No abstract

Fragile X syndrome is the result of the unstable expansion of a trinucleotide repeat in the 5'-untranslated region of the FMR1 gene. Fibroblast subclones from a mildly affected patient, each containing stable FMR1 alleles with 57 to 285 CGG repeats, were shown to exhibit normal steady-state levels of FMR1 messenger RNA. However, FMR protein was markedly diminished from transcript with more than 200 repeats. Such transcripts were associated with stalled 40S ribosomal subunits. These results suggest that a structural RNA transition beyond 200 repeats impedes the linear 40S migration along the 5'-untranslated region. This results in translational inhibition by trinucleotide repeat expansion.

Metabolic acidosis impairs protein and amino acid metabolism in rat muscle. To examine how extracellular acidification affects cellular protein turnover, we studied the BC3H1 myocyte. At pH 7.1 vs. 7.4, intracellular pH was lower; the decrease was greater in cells incubated in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-tris(hydroxymethyl)aminomethane compared with bicarbonate buffer. We monitored degradation of proteins labeled with L-[14C]phenylalanine by measuring radioactivity released into media containing an excess of unlabeled phenylalanine. Extracellular acidification increased degradation compared with incubation at pH 7.4. Adding a physiological concentration of insulin (1 nM) decreased protein degradation at pH 7.1 and 7.4; a supraphysiological (71 nM) insulin concentration decreased degradation at pH 7.1 to the same rate as cells incubated at pH 7.4 without insulin. Compared with pH 7.4, protein synthesis decreased 29% at pH 7.2; at pH 7.6 it increased 129%. Insulin stimulated protein synthesis at all pHs, but at pH 7.4 the insulin-induced increase was less than the rate at pH 7.6 without insulin. Dexamethasone did not change protein breakdown regardless of the pH; it had variable effects on protein synthesis. Thus extracellular acidification causes marked changes in protein turnover in BC3H1 myocytes.

Expansions of trinucleotide repeats within gene transcripts are responsible for fragile X syndrome, myotonic dystrophy and spinal and bulbar muscular atrophy. To identify other human genes with similar features as candidates for triplet repeat expansion mutations, we screened human cDNA libraries with repeat probes and searched databases for transcribed genes with repeats. From both strategies, 40 genes were identified and 14 characterized. Five were found to contain repeats which are highly polymorphic including the N-cadherin, BCR, glutathione-S-transferase and Na+/K+ ATPase (beta-subunit) genes. These data demonstrate the occurrence of other human loci which may undergo this novel mechanism of mutagenesis giving rise to genetic disease.