In-gel probing of individual RNA conformers within a mixed population reveals a dimerization structural switch in the HIV-1 leader

Definitive secondary structural mapping of RNAs in vitro can be complicated by the presence of more than one structural conformer or multimerization of some of the molecules. Until now, probing a single structure of conformationally flexible RNA molecules has typically relied on introducing stabilizing mutations or adjusting buffer conditions or RNA concentration. Here, we present an in-gel SHAPE (selective 2′OH acylation analysed by primer extension) approach, where a mixed structural population of RNA molecules is separated by non-denaturing gel electrophoresis and the conformers are individually probed within the gel matrix. Validation of the technique using a well-characterized RNA stem-loop structure, the HIV-1 trans-activation response element, showed that authentic structure was maintained and that the method was accurate and highly reproducible. To further demonstrate the utility of in-gel SHAPE, we separated and examined monomeric and dimeric species of the HIV-1 packaging signal RNA. Extensive differences in acylation sensitivity were seen between monomer and dimer. The results support a recently proposed structural switch model of RNA genomic dimerization and packaging, and demonstrate the discriminatory power of in-gel SHAPE.     

Linkage disequilibrium between an allele at the dopamine D4 receptor locus and Tourette syndrome, by the transmission-disequilibrium test.

Dopaminergic abnormalities are implicated in the pathogenesis of Tourette syndrome (TS) and chronic multiple tics. We used the transmission-disequilibrium test (TDT) method to test for linkage disequilibrium between a specific allele (the seven-repeat allele (DRD4*7R) of the exon 3 VNTR polymorphic site) at the D4 dopamine receptor locus (DRD4) and expression of chronic multiple tics and TS. This particular allele had been shown in functional studies to have different binding properties compared with the other common alleles in this DRD4 polymorphic system. We studied 64 family trios (consisting of an affected person and two parents, at least one heterozygous for DRD4*7R), including 12 nuclear family trios and 52 trios from four large TS kindreds. The DRD4*7R allele was transmitted significantly more frequently than expected (chi 2 TDT ranging from 8.47 [P < .004] to 10.80 [P = .001], depending on breadth of disease definition and inclusion or exclusion of inferred genotypes). Confirmation of this finding will depend on either replication in other samples or the identification of a transmitted functional mutation within this sample.     

Synergies between site protection and agri‐environment schemes for the conservation of waders on lowland wet grasslands

Two conservation strategies have been put in place in Europe to address precipitous population declines of wading birds that breed on lowland wet grasslands. These are site protection and agri‐environment schemes (AESs) and the two are rarely compared, or their synergy assessed. Increasingly, efforts to recover populations of previously widespread species follow a landscape‐scale approach whereby habitat improvement takes place at key sites through partially overlapping protected area management and AESs. To investigate whether site protection and AESs improve the conservation status of breeding waders and how these interact, we partially repeated a 2002 survey of breeding waders on protected areas (nature reserves and Sites of Special Scientific Interest) and sites with wader‐specific AES options in England in 2009 and 2010. We then assessed the individual and combined effects of these delivery mechanisms on field occupancy, breeding density and population change of four species of declining wader (Northern Lapwing Vanellus vanellus, Common Snipe Gallinago gallinago, Eurasian Curlew Arquata numenius and Common Redshank Tringa totanus). Although results for Curlew differed from the other species, in general field occupancy was positively influenced by conservation delivery mechanisms, with the highest occupancy and breeding densities on land where site protection was combined with wader‐specific AES options. Field occupancy varied between different types of AES, with higher occupancy associated with higher‐level options in fields, particularly those on nature reserves. Outside nature reserves, the history of AES management did not influence wader populations, but within nature reserves and on fields that gained AES management between 2002 and 2009–2010, populations of Curlew and Snipe were more likely to have persisted and population change in Snipe and Lapwing was more positive. We conclude that the conservation of breeding waders will be most effective when site protection and AES management are combined on the same land. Using limited AES money to support management for breeding waders on, around and between the existing network of protected sites will protect remaining populations while presenting opportunities for population expansion in future.