with the REPINs or a combination of both. Examination of the RAYT-encoding genes provides little evidence in support of the hypothesis that they are fossilized remnants. Indeed, evolutionary analyses of dN/dS show that approximately 60% of codons have a significant excess of syn- onymous substitutions and are thus sub- ject to negative (purifying) selection. This suggests that the amino acid sequence of RAYTs is constrained by virtue of function, and that whatever that function might be, that it is important for cell viability. While dN/dS analyses fail to find evi- dence of positive selection, the substantive diversity at non conserved codons makes such analyzes problematic. Indeed, high levels of polymorphism at non-conserved codons are to be expected where genes are subject to strong diversifying selection. Tests using evolutionary fingerprinting17 reveal that a number of sites have in fact experienced strong diversifying selection. Together, the signature of both purifying and diversifying selection suggest that RAYTs are, on one hand, functionally constrained, yet also subject to positive selection. Such a signature is reminiscent of genes involved in interactions with hosts, or more generally, genes involved in co-evolutionary processes.18,19 One possible explanation for the main- tenance of RAYTs is the existence of some kind of addiction system, which ensures that cells containing defective RAYTs (the defect being caused by a spontaneous mutation) are killed and thus eliminated. Such a scenario would be akin to well described plasmid addiction systems.20 To test this hypothesis the three RAYT genes from SBW25 were deleted from the genome, however, the resulting mutant was fully viable (Zhang XX, Bertels F and Rainey PB, unpublished). This suggests that addiction is not responsible for the maintenance of RAYT function. www.landesbioscience.com Mobile Genetic Elements 265