CLONING, CHARACTERIZATION, AND PROPERTIES OF 7 TRIPLET REPEAT DNA-SEQUENCES

Several neuromuscular and neurodegenerative diseases are caused by gen etically unstable triplet repeat sequences (CTG . CAG, CGG . CCG, or A AG . CTT) in or near the responsible genes. We implemented novel cloni ng strategies with chemically synthesized oligonucleotides to clone se ven of the triplet repeat sequences (GTA . TAC, GAT . ATC, GTT . AAC, CAC . GTG, AGG . CCT, TCG . CGA, and AAG . CTT), and the adjoining pap er (Ohshima, K., hang, S., Larson, J. E., and Wells, R. D. (1996) J. B iol. Chem. 271, 16784-16791) describes studies on TTA . TAA. This appr oach in conjunction with in vivo expansion studies in Escherichia coil enabled the preparation of at least 81 plasmids containing the repeat sequences with lengths of similar to 16 up to 158 triplets in both or ientations with varying extents of polymorphisms. The inserts were cha racterized by DNA sequencing as well as DNA polymerase pausings, two-d imensional agarose gel electrophoresis, and chemical probe analyses to evaluate the capacity to adopt negative supercoil induced non-B DNA c onformations. AAG . CTT and AGG . CCT form intramolecular triplexes, a nd the other five repeat sequences do not form any previously characte rized non-B structures. However, long tracts of TCG . CGA showed stron g inhibition of DNA synthesis at specific loci in the repeats as seen in the cases of CTG . CAG and CGG . CCG (Kang, S., Ohshima, K., Shimiz u, M., Amirhaeri, S., and Wells, R. D. (1995) J. Biol. Chem. 270, 2701 4-27021). This work along with other studies (Wells, R. D. (1996) J. B iol. Chem. 271, 2875-2878) on CTG . CAG, CGG . CCG, and TTA . TAA make s available long inserts of all 10 triplet repeat sequences for a vari ety of physical, molecular biological, genetic, and medical investigat ions, A model to explain the reduction in mRNA abundance in Friedreich 's ataxia based on intermolecular tripler formation is proposed.