800 research outputs found
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Targeted gene disruption of the endogenous c-abl locus by homologous recombination with DNA encoding a selectable fusion protein
We have introduced a substitution mutation into the c-abl locus of murine embryonic stem cells by homologous recombination between exogenously added DNA and the endogenous gene. Model constructs were initially generated that consisted of a promoterless selectable neomycin resistance marker inserted into the v-abl gene of the complete Abelson murine leukemia virus genome, designed to be expressed either as a fusion protein or by translational restart. Tests of these viral genomes for transmission of v-abl and neo markers showed more stable coexpression in a protein fusion construct. The neo fusion was subcloned from this v-abl construct into a promoterless c-abl fragment, and the resulting DNA was used to transform embryonic stem cells. Direct screening of genomic DNAs showed that a high proportion of drug-resistant clones arose from homologous recombination into the endogenous c-abl locus
The Drosophila snr1 and brm Proteins are Related to Yeast SWI/SNF Proteins and are Components of a Large Protein Complex
During most of Drosophila development the regulation of homeotic gene transcription is controlled by two groups of regulatory genes, the trithorax group of activators and the Polycomb group of repressors. brahma (brm), a member of the trithorax group, encodes a protein related to the yeast SWI2/SNF2 protein, a subunit of a protein complex that assists sequence-specific activator proteins by alleviating the repressive effects of chromatin. To learn more about the molecular mechanisms underlying the regulation of homeotic gene transcription, we have investigated whether a similar complex exists in flies. We identified the Drosophila snr1 gene, a potential homologue of the yeast SNF5 gene that encodes a subunit of the yeast SWI/SNF complex. The snr1 gene is essential and genetically interacts with brm and trithorax (trx), suggesting cooperation in regulating homeotic gene transcription. The spatial and temporal patterns of expression of snr1 are similar to those of brm. The snr1 and brm proteins are present in a large (> 2 x 10(6) Da) complex, and they co-immunoprecipitate from Drosophila extracts. These findings provide direct evidence for conservation of the SWI/SNF complex in higher eucaryotes and suggest that the Drosophila brm/snr1 complex plays an important role in maintaining homeotic gene transcription during development by counteracting the repressive effects of chromatin
The Drosophila snr1 and brm Proteins are Related to Yeast SWI/SNF Proteins and are Components of a Large Protein Complex
During most of Drosophila development the regulation of homeotic gene transcription is controlled by two groups of regulatory genes, the trithorax group of activators and the Polycomb group of repressors. brahma (brm), a member of the trithorax group, encodes a protein related to the yeast SWI2/SNF2 protein, a subunit of a protein complex that assists sequence-specific activator proteins by alleviating the repressive effects of chromatin. To learn more about the molecular mechanisms underlying the regulation of homeotic gene transcription, we have investigated whether a similar complex exists in flies. We identified the Drosophila snr1 gene, a potential homologue of the yeast SNF5 gene that encodes a subunit of the yeast SWI/SNF complex. The snr1 gene is essential and genetically interacts with brm and trithorax (trx), suggesting cooperation in regulating homeotic gene transcription. The spatial and temporal patterns of expression of snr1 are similar to those of brm. The snr1 and brm proteins are present in a large (> 2 x 10(6) Da) complex, and they co-immunoprecipitate from Drosophila extracts. These findings provide direct evidence for conservation of the SWI/SNF complex in higher eucaryotes and suggest that the Drosophila brm/snr1 complex plays an important role in maintaining homeotic gene transcription during development by counteracting the repressive effects of chromatin
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ERK Phosphorylates p66shcA on Ser36 and Subsequently Regulates p27^kip1 Expression via the Akt-FOXO3a Pathway: Implication of p27^kip1 in Cell Response to Oxidative Stress
Mice deficient for p66shcA represent an animal model to link oxidative stress and aging. p66shcA is implicated in oxidative stress response and mitogenic signaling. Phosphorylation of p66shcA on Ser36 is critical for its function in oxidative stress response. Here we report the identification of ERK as the kinase phosphorylating p66shcA on Ser36. Activation of ERKs was necessary and sufficient for Ser36 phosphorylation. p66shcA interacted with ERK and was demonstrated to be a substrate for ERK, with Ser36 being the major phosphorylation site. Furthermore, in response to H2O2, inhibition of ERK activation repressed p66shcA-dependent phosphorylation of FOXO3a and the down-regulation of its target gene p27kip1. Down-regulation of p27 might promote cell survival, as p27 played a proapoptotic role in oxidative stress response. As a feedback regulation, Ser36 phosphorylated p66shcA attenuated H2O2-induced ERK activation, whereas p52/46shcA facilitated ERK activation, which required tyrosine phosphorylation of CH1 domain. p66shcA formed a complex with p52/46ShcA, which may provide a platform for efficient signal propagation. Taken together, the data suggest there exists an interplay between ERK and ShcA proteins, which modulates the expression of p27 and cell response to oxidative stress
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Embryonic Lethality in Mice Homozygous for a Targeted Disruption of the N-myc Gene
The N-myc gene encodes a putative transcription factor that is thought to function in the regulation of gene expression during cell differentiation and/or growth. To examine the role of N-myc during development, we have used targeted mutagenesis in embryonic stem cells to produce a mouse line that carries an N-myc null allele. Mice homozygous for the mutation died between 10.5 and 12.5 days of gestation. Histological analysis of mutant embryos revealed that organs and tissues expected at these stages of development were present. However, multiple defects were observed, primarily in tissues and organs that normally express N-myc. In particular, mutant hearts were underdeveloped, often retaining the S-shape more typical of 9-day-old embryos. In addition, cranial and spinal ganglia were reduced in size and/or cellularity. Most of the noted defects were more consistent with a role of N-myc in proliferation of precursor populations than with a block in differentiation per se, at least at these early stages. These results demonstrate that N-myc plays an essential role during development and clearly confirm that N-myc has a physiological function that is distinct from that of the other myc-family genes
Detection of Ly\beta auto-correlations and Ly\alpha-Ly\beta cross-correlations in BOSS Data Release 9
The Lyman- forest refers to a region in the spectra of distant quasars
that lies between the rest-frame Lyman- and Lyman- emissions.
The forest in this region is dominated by a combination of absorption due to
resonant Ly and Ly scattering. When considering the 1D Ly
forest in addition to the 1D Ly forest, the full statistical
description of the data requires four 1D power spectra: Ly and
Ly auto-power spectra and the Ly-Ly real and imaginary
cross-power spectra. We describe how these can be measured using an optimal
quadratic estimator that naturally disentangles Ly and Ly
contributions. Using a sample of approximately 60,000 quasar sight-lines from
the BOSS Data Release 9, we make the measurement of the one-dimensional power
spectrum of fluctuations due to the Ly resonant scattering. While we
have not corrected our measurements for resolution damping of the power and
other systematic effects carefully enough to use them for cosmological
constraints, we can robustly conclude the following: i) Ly power
spectrum and Ly-Ly cross spectra are detected with high
statistical significance; ii) the cross-correlation coefficient is
on large scales; iii) the Ly measurements are contaminated by the
associated OVI absorption, which is analogous to the SiIII contamination of the
Ly forest. Measurements of the Ly forest will allow extension of
the usable path-length for the Ly measurements while allowing a better
understanding of the physics of intergalactic medium and thus more robust
cosmological constraints.Comment: 26 pages, 10 figures; matches version accepted by JCA
Radiation-induced Assembly of Rad51 and Rad52 Recombination Complex Requires ATM and c-Abl
Cells from individuals with the recessive cancer-prone disorder ataxia telangiectasia (A-T) are hypersensitive to ionizing radiation (I-R). ATM (mutated in A-T) is a protein kinase whose activity is stimulated by I-R. c-Abl, a nonreceptor tyrosine kinase, interacts with ATM and is activated by ATM following I-R. Rad51 is a homologue of bacterial RecA protein required for DNA recombination and repair. Here we demonstrate that there is an I-R-induced Rad51 tyrosine phosphorylation, and this induction is dependent on both ATM and c-Abl. ATM, c-Abl, and Rad51 can be co-immunoprecipitated from cell extracts. Consistent with the physical interaction, c-Abl phosphorylates Rad51 in vitro and in vivo. In assays using purified components, phosphorylation of Rad51 by c-Abl enhances complex formation between Rad51 and Rad52, which cooperates with Rad51 in recombination and repair. After I-R, an increase in association between Rad51 and Rad52 occurs in wild-type cells but not in cells with mutations that compromise ATM or c-Abl. Our data suggest signaling mediated through ATM, and c-Abl is required for the correct post-translational modification of Rad51, which is critical for the assembly of Rad51 repair protein complex following I-R
Structural basis of suppression of host translation termination by Moloney Murine Leukemia Virus
Retroviral reverse transcriptase (RT) of Moloney murine leukemia virus (MoMLV) is expressed in the form of a large Gag–Pol precursor protein by suppression of translational termination in which the maximal efficiency of stop codon read-through depends on the interaction between MoMLV RT and peptidyl release factor 1 (eRF1). Here, we report the crystal structure of MoMLV RT in complex with eRF1. The MoMLV RT interacts with the C-terminal domain of eRF1 via its RNase H domain to sterically occlude the binding of peptidyl release factor 3 (eRF3) to eRF1. Promotion of read-through by MoMLV RNase H prevents nonsense-mediated mRNA decay (NMD) of mRNAs. Comparison of our structure with that of HIV RT explains why HIV RT cannot interact with eRF1. Our results provide a mechanistic view of how MoMLV manipulates the host translation termination machinery for the synthesis of its own proteins
Surface Zeta Potential and Diamond Seeding on Gallium Nitride Films.
The measurement of ζ potential of Ga-face and N-face gallium nitride has been carried out as a function of pH. Both of the faces show negative ζ potential in the pH range 5.5-9. The Ga-face has an isoelectric point at pH 5.5. The N-face shows a more negative ζ potential due to larger concentration of adsorbed oxygen. The ζ potential data clearly showed that H-terminated diamond seed solution at pH 8 will be optimal for the self-assembly of a monolayer of diamond nanoparticles on the GaN surface. The subsequent growth of thin diamond films on GaN seeded with H-terminated diamond seeds produced fully coalesced films, confirming a seeding density in excess of 1011 cm-2. This technique removes the requirement for a low thermal conduction seeding layer like silicon nitride on GaN
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