CLONING, CHARACTERIZATION AND PROPERTIES OF PLASMIDS CONTAINING CGG TRIPLET REPEATS FROM THE FMR-1 GENE

The FMR-1 gene for the human fragile-X: syndrome, a mental retardation disease inherited hy non-Mendelian transmission, contains a genetical ly unstable CGG region in the 5' non-translated region. The severity o f the disease is correlated with the length of the CGG tract, The clon ing of 28 stable plasmids containing (CGG)(n) inserts (where n=6 to 24 0) with different extents and types of sequence interruptions (polymor phisms), and in different orientations tvas accomplished by three stra tegies in Escherichia coli, Some shorter tracts were prepared by the d irect cloning of synthetic oligonucleotides, and longer runs were clon es of multimers of (CGG)(81), (CGG)(11)AGG(CGG)(60)CAG(CGG)(8), from a cDNA from a fragile-X patient or from expansions or deletions of thes e sequences in E. coli. The genetic stability of the inserts, especial ly for the longer tracts, was dependent on the sequence length, the pr esence of polymorphisms, the host cell genotypes, the orientation of t he inserts in the vector and the position of cloning in a vector. Two- dimensional agarose gel electrophoresis studies on fully methylated an d on non-methylated plasmids as well as chemical probe studies reveale d the absence of underwound structures or accessible base-pairs. These DNAs enable a range of genetic and biochemical investigations into th e molecular basis of the fragile-X syndrome. (C) 1996 Academic Press L imited