Disrupted phylogeographical microsatellite and chloroplast DNA patterns indicate a vicariance rather than long‐distance dispersal origin for the disjunct distribution of the Chilean endemic Dioscorea biloba (Dioscoreaceae) around the Atacama Desert

Aim The Chilean endemic Dioscorea biloba (Dioscoreaceae) is a dioecious geophyte that shows a remarkable 600 km north–south disjunction in the peripheral arid area of the Atacama Desert. Its restricted present‐day distribution and probable Neogene origin indicate that its populations have a history linked to that of the Atacama Desert, making this an ideal model species with which to investigate the biogeography of the region. Location Chile, Atacama Desert and peripheral arid area. Methods Two hundred and seventy‐five individuals from nine populations were genotyped for seven nuclear microsatellite loci, and plastid trnL–F and trnT–L sequences were obtained for a representative subset of these. Analyses included the estimation of genetic diversity and population structure through clustering, Bayesian and analysis of molecular variance analyses, and statistical parsimony networks of chloroplast haplotypes. Isolation by distance was tested against alternative dispersal hypotheses. Results Microsatellite markers revealed moderate to high levels of genetic diversity within populations, with those from the southern Limarí Valley showing the highest values and northern populations showing less exclusive alleles. Bayesian analysis of microsatellite data identified three genetic groups that corresponded to geographical ranges. Chloroplast phylogeography revealed no haplotypes shared between northern and southern ranges, and little haplotype sharing between the two neighbouring southern valleys. Dispersal models suggested the presence of extinct hypothetical populations between the southern and northern ranges. Main conclusions Our results are consistent with prolonged isolation of the northern and southern groups, mediated by the life‐history traits of the species. Significant isolation was revealed at both large and moderate distances as gene flow was not evident even between neighbouring valleys. Bayesian analyses of microsatellite and chloroplast haplotype diversity identified the southern area of Limarí as the probable area of origin of the species. Our data do not support recent dispersal of D. biloba from the southern range into Antofagasta, but indicate the fragmentation of an earlier wider range, concomitant with the Pliocene–Pleistocene climatic oscillations, with subsequent extinctions of the Atacama Desert populations and the divergence of the peripheral ones as a consequence of genetic drift.     

Manganese-54 accumulation by Chlorella spp., Daphnia magna and yellow perch (Perca flavescens)

Concentration factor and biological half-life of ⁵⁴Mn were determined in three species representing an ecologically and economically important food chain. Green algae (Chlorella spp.), Daphnia magna and yellow perch (Perca flavescens) were exposed to ⁵⁴Mn in water and assayed for ⁵⁴Mn uptake. Steady state concentration factors computed from the laboratory data for algae, Daphnia and perch were 4230, 17 000 and 11, respectively. Respective biological half-lives were 1.6, 1.2 and 8.3 days.     

MODY 2: Mutation identification and molecular ancestry in a Brazilian family

Maturity Onset Diabetes of the Young (MODY) is a heterogeneous group of genetic diseases characterized by a primary defect in insulin secretion and hyperglycemia, non-ketotic disease, monogenic autosomal dominant mode of inheritance, age at onset less than 25 years, and lack of auto-antibodies. It accounts for 2–5% of all cases of non-type 1 diabetes. MODY subtype 2 is caused by mutations in the glucokinase (GCK) gene. In this study, we sequenced the GCK gene of two volunteers with clinical diagnosis for MODY2 and we were able to identify four mutations including one for a premature stop codon (c.76C>T). Based on these results, we have developed a specific PCR-RFLP assay to detect this mutation and tested 122 related volunteers from the same family. This mutation in the GCK gene was detected in 21 additional subjects who also had the clinical features of this genetic disease. In conclusion, we identified new GCK gene mutations in a Brazilian family of Italian descendance, with one due to a premature stop codon located in the second exon of the gene. We also developed a specific assay that is fast, cheap and reliable to detect this mutation. Finally, we built a molecular ancestry model based on our results for the migration of individuals carrying this genetic mutation from Northern Italy to Brazil.     

Generation of a purely clonal defective interfering influenza virus

Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals.     

Structural Insight into Chromatin Recognition by Multiple Domains of the Tumor Suppressor RBBP1

Retinoblastoma-binding protein 1 (RBBP1) is involved in gene regulation, epigenetic regulation, and disease processes. RBBP1 contains five domains with DNA-binding or histone-binding activities, but how RBBP1 specifically recognizes chromatin is still unknown. An AT-rich interaction domain (ARID) in RBBP1 was proposed to be the key region for DNA-binding and gene suppression. Here, we first determined the solution structure of a tandem PWWP-ARID domain mutant of RBBP1 after deletion of a long flexible acidic loop L12 in the ARID domain. NMR titration results indicated that the ARID domain interacts with DNA with no GC- or AT-rich preference. Surprisingly, we found that the loop L12 binds to the DNA-binding region of the ARID domain as a DNA mimic and inhibits DNA binding. The loop L12 can also bind weakly to the Tudor and chromobarrel domains of RBBP1, but binds more strongly to the DNA-binding region of the histone H2A-H2B heterodimer. Furthermore, both the loop L12 and DNA can enhance the binding of the chromobarrel domain to H3K4me3 and H4K20me3. Based on these results, we propose a model of chromatin recognition by RBBP1, which highlights the unexpected multiple key roles of the disordered acidic loop L12 in the specific binding of RBBP1 to chromatin.     

Site-Specific Structural Variations Accompanying Tubular Assembly of the HIV-1 Capsid Protein

The 231-residue capsid (CA) protein of human immunodeficiency virus type 1 (HIV-1) spontaneously self-assembles into tubes with a hexagonal lattice that is believed to mimic the surface lattice of conical capsid cores within intact virions. We report the results of solid-state nuclear magnetic resonance (NMR) measurements on HIV-1 CA tubes that provide new information regarding changes in molecular structure that accompany CA self-assembly, local dynamics within CA tubes, and possible mechanisms for the generation of lattice curvature. This information is contained in site-specific assignments of signals in two- and three-dimensional solid-state NMR spectra, conformation-dependent ¹⁵N and ¹³C NMR chemical shifts, detection of highly dynamic residues under solution NMR conditions, measurements of local variations in transverse spin relaxation rates of amide ¹H nuclei, and quantitative measurements of site-specific ¹⁵N–¹⁵N dipole–dipole couplings. Our data show that most of the CA sequence is conformationally ordered and relatively rigid in tubular assemblies and that structures of the N-terminal domain (NTD) and the C-terminal domain (CTD) observed in solution are largely retained. However, specific segments, including the N-terminal β-hairpin, the cyclophilin A binding loop, the inter-domain linker, segments involved in intermolecular NTD–CTD interactions, and the C-terminal tail, have substantial static or dynamical disorder in tubular assemblies. Other segments, including the 3₁₀-helical segment in CTD, undergo clear conformational changes. Structural variations associated with curvature of the CA lattice appear to be localized in the inter-domain linker and intermolecular NTD–CTD interface, while structural variations within NTD hexamers, around local 3-fold symmetry axes, and in CTD–CTD dimerization interfaces are less significant.