My long term goal is to understand the functions of interspersed repeats in human DNA. Studies proposed here define the properties of a family of repeated sequences called THE-1, which consists of 10,000 genomic copies and has hallmarks of transposable elements. The structure of the 2 kb THE-1 repeats resembles copia-like/proviral elements. THE-1 repeats hybridize to both discrete length poly A plus RNA and a number of discrete length extrachromosomal circular DNAs. The following experiments will describe the structure, transcription and transposition of THE-1 repeats. Structure: Sequence studies of the ends of THE-1 repeats will test preliminary findings that THE-1 elements insert into specific sequences. Other transporon-like families which are related to THE-1 will be identified by several alternative screening procedures. Transcription: Although most polymorphic THE-1 repeats are pseudogenes, one or more members probably code for an mRNA. The relationship of discrete length RNAs to THE-1 will be mapped and significant open reading frames will be identified by sequencing cDNA clones. The in vivo translation of THE-1 RNA will be tested. The nearly single copy THE-1 sequences present in prosimian will be isolated as probable examples of an authentic THE-1 gene structure. The prosimian gene in turn would be employed to isolate the human THE-1 gene. Transposition: Restriction fragment length polymorphism in human and non human primates of single copy sequences flanking THE-1 repeats will be determined to measure the mobility of THE-1 sequences. Since the discrete length extrachromosomal circular DNAs are far larger than the 2 kb THE-1 sequence to which they hybridize, recombinant DNAs will be isolated from these circular DNAs for: 1) structural analysis to decide their relationship to THE-1 and whether they are transpositional intermediates and 2) as a source of hybridization probes to isolate the transpositionally active sequences.
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