Cortical NMDA receptor expression in human chronic alcoholism: influence of the TaqIA allele of ANKK1.

Real-time RT-PCR normalized to GAPDH was used to assay N-methyl-D-aspartate (NMDA) receptor NR1, NR2A and NR2B subunit mRNA in human autopsy cortex tissue from chronic alcoholics with and without comorbid cirrhosis of the liver and matched controls. Subunit expression was influenced by the subject's genotype. The TaqIA polymorphism selectively modulated NMDA receptor mean transcript expression in cirrhotic-alcoholic superior frontal cortex, in diametrically opposite ways in male and female subjects. Genetic make-up may differentially influence vulnerability to brain damage by altering the excitation: inhibition balance, particularly in alcoholics with comorbid cirrhosis of the liver. The TaqIA polymorphism occurs within the poorly characterised ankyrin-repeat containing kinase 1 (ANKK1) gene. Using PCR, ANKK1 mRNA transcript was detected in inferior temporal, occipital, superior frontal and primary motor cortex of control human brain. ANKK1 expression may mediate the influence of the TaqIA polymorphism on phenotype.

Arg660Ser mutation in Thermus aquaticus DNA polymerase I suppresses T-->C transitions: implication of wobble base pair formation at the nucleotide incorporation step.

We examined the replication fidelity of an Arg660Ser (R660S) mutant of Thermus aquaticus DNA polymerase I (Taq pol I). In a forward mutation assay, R660S showed a marked reduction in T-->C transitions, one of the most frequent errors made by the wild-type enzyme. Steady-state kinetics showed that R660S discriminates against dGTP incorporation at a template T 13-fold better than the wild-type. R660S was also 3.2-fold less efficient than the wild-type at extending a T:dG mismatch. These results indicate that R660S has enhanced fidelity during incorporation and extension, which reduces its T-->C transition frequency. Interestingly, R660S also discriminated correct from incorrect nucleotides at the incorporation step of C:dATP, A:dATP, G:dATP and C:8-OH-dGTP mispairs 28-, 6.0-, 4.1- and 6.8-fold better, respectively, than the wild-type, although it may not always be as accurate as the wild-type at the extension step. A structural model suggests that Arg660 may participate in two interactions that influence fidelity; the guanidinium group of Arg660 might interact with the incoming guanine base at the major groove and it might compete for forming another interaction with the primer terminus. Substituting Arg with Ser may eliminate or alter these interactions and destabilize the closed complex with incorrect substrates. Our data also suggest that T:dGTP and C:dATP base pairs form 'wobble' structures at the incorporation step of Taq pol I.