Some pubs

Brief summaries of a few papers that caught my attention this morning.

Molecular breeding of polymerases for amplification of ancient DNA (abstract)

This research provides a novel strategy for recovering (via replicative means) ancient DNA. In the absence of routine molecular maintenance actors, nucleic matter will undergo progressive time-dependent degradation and its structural character will suffer from any given environment. The accumulation of damage greatly harms the replicative integrity of DNA. Traditionally, Taq polymerase has been the machinery of choice for PCR-based amplification. An engineered class of polymerases were selected with high tolerance for template damage. Technical information about the forms of DNA damage an ancient sample may have undergone is lacking, therefore selection for damage tolerance was necessarily generic. Some lesions (aside from miscoding lesions [uracil]) abrogate pairing and produce distorted, non-cognate 3′ structures similar to transversion mismatches. A-family polymerases may stall up to four bases upstream of of such lesions. To optimize tolerance for such distortion, polymerases were selected capable of extending a 3′ terminus preceded by up to four mismatched bases. Previous attempts had been made with random mutagenesis libraries of polymerase genes, but proved largely unproductive. Here, a new ‘molecular breeding’ library was prepared via recombination of three A-family polymerases. Glossing over their methods, an advanced selection process garnered polymerases capable of extending [single, double, or quadruple] mismatches, extending non-canonical primer-template duplexes, and bypassing structural lesions. Pretty cool!

Ligand screening system using fusion proteins of G protein-coupled receptors with G protein alpha subunits. (abstract)

The importance of this paper may be underlined by the crucial role of GPCRs in the drug discovery world. The general flow has been to first bioinformatically identify these receptors. This has traditionally been accomplished using various HMMs, gene prediction algorithms, hydropathicity constraints, homology to previously identified receptors, and even chomsky-like grammar rules. Once a receptor has been characterized in silico, its primary gene structure requires validation with 3′,5′-RACE PCR. The cloned receptor is then usually placed in a vector for either stable or transient modes of expression. Deorphanization of the receptor is next on the agenda, and this may be helped along with some creative bioinformatic exercises. To identify an agonist for a receptor, you need an end-point by which you can measure receptor activation. The G-protein signaling cascade through which a given GPCR may operate, is wholly dependent on the Gα subunit the receptor couples to. Only one of these subunits (G_q), leads to release of intracelullar Ca(2+) stores, for which a fluorescent-based detection scheme has been developed. To cope with this, a class of Gα chimeras were introduced (Gα_q/i, for example) which serve to ‘divert’ the signal cascade towards Ca(2+) release upon co-transfection. This paper introduces the idea of using GPCR-Gα fusions as means to the same end. The function of these fusion products may be confirmed by measuring agonist stimulation of guanosine 5′-O-(3-[³⁵S]thiotriphosphate) binding or GTP hydrolysis.