The human genetic disorder, Nijmegen breakage syndrome, is characterized by radiosensitivity, immunodeficiency, chromosomal instability and an increased risk for cancer of the lymphatic system. The NBS1 gene codes for a protein, nibrin, involved in the processing/repair of DNA double strand breaks and in cell cycle checkpoints. Most patients are homozygous for a founder mutation, a 5 bp deletion, which might not be a null mutation, as functionally relevant truncated nibrin proteins are observed, at least in vitro. In agreement with this hypothesis, null mutation of the homologous gene, Nbn, is lethal in mice. Here, we have used Cre recombinase/loxP technology to generate an inducible Nbn null mutation allowing the examination of DNA-repair and cell cycle-checkpoints in the complete absence of nibrin. Induction of Nbn null mutation leads to the loss of the G2/M checkpoint, increased chromosome damage, radiomimetic-sensitivity and cell death. In vivo, this particularly affects the lymphatic tissues, bone marrow, thymus and spleen, whereas liver, kidney and muscle are hardly affected. In vitro, null mutant murine fibroblasts can be rescued from cell death by transfer of human nibrin cDNA and, more significantly, by a cDNA carrying the 5 bp deletion. This demonstrates, for the first time, that the common human mutation is hypomorphic and that the expression of a truncated protein is sufficient to restore nibrin's vital cellular functions.

The genome of hepatitis B virus (HBV) encodes two transcriptional activators: the HBx protein and the PreS2-activator large surface protein (LHBs). Both proteins trigger activation of c-Raf-1/MEK kinase cascade. In case of HBx this can be mediated by a PKC-independent and Ras-dependent mechanism, in case of LHBs activation is PKC-dependent and does not require Ras. Selective destruction of either LHBs-or of HBx-specific activation does not result in significant decrease of viral production from transfected HepG2 cells. Simultaneous inhibition of LHBs-and HBx-dependent activation by blocking signaling steps common to both activators, using trans dominant negative c-Raf-1-or MEK-specific inhibitors, abolished HBV gene expression. In accordance with this no HBV propagation was observed after transfection of a mutated HBV genome defective for HBx-and PreS2-activator function. A detailed analysis revealed that the observed inhibition of HBV-propagation is because of a significant reduction of HBV-specific RNA resulting in an inhibition of the de novo synthesis of viral compounds (viral proteins and nucleic acid) and not by blocking secretion or assembly of the virus. Based on these results we conclude that transcriptional-activator function, mediated by the cRaf-1/MEK signaling cascade, is essential for HBV gene expression.

R ecently, a variety of protein transduction domains (PTDs) have been identified allowing the transfer of peptides, proteins, or nucleic acids across cellular membranes into cells. 1-9 PTDs have been used to successfully treat preclinical models of human disease such as cancer, psoriasis, and stroke. 10-13 Transfer of nano-particular structures across cellular membranes is of increasing importance for the development of novel diagnostic and therapeutic tools. The capacity of PTDs to mediate an endocytosis-independent transfer of particles across the plasma membrane into the cytoplasm is still unclear. To investigate the potential of PTD to mediate the transfer of nano-particles across the cell membrane, virus-like particles (VLPs) harboring a marker gene were instrumental.Hepatitis B virus (HBV) core particles represent a very well-characterized VLP model system. 14,15 The HBV core particle (capsid) is assembled by 180 or 240 core protein monomers (HBcAg), resulting in an icosahedral particle 30 or 34 nm in diameter, respectively. A characteristic of the core protein is a basic arginine-rich C-terminal region, which is responsible for the packaging of DNA 16 and for guidance of the capsid to the nucleus. 17 Based on extensive structural analysis, 18 the hepatitis B capsid was used as a carrier for foreign epitopes to develop new vaccines. 19,20 In the viral context, the core particle harbors the viral genome (nucleocapsid). Efficient in vivo packaging of nucleic acids into capsids requires HBV polymerase and its interaction with a defined secondary structure (termed epsilon) at the 5Ј end of the RNA to be packaged. 21 The ⑀ signal is the primary element of the hepadnaviral packag-

Hypomorphic mutations of the NBS1 gene are responsible for Nijmegen breakage syndrome (NBS), characterized by microcephaly, chromosomal instability, radiosensitivity, immunodeficiency and high cancer predisposition. Over 90% of NBS patients are homozygous for the 657Delta5 mutation and are of Slavic origin; however, 10 further truncating mutations have been identified in patients of other ethnic origin. Partially functional proteins produced by alternative initiation of translation, and possibly diminishing the severity of the NBS phenotype, have been described for several NBS1 mutations. Here, we report a 53-year-old NBS patient, homozygous for the NBS1 mutation, 742insGG, in exon 7 and who presents with a particularly mild phenotype. In an attempt to find a potential molecular explanation for the mild phenotype observed, we carried out a conventional semi-quantitative and quantitative RT-PCR analyses which revealed two transcripts of almost equal amounts in the patient and her parents--the expected full-length transcript carrying the 742insGG mutation and a second transcript with deleted exons 6 and 7. The transcript was also observed in controls and other NBS patients, however, at quantities more than 100-fold lower than that in the patient described here. Because the skipping of exons 6 and 7 results in an internal in-frame deletion, which eliminates the truncating GG-insertion, we propose that this transcript may code for a partially functional protein of approximately 70 kDa that could be responsible for the unusually mild NBS phenotype observed in this patient. Indeed, complementation analysis of null-mutant mouse cells indicates that the alternatively spliced mRNA codes for a protein with significant functional capacity.