Photobleaching of GFP-labeled H2AX in chromatin: H2AX has low diffusional mobility in the nucleus

The Ser-139 phosphorylated form of replacement histone H2AX (gamma-H2AX) is induced within large chromatin domains by double-strand DNA breaks (DSBs) in mammalian chromosomes. This modification is known to be important for the maintenance of chromosome stability. However, the mechanism of gamma-H2AX formation at DSBs and its subsequent elimination during DSB repair remains unknown. gamma-H2AX formation and elimination could occur by direct phosphorylation and dephosphorylation of H2AX in situ in the chromatin. Alternatively, H2AX molecules could be phosphorylated freely in the nucleus, diffuse into chromatin regions containing DSBs and then diffuse out after DNA repair. In this study we show that free histone H2AX can be efficiently phosphorylated in vitro by nuclear extracts and that free gamma-H2AX can be dephosphorylated in vitro by the mammalian protein phosphatase 1-alpha. We made N-terminal fusion constructs of H2AX with green fluorescent protein (GFP) and studied their diffusional mobility in transient and stable cell transfections. In the absence or presence of DSBs, only a small fraction of GFP-H2AX is redistributed after photobleaching, indicating that in vivo this histone is essentially immobile in chromatin. This suggests that gamma-H2AX formation in chromatin is unlikely to occur by diffusion of free histone and gamma-H2AX dephosphorylation may involve the mammalian protein phosphatase 1alpha.     

Cerebral Developmental Abnormalities in a Mouse with Systemic Pyruvate Dehydrogenase Deficiency

Pyruvate dehydrogenase (PDH) complex (PDC) deficiency is an inborn error of pyruvate metabolism causing a variety of neurologic manifestations. Systematic analyses of development of affected brain structures and the cellular processes responsible for their impairment have not been performed due to the lack of an animal model for PDC deficiency. METHODS: In the present study we investigated a murine model of systemic PDC deficiency by interrupting the X-linked Pdha1 gene encoding the α subunit of PDH to study its role on brain development and behavioral studies. RESULTS: Male embryos died prenatally but heterozygous females were born. PDC activity was reduced in the brain and other tissues in female progeny compared to age-matched control females. Immunohistochemical analysis of several brain regions showed that approximately 40% of cells were PDH⁻. The oxidation of glucose to CO₂ and incorporation of glucose-carbon into fatty acids were reduced in brain slices from 15 day-old PDC-deficient females. Histological analyses showed alterations in several structures in white and gray matters in 35 day-old PDC-deficient females. Reduction in total cell number and reduced dendritic arbors in Purkinje neurons were observed in PDC-deficient females. Furthermore, cell proliferation, migration and differentiation into neurons by newly generated cells were reduced in the affected females during pre- and postnatal periods. PDC-deficient mice had normal locomotor activity in a novel environment but displayed decreased startle responses to loud noises and there was evidence of abnormal pre-pulse inhibition of the startle reflex. CONCLUSIONS: The results show that a reduction in glucose metabolism resulting in deficit in energy production and fatty acid biosynthesis impairs cellular differentiation and brain development in PDC-deficient mice.     

Immunoaffinity enrichments followed by mass spectrometric detection for studying global protein tyrosine phosphorylation

Phosphorylation of protein tyrosine residues regulates important cell functions and is, when dysregulated, often crucially involved in oncogenesis. It is therefore important to develop and evaluate methods for identifying and studying tyrosine phosphorylated (P-Tyr) proteins. P-Tyr proteins are present at very low concentrations within cells, requiring highly selective enrichment methods to be detected. In this study, we applied immunoaffinity as enrichment step for P-Tyr proteins. Five selected anti-phosphotyrosine antibodies (monoclonal antibodies 4G10, PY100, PYKD1, 13F9 and one polyclonal antiserum) were evaluated with respect to their capability to enrich P-Tyr proteins from cell extracts of the K562 leukemia cell line. The enrichment resulted in the detection of a group of proteins that potentially were tyrosine-phosphorylated (putative P-Tyr proteins). High accuracy identification of actual P-Tyr sites were performed using a highly selective and sensitive liquid chromatography Fourier transform mass spectrometer (LC-FTMS) setup with complementary collision activated dissociation (CAD) and electron capture dissociation (ECD) fragmentations. 4G10 and PY100 antibodies recognized the greatest number of putative P-Tyr proteins in initial screening experiments and were therefore further evaluated and compared in immunoaffinity enrichment of both P-Tyr proteins and peptides. Using the 4G10 antibody for enrichment of proteins, we identified 459 putative P-Tyr proteins by MS. Out of these proteins, 12 were directly verified as P-Tyr proteins by MS analysis of the actual site. Using the PY100 antibody for enrichment of peptides, we detected 67 P-Tyr peptides (sites) and 89 putative P-Tyr proteins. Generally, enrichment at the peptide level made it difficult to reliably determine the identity of the proteins. In contrast, protein identification following immunoaffinity enrichment at the protein level gave greater sequence coverage and thus a higher confidence in the protein identification. By combining all available information, 40 proteins were identified as true P-Tyr proteins from the K562 cell line. In conclusion, this study showed that a combination of immunoaffinity enrichment using multiple antibodies of both intact and digested proteins in parallel experiments is required for best possible coverage of all possible P-Tyr proteins in a sample.     

Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes

A general purpose transformation vector, designated pPha-T1, was constructed for use with the diatom Phaeodactylum tricornutum Bohlin. This vector harbors the sh ble cassette for primary selection on medium containing the antibiotic zeocin, and a multiple cloning site flanked by the P. tricornutum fcp A promoter. pPha-T1 was used to establish the utility of three selectable marker genes and two reporter genes for P. tricornutum transformation. The nat and sat-1 genes confer resistance to the antibiotic nourseothricin, and npt II confers resistance to G418. Each of these genes was effective as a selectable marker for identifying primary transformants. These markers could also be used for dual selections in combination with the sh ble gene. The reporter genes uid A and gfp were also introduced into P. tricornutum using pPha-T1. Gus expression in some transformants reached 15 μg·μg⁻¹ of total soluble protein and permitted excellent cell staining, while GFP fluorescence was readily visible with standard fluorescence microscopy. The egfp gene, which has optimal codon usage for expression in human cells, was the only version of gfp that produced a strong fluorescent signal in P. tricornutum. The codon bias of the egfp gene is similar to that of P. tricornutum genes. This study suggests that codon usage has a significant effect on the efficient expression of reporter genes in P. tricornutum. The results presented here demonstrate that a variety of selectable markers and reporter genes can be expressed in P. tricornutum , enhancing the potential of this organism for exploring basic biological questions and industrial applications.