Developmental stage-specific proteins bind to sequences within the long repeat of the Tetrahymena thermophila Tlr1 DNA rearrangement
Abstract
DNA rearrangement is a fundamental biological process that generates DNA sequence diversity in a variety of prokaryotes and eukaryotes. Extensive programmed DNA rearrangement occurs during the development of the somatic macronucleus from the germline micronucleus in ciliated protozoans. The Tlr1 (Tetrahymena long repeat1) rearrangement in Tetrahymena thermophila is an intrachromosomal DNA rearrangement in which the micronuclear junctions are separated by $\ge$13 kb of DNA. There is a long, 825-bp, inverted repeat near the Tlr1 micronuclear junctions. The long repeat contains two different 19-bp tandem repeats, designated 19A and 19B. The 19-bp repeats are associated with each other and with DNA rearrangement at seven locations in the micronuclear genome. Southern blot analysis is consistent with the occurrence of the 19-bp repeats within pairs of longer repeated sequences. The 19-bp repeats are proximal to at least one additional DNA rearrangement junction, Tlr2. Sequences within the long repeat bind developmental stage-specific proteins present exclusively at the time of DNA rearrangement. Southern hybridization analysis of DNA isolated from developing macronuclei indicates that the DNA rearrangement occurs at approximately 12 hrs. of conjugation (sexual reproduction). Proteins that specifically bind to a 237-bp fragment containing the 19A tandem repeats are detectable in extracts prepared from conjugating cells 10-12 hrs. post mixing by a DNA mobility shift assay. Extracts prepared from non-mating cells and cells at earlier or later stages of conjugation do not show this DNA-binding activity. There is no detectable protein binding to a 148-bp fragment containing the 19B tandem repeats. DNase I footprinting analysis of a fragment containing the 19A and the 19B tandem repeats shows protection in the regions of both 19mers, occurring with 19-bp periodicity in both tandem repeats. The same extracts do not produce a footprint on the 148-bp fragment containing the 19B tandem repeats. The data support a model for binding of developmental stage-specific proteins to sequences within the Tlr1 long repeat in which both 19A and 19B tandem repeats must be on a contiguous DNA fragment in order for proteins to bind the 19B region. The close proximity of the long repeated sequence to DNA rearrangement junctions, and the binding of developmental stage-specific proteins to these sequences, suggests they are cis-acting sequences for DNA rearrangement.
This paper has been withdrawn.