Ka-Lok Tong

Positive Selection within the Schizophrenia-Associated GABAA Receptor β2 Gene

The gamma-aminobutyric acid type-A (GABAA) receptor plays a major role in inhibitory neurotransmissions. Intronic SNPs and haplotypes in GABRB2, the gene for GABAA receptor β2 subunit, are associated with schizophrenia and correlated with the expression of two alternatively spliced β2 isoforms. In the present study, using chimpanzee as an ancestral reference, high frequencies were observed for the derived (D) alleles of the four SNPs rs6556547, rs187269, rs1816071 and rs1816072 in GABRB2, suggesting the occurrence of positive selection for these derived alleles. Coalescence-based simulation showed that the population frequency spectra and the frequencies of H56, the haplotype having all four D alleles, significantly deviated from neutral-evolution expectation in various demographic models. Haplotypes containing the derived allele of rs1816072 displayed significantly less diversity compared to haplotypes containing its ancestral allele, further supporting positive selection. The variations in DD-genotype frequencies in five human populations provided a snapshot of the evolutionary history, which suggested that the positive selections of the D alleles are recent and likely ongoing. The divergence between the DD-genotype profiles of schizophrenic and control samples pointed to the schizophrenia-relevance of positive selections, with the schizophrenic samples showing weakened selections compared to the controls. These DD-genotypes were previously found to increase the expression of β2, especially its long isoform. Electrophysiological analysis showed that this long β2 isoform favored by the positive selections is more sensitive than the short isoform to the inhibition of GABAA receptor function by energy depletion. These findings represent the first demonstration of positive selection in a schizophrenia-associated gene.

NMR Analysis of Bovine tRNATrp CONFORMATION DEPENDENCE OF Mg2+ BINDING

NMR was used to study the solution structure of bovine tRNATrp hyperexpressed inEscherichia coli. With the use of 15N labeling and site-directed mutagenesis to assign overlapping resonances through the base pair replacement of U71A2 by G2C71, U27A43 by G27C43, and G12C23 by U12A23, the resonances of all 26 observable imino protons in the helical regions and in the tertiary interactions were assigned unambiguously by means of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear single quantum coherence methods. When the discriminator base A73and the G12C23 base pair on the D stem, two identity elements on bovine tRNATrp that are important for effective recognition by tryptophanyl-tRNA synthetase, were mutated to the ineffective forms of G73 and U12A23, respectively, NMR analysis revealed an important conformational change in the U12A23mutant but not in the G73 mutant molecule. Thus A73 appears to be directly recognized by tryptophanyl-tRNA synthetase, and G12C23 represents an important structural determinant. Mg2+ effects on the assigned resonances of imino protons allowed the identification of strong, medium, and weak Mg2+ binding sites in tRNATrp. Strong Mg2+ binding modes were associated with the residues G7, s4U8 (where s4U is 4-thiouridine), G12, and U52. The observations that G42 was associated with strong Mg2+ binding in only the U12A23mutant tRNATrp but not the wild type or G73mutant tRNATrp and that the G7, s4U8, G24, and G22imino protons are associated with a two-site Mg2+ binding mode in wild type and G73 mutant but only a one-site mode in the U12A23 mutant established the occurrence of conformational change in the U12A23 mutant tRNATrp. These observations also established the dependence of Mg2+ binding on tRNA conformation and the usefulness of Mg2+ binding sites as conformational probes. The thermal titration of tRNATrp in the presence and absence of 10 mm Mg2+ indicated that overall tRNATrp structure stability was increased by more than 15 °C by the presence of Mg2+.