In vivo stimulation of I kappa B phosphorylation is not sufficient to activate NF-kappa B.

NF-kappa B is a major inducible transcription factor in many immune and inflammatory reactions. Its activation involves the dissociation of the inhibitory subunit I kappa B from cytoplasmic NF-kappa B/Rel complexes, following which the Rel proteins are translocated to the nucleus, where they bind to DNA and activate transcription. Phosphorylation of I kappa B in cell-free experiments results in its inactivation and release from the Rel complex, but in vivo NF-kappa B activation is associated with I kappa B degradation. In vivo phosphorylation of I kappa B alpha was demonstrated in several recent studies, but its role is unknown. Our study shows that the T-cell activation results in rapid phosphorylation of I kappa B alpha and that this event is a physiological one, dependent on appropriate lymphocyte costimulation. Inducible I kappa B alpha phosphorylation was abolished by several distinct NF-kappa B blocking reagents, suggesting that it plays an essential role in the activation process. However, the in vivo induction of I kappa B alpha phosphorylation did not cause the inhibitory subunit to dissociate from the Rel complex. We identified several protease inhibitors which allow phosphorylation of I kappa B alpha but prevent its degradation upon cell stimulation, presumably through inhibition of the cytoplasmic proteasome. In the presence of these inhibitors, phosphorylated I kappa B alpha remained bound to the Rel complex in the cytoplasm for an extended period of time, whereas NF-kappa B activation was abolished. It appears that activation of NF-kappa B requires degradation of I kappa B alpha while it is a part of the Rel cytoplasmic complex, with inducible phosphorylation of the inhibitory subunit influencing the rate of degradation.

Molecular diagnosis of Prader-Willi syndrome: parent-of-origin dependent methylation sites and non-isotopic detection of (CA)n dinucleotide repeat polymorphisms.

We describe our experience in the molecular diagnosis of 22 patients suspected of Prader-Willi syndrome (PWS) using a DNA probe PW71 (D15S63) which detects a parent-of-origin specific methylated site in the PWS critical region. The cause of the syndrome was determined as deletion or uniparental disomy according to the segregation of (CA)n dinucleotide repeat polymorphisms of the PWS/AS region and more distal markers of chromosome 15. In 10 patients the clinical diagnosis was confirmed by this approach, 6 with paternal deletion and 4 with maternal disomy. In one patient, the aberrant methylation pattern that was detected by PW71 could not be confirmed by the segregation of (CA)n, probably due to paternal microdeletion in the PWS critical region which did not include the loci D15S97, D15S113, GABRB3, and GABRA5. This case demonstrates the advantage of the DNA probe PW71 in the diagnosis of PWS.

Myotonic dystrophy: molecular analysis of Israeli patients.

Myotonic dystrophy (DM) is an autosomal dominant, multisystem disorder and the most common adult form of muscular dystrophy. The age of onset and degree of severity of DM is highly variable. The biochemical defect in DM is unknown. DM was the first autosomal disorder to be localised by genetic linkage to protein markers (Lu, Se, C3), and assigned to chromosome 19. Linkage studies in DM families using RFLPs as polymorphic markers refined the mapping position to 19q13.1-13.2 distal to the BCL3, apoCII, CKM and ERCCI genes. Based on the linkage data, healthy individuals from DM families request a pre-symptomatic test. The information is of use in planning their family, if at high risk they can choose to have prenatal diagnosis. We have studied ten unrelated DM families by linkage analysis. The DNA probes to detect the various RFLPS were either from the vicinity of BCL3, ApoCII, CKMM and ECCRI genes or anonymous DNA probes. Linkage analysis in the DM families enabled us to determine the carrier status of healthy individuals and to perform prenatal diagnosis at a confidence of > 99%. In two families the DM diagnosis was in doubt and we did not include them in the combined analysis. Linkage disequilibrium was noted with two RFLPs pDIO/Pst1 and p37.1/BamH1. Both DNA probes were isolated by Shaw and his group in Cardiff. In six out of eight families, the DM chromosome was associated with allele 3 of pDIO/Pst1 and allele 1 of p37.1/BamH1.(ABSTRACT TRUNCATED AT 250 WORDS)

Negative expansion of the myotonic dystrophy unstable sequence.

We have analyzed the unstable fragment of the myotonic dystrophy (DM) gene in a pregnancy at 50% risk for DM. The affected father in this family had a 3.0-kb expansion of the DM unstable region. The fetus inherited the mutated gene, but with an expansion of 0.5 kb. This case represented a counseling problem in light of the absence of data concerning "negative expansion." Analysis of the DM gene in 17 families with 72 affected individuals revealed four more cases of negative expansions, all of them in paternal transmissions. The possible significance of this finding is discussed.