A tight coupling between β\u3csub\u3e2\u3c/sub\u3eY97 and β\u3csub\u3e2\u3c/sub\u3eF200 of the GABA\u3csub\u3eA\u3c/sub\u3e receptor mediates GABA binding

The GABAA receptor is an oligopentameric chloride channel that is activated via conformation changes induced upon the binding of the endogenous ligand, GABA, to the extracellular inter-subunit interfaces. Although dozens of amino acid residues at the α/β interface have been implicated in ligand binding, the structural elements that mediate ligand binding and receptor activation are not yet fully described. In this study, double-mutant cycle analysis was employed to test for possible interactions between several arginines (α1R67, α1R120, α1R132, and β2R207) and two aromatic residues (β2Y97 and β2F200) that are present in the ligand-binding pocket and are known to influence GABA affinity. Our results show that neither α1R67 nor α1R120 is functionally coupled to either of the aromatics, whereas a moderate coupling exists between α1R132 and both aromatic residues. Significant functional coupling between β2R207 and both β2Y97 and β2F200 was found. Furthermore, we identified an even stronger coupling between the two aromatics, β2Y97 and β2F200, and for the first time provided direct evidence for the involvement of β2Y97 and β2F200 in GABA binding. As these residues are tightly linked, and mutation of either has similar, severe effects on GABA binding and receptor kinetics, we believe they form a single functional unit that may directly coordinate GABA

High-efficiency receptor-mediated delivery of small and large (48 kilobase gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles.

One limit to successful receptor-mediated gene delivery is the exit of the endocytosed material from the endosome. We demonstrate here the delivery of marker genes to tissue culture cells using a modification of the receptor-mediated gene delivery technique that exploits the endosomolytic activity of defective adenovirus particles. In particular, greater than 90% of the transfected-cell population is found to express a beta-galactosidase gene, and, most importantly, this high level of expression can be obtained with psoralen-inactivated virus particles. Furthermore, because the delivered gene is not carried within the genome of the adenovirus particle, the size constraints are relieved, and we can, therefore, show the delivery of a 48-kilobase cosmid DNA molecule

Binding-incompetent adenovirus facilitates molecular conjugate-mediated gene transfer by the receptor-mediated endocytosis pathway

Molecular conjugate vectors may be constructed that accomplish high efficiency gene transfer by the receptor-mediated endocytosis pathway. In order to mediate escape from lysosomal degradation, we have incorporated adenoviruses into the functional design of the conjugate. In doing so, however, we have introduced an additional ligand, which can bind to receptors on the cell surface, undermining the potential for cell specific targeting. To overcome this, we have treated the adenovirus with a monoclonal anti-fiber antibody, which renders the virus incapable of binding to its receptor. The result is a multi-functional molecular conjugate vector, which has preserved its binding specificity while at the same time being capable of preventing lysosomal degradation of endosome-internalized conjugate-DNA complexes. This finding indicates that adenoviral binding is not a prerequisite for adenoviral-mediated endosome disruption

Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes.

We are developing efficient methods for gene transfer into tissue culture cells. We have previously shown that coupling of a chimeric adenovirus with polylysine allowed the construction of an adenovirus-polylysine-reporter-gene complex that transferred the transporter gene with great efficiency into HeLa cells. We have now explored simpler, biochemical means for coupling adenovirus to DNA/polylysine complexes and show that such complexes yield virtually 100% transfection in tissue culture cell lines. In these methods adenovirus is coupled to polylysine, either enzymatically through the action of transglutaminase or biochemically by biotinylating adenovirus and streptavidinylating the polylysine moiety. Combination complexes containing DNA, adenovirus-polylysine, and transferrin-polylysine have the capacity to transfer the reporter gene into adenovirus-receptor- and/or transferrin-receptor-rich cells

Dual gene activation and knockout screen reveals directional dependencies in genetic networks.

Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyze the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair. Based on the results from over 100,000 perturbed gene pairs, we reconstruct a directional dependency network for human K562 leukemia cells and demonstrate how our approach allows the determination of directionality in activating genetic interactions. Our interaction network connects previously uncharacterized genes to well-studied pathways and identifies targets relevant for therapeutic intervention

Transferrinfection. A Highly Efficient Way to Express Gene Constructs in Eukaryotic Cells

No abstract available