Adeno-associated virus (AAV) site-specific integration: Formation of AAV–AAVS1 junctions in an in vitro system

An in vitro system to study the mechanism of site-specific integration of adeno-associated virus (AAV) was developed. This system is based on two substrates, a linear or circular AAV donor and a circular acceptor containing the preintegration locus AAVS1. In the presence of HeLa extract and the His-Tag-purified Rep68 protein, specific covalent junctions between AAV and AAVS1 were formed and detected by PCR. The majority of the junctions were located within the Rep binding site of both the AAV and the AAVS1 substrates, underlining the involvement of the Rep protein. A limited amount of replication and the presence of nuclear factors promoted the efficiency of the reaction. The process was ATP-dependent, indicating that the helicase activity of Rep may be important in the formation of the junctions. According to current models of integration, the formation of the junctions would represent a first step in the process of AAV integration. This step could be crucial for the site specificity of the recombination event that leads to the integration of AAV into human chromosome 19 in vivo.

Unexpected signals in a system subject to kinetic proofreading

When multivalent ligands attach to IgEs bound to the receptors with high affinity for IgE on mast cells, the receptors aggregate, tyrosines on the receptors become phosphorylated, and a variety of cellular responses are stimulated. Prior studies, confirmed here, demonstrated that the efficiency with which later events are generated from earlier ones is inversely related to the dissociation rate of the aggregating ligand. This finding suggests that the cellular responses are constrained by a “kinetic proofreading” regimen. We have now observed an apparent exception to this rule. Doses of the rapidly or slowly dissociating ligands that generated equivalent levels of tyrosine-phosphorylated receptors comparably stimulated a putatively distal event: transcription of the gene for monocyte chemoattractant protein 1. Possible explanations of this apparent anomaly were explored.

A role for ciliary neurotrophic factor as an inducer of reactive gliosis, the glial response to central nervous system injury.

Within the central nervous system (CNS) ciliary neurotrophic factor (CNTF) is expressed by astrocytes where it remains stored as an intracellular protein; its release and function as an extracellular ligand are thought to occur in the event of cellular injury. We find that overexpression of CNTF in transgenic mice recapitulates the glial response to CNS lesion, as does its injection into the uninjured brain. These results demonstrate that CNTF functions as an inducer of reactive gliosis, a condition associated with a number of neurological diseases of the CNS.