Cloning, Characterization and Properties of Plasmids Containing CGG Triplet Repeats from the FMR-1 Gene

• Published in: Journal of molecular biology 1996-05, Vol.258 (4), p.614-626
• Main Authors: Shimizu, Miho, Gellibolian, Robert, Oostra, Ben A., Wells, Robert D.
• Format: Article
• Language: English
• Subjects: Base Sequence; CGG; Cloning, Molecular - methods; DNA structure; DNA, Recombinant; Escherichia coli - genetics; expansion; Fragile X Mental Retardation Protein; Fragile X Syndrome - genetics; fragile-X syndrome; Humans; Molecular Sequence Data; Nerve Tissue Proteins - genetics; Plasmids - genetics; Polymorphism, Genetic; RNA-Binding Proteins; Trinucleotide Repeats; triplet repeat
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1996.0273

The FMR-1 gene for the human fragile-X syndrome, a mental retardation disease inherited by non-Mendelian transmission, contains a genetically unstable CGG region in the 5′ non-translated region. The severity of the disease is correlated with the length of the CGG tract. The cloning of 28 stable plasmids containing (CGG) n inserts (where n=6 to 240) with different extents and types of sequence interruptions (polymorphisms), and in different orientations was accomplished by three strategies in Escherichia coli. Some shorter tracts were prepared by the direct cloning of synthetic oligonucleotides, and longer runs were clones of multimers of (CGG) 81, (CGG) 11AGG(CGG) 60CAG(CGG) 8, from a cDNA from a fragile-X patient or from expansions or deletions of these sequences in E. coli. The genetic stability of the inserts, especially for the longer tracts, was dependent on the sequence length, the presence of polymorphisms, the host cell genotypes, the orientation of the inserts in the vector and the position of cloning in a vector. Two-dimensional agarose gel electrophoresis studies on fully methylated and on non-methylated plasmids as well as chemical probe studies revealed the absence of underwound structures or accessible base-pairs. These DNAs enable a range of genetic and biochemical investigations into the molecular basis of the fragile-X syndrome.