Detection of DNA Double-Strand Breaks byγ-H2AX Immunodetection

DNA double-strand breaks (DSBs) are the most deleterious type of DNA damage and a cause of geneticinstability as they can lead to mutations, genome rearrangements, or loss of genetic material when notproperly repaired. Eukaryotes from budding yeast to mammalian cells respond to the formation of DSBswith the immediate phosphorylation of a histone H2A isoform. The modified histone, phosphorylated inserine 139 in mammals (S129 in yeast), is namedγ-H2AX. Detection of DSBs is of high relevance inresearch on DNA repair, aging, tumorigenesis, and cancer drug development, given the tight association ofDSBs with different diseases and its potential to kill cells. DSB levels can be obtained by measuring levels ofγ-H2AX in extracts of cell populations or by counting foci in individual nuclei. In this chapter sometechniques to detectγ-H2AX are described

The CBRB regulon: Promoter dissection reveals novel insights into the CbrAB expression network in Pseudomonas putida

CbrAB is a high ranked global regulatory system exclusive of the Pseudomonads that responds to carbon limiting conditions. It has become necessary to define the particular regulon of CbrB and discriminate it from the downstream cascades through other regulatory components. We have performed in vivo binding analysis of CbrB in P. putida and determined that it directly controls the expression of at least 61 genes; 20% involved in regulatory functions, including the previously identified CrcZ and CrcY small regulatory RNAs. The remaining are porines or transporters (20%), metabolic enzymes (16%), activities related to protein translation (5%) and orfs of uncharacterised function (38%). Amongst the later, we have selected the operon PP2810-13 to make an exhaustive analysis of the CbrB binding sequences, together with those of crcZ and crcY. We describe the implication of three independent non-palindromic subsites with a variable spacing in three different targets; CrcZ, CrcY and operon PP2810-13 in the CbrAB activation. CbrB is a quite peculiar σN—depen-dent activator since it is barely dependent on phosphorylation for transcriptional activation. With the depiction of the precise contacts of CbrB with the DNA, the analysis of the multi-merisation status and its dependence on other factors such as RpoN o IHF, we propose a model of transcriptional activation.Ministerio de Economía y Competitividad BIO2014-57545-

Spectroscopic characterisation of the stellar content of ultra diffuse galaxies

Understanding the peculiar properties of Ultra Diffuse Galaxies (UDGs) via spectroscopic analysis is a challenging task requiring very deep observations and exquisite data reduction. In this work we perform one of the most complete characterisations of the stellar component of UDGs to date using deep optical spectroscopic data from OSIRIS at GTC. We measure radial and rotation velocities, star formation histories (SFH) and mean population parameters, such as ages and metallicities, for a sample of five UDG candidates in the Coma cluster. From the radial velocities, we confirm the Coma membership of these galaxies. We find that their rotation properties, if detected at all, are compatible with dwarf-like galaxies. The SFHs of the UDG are dominated by old (~ 7 Gyr), metal-poor ([M/H] ~ -1.1) and alpha-enhanced ([Mg/Fe] ~ 0.4) populations followed by a smooth or episodic decline which halted ~ 2 Gyr ago, possibly a sign of cluster-induced quenching. We find no obvious correlation between individual SFH shapes and any UDG morphological properties. The recovered stellar properties for UDGs are similar to those found for DDO44, a local UDG analogue resolved into stars. We conclude that the UDGs in our sample are extended dwarfs whose properties are likely the outcome of both internal processes, such as bursty SFHs and/or high-spin haloes, as well as environmental effects within the Coma cluster.Comment: Accepted for publication in MNRA