DNaseI Hypersensitivity and Ultraconservation Reveal Novel, Interdependent Long-Range Enhancers at the Complex Pax6 Cis-Regulatory Region

The PAX6 gene plays a crucial role in development of the eye, brain, olfactory system and endocrine pancreas. Consistent with its pleiotropic role the gene exhibits a complex developmental expression pattern which is subject to strict spatial, temporal and quantitative regulation. Control of expression depends on a large array of cis-elements residing in an extended genomic domain around the coding region of the gene. The minimal essential region required for proper regulation of this complex locus has been defined through analysis of human aniridia-associated breakpoints and YAC transgenic rescue studies of the mouse smalleye mutant. We have carried out a systematic DNase I hypersensitive site (HS) analysis across 200 kb of this critical region of mouse chromosome 2E3 to identify putative regulatory elements. Mapping the identified HSs onto a percent identity plot (PIP) shows many HSs correspond to recognisable genomic features such as evolutionarily conserved sequences, CpG islands and retrotransposon derived repeats. We then focussed on a region previously shown to contain essential long range cis-regulatory information, the Pax6 downstream regulatory region (DRR), allowing comparison of mouse HS data with previous human HS data for this region. Reporter transgenic mice for two of the HS sites, HS5 and HS6, show that they function as tissue specific regulatory elements. In addition we have characterised enhancer activity of an ultra-conserved cis-regulatory region located near Pax6, termed E60. All three cis-elements exhibit multiple spatio-temporal activities in the embryo that overlap between themselves and other elements in the locus. Using a deletion set of YAC reporter transgenic mice we demonstrate functional interdependence of the elements. Finally, we use the HS6 enhancer as a marker for the migration of precerebellar neuro-epithelium cells to the hindbrain precerebellar nuclei along the posterior and anterior extramural streams allowing visualisation of migratory defects in both pathways in Pax6(Sey/Sey) mice

Pyridylbenzamidines: versatile ligands for palladium(II)

Recently, we have demonstrated that in copolymerization reactions palladium complexes with nonsymmetric\uf020\uf061-diimines generate more productive catalysts than those having the corresponding symmetric counterparts, highlighting the positive effect of an unbalance in the steric and electronic properties of the N-donor atoms.1 With the aim to verify the more general validity of this principle, we have now studied bidentate N-donor ligands belonging to the family of pyridylbenzamidines (Figure). While the two molecules with R1 = H were reported in literature,2 the N-methyl substituted derivative is new. The NMR characterization of the molecules pointed out the presence of dynamic phenomena in solution, due to the interconversion of several isomers, including tautomers. The coordination chemistry to Pd(II) was studied on [Pd(cod)(CH3)Cl] and [Pd(cod)(CH3)(CH3CN)][PF6]: depending on the precursor and on the ligand, different coordination compounds were obtained, indicating that these molecules can act both as mono- and bidentate ligands. None of the isolated complexes generated active catalysts either for ethylene homopolymerization or for ethylene/methyl acrylate copolymerization. The deactivation pathway was unraveled

The method of the Italian network POLLnet for counting and evaluating the concentration of airborne particles in a daily sample

Airborne pollen monitoring is of concern for health purposes as well as for studies on climate changes and biodiversity. The reliability of data estimates depends on the accuracy and precision of pollen counts (Gottardini et al., 2008). An important aspect of the microscope analysis, heavily influencing this reliability, is the choice of the minimal surface to be read on a microscope slide. At the moment, operators seem to argue among 10%, 15% and 20% of the target surface. The Italian network POLLnet searched a criterion both on experimental and statistics basis. For this purpose, three microscope slides, with low, medium and high amounts of particles, were read by twenty experienced aerobiologists. Each slide was analyzed on 100% of target surface, as well as 10%, 15% and 20%. The statistic approach applied the Poisson distribution to search the minimal surface to examine in order to find at least 1 pollen grain if on the total target sample there are 5 grains, or 0.5 particles/m3 that is the threshold concentration for the detection of most of the monitored families. This study confirmed that the variability of data decreases as the examined area increases. In particular, when 10% of the target area is examined, up to 45% of the pollen species can escape the analysis; when the slides were examined on the wider areas (15% and 20%), this lost of taxa decreased up to 15%. Also, the repeatability and the reproducibility of the analysis varied significantly on the basis of the percentages of the target area examined. Moreover, the statistic approach indicated that 14% is the minimal area to examine to find at least 1 airborne particle on a total of 5, with a confidence of 50%. Even if the experimental data suggest that the ideal surface would be at least 20%, both the experimental and the statistical approach indicate that adopting as a minimum reading limit 15% of the sampling surface is still acceptable