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Biology subjects

Tessier, C. J. G.

Publications and source records attributed to Tessier, C. J. G..

3 recordsLinked to original sources

pUdOs: concise plasmids for bacterial and mammalian cells

The pUdOs are 28 plasmids of small size combining four different origins of replication and seven selection markers, which together afford flexible use in Escherichia coli and several related gram- negative bacteria. The promoterless multicloning site is insulated from upstream spurious promoters by strong transcription terminators, and contains type IIP or IIS restriction sites for conventional or Golden-gate cloning. pUdOs can be converted into efficient expression vectors through the insertion of a promoter at the users discretion. For example, we demonstrate the utility of pUdOs as the backbone for an improved version of a Type III Secretion System reporter in Shigella. In addition, we derive a series of pUdO-based mammalian expression vectors affording distinct levels of expression and transfection efficiencies comparable to commonly used mammalian expression plasmids. Thus, pUdOs could advantageously replace traditional plasmids in a wide variety of cell types and applications.

synthetic biology↗

Derepression masquerades as activation in a pentameric ligand-gated ion channel

Agonists are ligands that bind to receptors and activate them. In the case of ligand-gated ion channels, such as the muscle-type nicotinic acetylcholine receptor, mechanisms of agonist activation have been studied for decades. Taking advantage of a reconstructed ancestral muscle-type {beta}-subunit that forms spontaneously activating homopentamers, here we show that incorporation of human muscle-type -subunits represses spontaneous activity, and furthermore that the presence of agonist relieves this -subunit-dependent repression. Our results demonstrate that rather than provoking channel activation/opening, agonists may instead inhibit the inhibition of intrinsic spontaneous activity. Thus, agonist activation may be the apparent manifestation of agonist-induced derepression. These results provide insight into intermediate states that precede channel opening and have implications for the interpretation of agonism in ligand-gated ion channels.

biophysics↗

Ancestral acetylcholine receptor β-subunit forms homopentamers that prime before opening spontaneously

Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from two -subunits, and one each of the {beta}-, {delta}-, and {varepsilon}-subunits. To form functional channels, the subunits must assemble with one another in a precise stoichiometry and arrangement. Despite being different, the four subunits share a common ancestor that is presumed to have formed homopentamers. The extent to which the properties of the modern-day receptor result from its subunit complexity is unknown. Here we show that a reconstructed ancestral muscle-type {beta}-subunit can form homopentameric ion channels. These homopentamers open spontaneously and display single-channel hallmarks of muscle-type acetylcholine receptor activity. Our findings demonstrate that signature features of muscle-type acetylcholine receptor function are independent of agonist, and do not necessitate the complex heteropentameric architecture of the modern-day receptor.

biophysics↗