TBE1 transposons of the ciliate Oxytricha are excised from its developing somatic nucleus, and as such are Internal Eliminated Sequences. We believe all IESs are transposon relics, not intrinsic components of host regulatory mechanisms. Our long-term goal is to develop the TBE1/IES system as a model for studying transposon host co-evolution. Because TBE1s form a large and coherent IES family, they provide large signals in developmental experiments and unlimited opportunities for comparisons of specific cases. Individual TBE1s have diverged widely, but nearly all are intact, both in gross structure and in the function of their three genes (encoding a DD35E transposase, a probably DNA-binding protein kinase, and an unidentified 22 kD protein). This is unprecedented among eukaryotic DNA transposons. An unusual form of selection must maintain TBE1s intact and functional. E.g., TBE1 transposase may act developmentally as excisase. if so, genes for developmental excision already are cloned, opening the door to mechanism description. """"""""This communal excision"""""""" hypothesis will be tested by a study of divergence between alleles of TBE1 insertions (to distinguish it from the alternative that selection is imposed during """"""""private"""""""" transposition). These analyses will help define the dynamics of the relationship between an unusual transposon family and its unusual host. Developmental expression of TBE1 transposase is indicated by preliminary 3' RACE PCR of putative mRNA transposase, and preliminary immunological results. Polysomal mRNAs will be sought for two other TBE1 genes and will be mapped, and potential element promoters identified. New antisera will be raised against whole transposase protein expressed in E. coli from a synthetic gene. Besides rare mRNAs, copious TBE1 RNAs appear during the time of excision; transcription enters elements from flanking promoters, potentially those of genes from which the elements must be excised to allow somatic gene function. We will test if such transcription is provoked by TBE1 presence, possibly by an potential enhancer at its center; such transcription may serve to improve access of DNA sites in chromatin to excisase, and facilitate precise excision. Developmental transposase activity (disintegration) will be assayed in extracts. The excisase mechanism will be further characterized. Transient breaks at TBE1 ends will be mapped and characterized. A possible TBE1 DNA binding site of TBE1 zinc-finger protein kinase will be mapped. Roles of it and conserved sites will be tested in a transient transfection assay for excision. A future is use an in vitro excision system to study TBE1/IES excision mechanisms. This work should illuminate the co-evolution of ciliates and their DNA parasites, as well as human relationships with their transposon and retroviral parasites.
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