No abstract

Most individuals with the fragile X premutation are clinically unaffected; however, some show clinical manifestations, including learning difficulties, emotional problems, or even mental retardation. The basis of clinical involvement in these individuals is unknown. Premutation alleles are reportedly associated with normal levels of mRNA and protein (FMRP). To examine this issue in more detail, we studied six individuals with a premutation. We are reporting these cases to demonstrate a spectrum of phenotypic involvement which can be seen clinically. These cases include one individual with the premutation who has no evidence of FMR1 gene dysfunction but has mental retardation from other causes. Other cases presented here show varying degrees of FMR1 gene dysfunction as assessed by FMRP and FMR1 mRNA levels and various clinical features of fragile X. In two cases we observed a significant reduction in FMRP expression and an elevated FMR1 mRNA expression level associated with moderate cognitive deficit. Thus, the utilization of FMRP measures can be helpful in understanding for which premutation patients clinical involvement is caused by dysfunction of the FMR1 gene.

The influence of single-stranded nicks and gaps on the flexibility of DNA has been investigated by subjecting to gel electrophoresis sets of molecules containing single-stranded regions of defined position and length. The DNA molecules were produced by ligating together synthetic oligomers that contained either nicks or single-stranded gaps of 1-4 nucleotides; the oligomer repeat lengths were 20, 21, 22, 23, or 26 bp, in order to produce nicks or gaps that were either in- or out-of-phase with the helix repeat of DNA. Nick-containing DNA molecules displayed nearly normal electrophoretic behavior, with maximum reductions in gel mobility (41 degrees C; 12% polyacrylamide gels) of approximately 10% for 230-bp molecules containing 10 nicks. In contrast, molecules containing gaps of 2-4 nucleotides demonstrated dramatic reductions in mobility, approaching one-half of the values of their full-duplex counterparts; molecules containing 1-nucleotide gaps displayed intermediate behavior. The observed (relative) mobilities of molecules containing gaps of more than 1 nucleotide were remarkably insensitive to temperature and to the presence of magnesium ions in the electrophoresis buffer. The central conclusion of the current study is that single-stranded gaps represent points of swivel-like character, whereas nicks retain much of the rigid character of double-helical DNA.

The intrinsic rigidities of DNA and RNA helices are generally thought to arise from some combination of vertical base-stacking interactions and intra-helix phosphate-phosphate charge repulsion; however, the relative contributions of these two types of interaction to helix rigidity have not been quantified. To address this issue, we have measured the rotational decay times of a 'gapped-duplex' DNA molecule possessing a central, single-stranded region, dT24, before and after addition of the free purine base, N6-methyladenine ((me)A). Upon addition of (me)A, the bases pair with the T residues, forming a continuous stack within the gap region. Formation of the gapped duplex is accompanied by a nearly 2-fold increase in decay time, to values that are indistinguishable from the full duplex control for monovalent salt concentrations up to 90 mM. These results indicate that at least 90% of the rigidity of the dT(n)-dA(n) homopolymer derives from base pair stacking effects, with phosphate-phosphate interactions contributing relatively little to net helix rigidity at moderate salt concentrations.