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

Abstract

The FMR-1 gene for the human fragile-chi 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)61, (CGG)11AGG(CGG)60CAG(CGG)8, from a cDNA from a fragile-chi 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-chi syndrome.

Hairpin Structure-forming Propensity of the (CCTG·CAGG) Tetranucleotide Repeats Contributes to the Genetic Instability Associated with Myotonic Dystrophy Type 2

“…Furthermore, no rearrangements were observed on analyzing individual colonies obtained from transforming the parental plasmids into E. coli HB101. Likewise, rearrangements were not observed previously in other prior instability studies with triplet repeat inserts in E. coli HB101 (10,16,18,19,39,86,(91)(92)(93). This confirms that the rearrangements were because of an intrinsic property of the pcDNA3.1 shuttle vector that carries the SV40 origin of replication.…”

“…Previous studies showed a marked effect of orientation of trinucleotide repeats (CTG⅐CAG, CGG⅐CCG, and GAA⅐TTC) with respect to the origin of replication (10,16,18,19,21,39,85,86). Therefore, we studied plasmids carrying the (CCTG⅐CAGG) repeats in both orientations.…”

“…Length-dependent Instability of the (CCTG⅐CAGG) n Tetranucleotide Repeats-For CTG⅐CAG, CGG⅐CCG, and GAA⅐TTC trinucleotide repeats, the number of repeat units plays an important role in genetic instability (10,18,21,39,93,94). To evaluate the effect of the length of the (CCTG⅐CAGG) n tetranucleotide repeats (where n ϭ 30, 114, or 200) on the genetic instability, three different lengths of the repeats were studied.…”

“…the direction of replication) with respect to the types and amount of products (10,16,18,22,52,85,95,96). To determine whether a similar orientation effect was observed for the tetranucleotide repeats, (CCTG⅐CAGG) n repeats were cloned in both orientations with FIG.…”

Triplet Repeat Instability and DNA Topology: An Expansion Model Based on Statistical Mechanics

“…Also, the extent of instability for a (CTG) n or (CGG) n tract depends on its length and orientation relative to the origin of replication in Escherichia coli (30,31). We propose that high levels of supercoiling (writhe) accumulate within the TRS during processes such as transcription and replication, which lead to instability.…”