Fully deleted adenovirus vectors (FD-AdVs) would appear to be promising tools for gene therapy. Since these vectors are deleted of all adenoviral genes, they require a helper adenovirus for their propagation. The contamination of the vector preparation by the helper limits the utility of currently existing FD-AdVs in gene therapy applications. We have developed an alternative system for the propagation of FD-AdVs, in which the adenoviral genes essential for replication and packaging of the vector are delivered into producer cells by a baculovirus-adenovirus hybrid. A hybrid baculovirus Bac-B4 was constructed to carry a Cre recombinase-excisable copy of the packaging-deficient adenovirus genome. Although the total size of the DNA insert in Bac-B4 was 38 kb, the genetic structure of this recombinant baculovirus was stable. Bac-B4 gave high yields in Sf9 insect cells, with titers of 5 × 10 8 p.f.u./ml before concentration. Transfection
Cyclic nucleotide-gated (CNG) channels open in response to direct binding of cyclic nucleotide messengers. Every subunit in a tetrameric CNG channel contains a cytoplasmic ligand-binding domain (BD) that includes a -roll (flanked by short helices) and a single C-terminal helix called the C-helix that was previously found to control efficacy (maximal open probability) and selectivity for cGMP versus cAMP. We constructed a series of chimeric CNG channel subunits, each containing a distinct BD sequence (chosen from among six phylogenetically divergent isoforms) fused to an invariant non-BD sequence. We assayed these "BD substitution" chimeras as homomeric CNG channels in Xenopus oocytes to compare their functions and found that the most efficient activation by both cAMP and cGMP derived from the BD of the catfish CNGA4 olfactory modulatory subunit (fCNGA4). We then tested the effects of replacing subregions of the bovine CNGA1 BD with corresponding fCNGA4 sequence and hence identified parts of the fCNGA4 BD producing efficient activation. For instance, replacing either the "hinge" that connects the roll and C-helix subdomains or the BD sequence N-terminal to the hinge greatly enhanced cAMP efficacy. Replacing the "loop-8" region (the C-terminal end of the -roll) improved agonist sensitivity for cGMP selectively over cAMP. Our results thus identify multiple BD elements outside the C-helix that control selective ligand interaction and channel gating steps by distinct mechanisms. This suggests that the purine ring of the cyclic nucleotide may interact with both the -roll and the C-helix at different points in the mechanism. Cyclic nucleotide-gated (CNG)1 channels conduct mono-and divalent cations upon activation by the direct binding of the cytoplasmic messengers cAMP and cGMP. These channels are widespread in the nervous system and in a variety of other tissues, and most notably they are essential signaling components in visual and olfactory transduction, where their activation leads both to changes in membrane potential and to the influx of calcium into the cytoplasm (reviewed in Refs. 1 and 2). Functional CNG channels are tetramers (homomeric or heteromeric) of homologous subunits; vertebrates contain a family of six paralogous CNG channel subunit genes in two phylogenetic subfamilies (3), CNGA and CNGB. Distinct combinations of these paralogues are expressed in each tissue type to produce CNG channels whose response parameters are presumably adapted to the physiological requirements of the tissue; these parameters include sensitivity and efficacy (maximal ligandgated open probability) for cAMP and cGMP and selectivity for one agonist over the other. Thus, CNG channels hold promise as targets for tissue-specific pharmacological regulation of signaling through cyclic nucleotide-dependent, electrical, and calcium-dependent pathways. It is therefore important to understand how CNG channel function is determined by the sequences of individual subunits and more generally how structural elements in the channel work t...
Together these studies demonstrate the feasibility of this approach for the stabilization of transgenes delivered to dividing cells by adenovirus vectors.
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