Intra-specific variability and unusual organization of the repetitive units in a satellite DNA from Rana dalmatina: molecular evidence of a new mechanism of DNA repair acting on satellite DNA.

We have characterized the S1 satellite from eight European populations of Rana dalmatina by Southern blot, cloning and a new method that determines the sequence variability of repetitive units in the genome. This report completes our previous studies on this satellite DNA family, thus providing the first characterization of the overall variability of the structure and genomic organization of a satellite DNA within a species and among related species. The S1 satellite from R. dalmatina has a pericentromeric location on ten chromosome pairs and presents two homologous repeats S1a (494 bp) and S1b (332 bp), mostly organized as composite S1a-S1b repetitive units. In other brown frog species, both repeats have different sequences and locations, and are usually organized as separate arrays, although composite S1a-S1b repeats represent a minor, widely variable component in Rana italica. The average genomic sequences indicate that the species contains an enormous number of variants of each repeat derived from a unique, species-specific common sequence. The repeat variability is restricted to specific base changes in specific sequence positions in all population samples. Our data show that the structure and evolution of S1 satellite family is not due to crossing-over and gene conversion, but to a mechanism that maintains the ability of the satellite DNA to assemble in constitutive heterochromatin by replacing altered satellite segments with new arrays generated by rolling circle amplification. The mode of action of this repair process not only directly explains the intra- and inter-specific variability of the structure and organization of the S1 satellite repeats from European brown frogs, but also accounts for all general features of satellite DNA in eukaryotes, including its discontinuous evolution. This repair mechanism can maintain the satellite structure in a species indefinitely, but also promote a rapid generation of new variants or types of satellite DNA when environmental conditions favor the formation of new species

The first characterisation of the overall variability of repetitive units in a species reveals unexpected features of satellite DNA.

We investigated the overall variability of the S1a satellite DNA repeats in ten European populations of Rana temporaria by a new procedure that determines the average sequence of the repeats in a genome. The average genomic sequences show that only 17% of the S1a repeat sequence (494 bp) is variable. The variable positions contain the same major and minor bases in all or many of the population samples tested, but the percentages of these bases can greatly vary among populations. This indicates the presence in the species of an enormous number of repeats having a different distribution of bases in these variable positions. Individual genomes contain thousands of repeat variants, but these mixtures have very similar characteristics in all populations because they present the same type of restricted and species-specific variability. Southern blots analyses and sequences of cloned S1a repeats fully support this conclusion. The S1 satellite DNA of other European brown frog species also presents properties indicating the same type of variability. This first characterisation of the overall repeat variability of a satellite DNA in a species has revealed features that cannot be determined by gene conversion and crossing over. Our results suggest that a specific directional process based on rolling circle amplification should play a relevant role in the evolution of satellite DNA