Ataxia-telangiectasia mutated (ATM) is required for resistance to radiation-induced DNA breaks. Here we use chromatin immunoprecipitation to show that ATM also localizes to breaks associated with V(D)J recombination. ATM recruitment to the recombining locus correlates approximately with recruitment of the break-initiating factor RAG1 and precedes efficient break repair, consistent with localization of ATM to normal recombination intermediates. A product of ATM kinase activity, Ser 18-phosphorylated p53, was detected similarly at these breaks, arguing that ATM phosphorylates target proteins in situ. We suggest routine surveillance of intermediates in V(D)J recombination by ATM helps suppress potentially oncogenic translocations when repair fails. Ataxia-telangiectasia is characterized by cerebellar degeneration, immunodeficiency, and a high frequency of malignancy, usually lymphoid in origin (Morrell et al. 1986). The gene mutated in this disease (ataxia-telangiectasia mutated, or ATM) is required for resistance to DNA double-strand break (DSB) inducing agents such as ionizing radiation, and is an important trigger for the cellular response to DSBs (for review, see Rotman and Shiloh 1999). ATM may act as a primary sensor of DSBs, first binding directly to DSBs and consequently activating through phosphorylation several downstream effectors of the DNA damage response, including Nbs1, BRCA1, Chk2, and p53 (for review, see Durocher and Jackson 2001). For example, after treatment with ionizing radiation, phosphorylation of p53 at Ser 15 (Ser 18 in mice) in cells is largely ATM dependent (for review, see Giaccia and Kastan 1998) and is required for effective p53-dependent responses to this stimulus (Chao et al. 2000).In contrast to its important role in mediating resistance to exogenous DSB-inducing agents, genetic evidence for a role for ATM in repair of DSB intermediates in V(D)J recombination is less clear. V(D)J recombination is an essential step in the generation of a diverse repertoire of immunoglobulins and T-cell receptors (for review, see Gellert 1997). RAG1 and RAG2 proteins initiate V(D)J recombination by introducing DSBs precisely adjacent to recombination-targeting signals that flank segments of immunoglobulin and T-cell receptor coding sequence. Efficient resolution of broken species requires factors implicated in end-joining DSB repair, including Ku, XRCC4, ligase IV, and the ATM-related kinase DNA-PKcs, but not ATM. Cells from patients with AT support normal levels of V(D)J recombination using an extra-chromosomal substrate assay (Hsieh et al. 1993), and mature antigen receptor-bearing lymphocytes are readily observed in ATM-deficient mice (Barlow et al. 1996;Elson et al. 1996;Xu et al. 1996).Are V(D)J recombination intermediates recognized by ATM as DNA damage? The lack of requirement for ATM in V(D)J recombination might suggest that ATM is excluded from breaks associated with this pathway, possibly through masking of ends by RAG1 and RAG2, to avoid counter-productive apoptotic responses during this n...

BackgroundThe most common approach used in generating cell lines for the production of therapetic proteins relies on gene amplification induced by a drug resistance gene e. g., DHFR and glutamine synthetase. Practically, this results in screening large number of clones for the one that expresses high levels of the biologic in a stable manner. The inefficiency of mammalian vector systems to express proteins in a stable manner typically involves silencing of the exogenous gene resulting from modifications such as methylation of CpG DNA sequences, histone deacetylation and chromatin condensation. The use of un-methylated CpG island fragments from housekeeping genes referred to as UCOE (ubiquitous chromatin opening elements) in plasmid vectors is now well established for increased stability of transgene expression. However, few UCOE-promoter combinations have been studied to date and in this report we have tested 14 different combinations.FindingsIn this report we describe studies with two different UCOEs (the 1.5 Kb human RNP fragment and the 3.2 Kb mouse RPS3 fragment) in combination with various promoters to express a large protein (B domain deleted factor VIII; BDD-FVIII) in a production cell line, BHK21. We show here that there are differences in expression of BDD-FVIII by the different UCOE-promoter combinations in both attached and serum free suspension adapted cells. In all cases, the 1.5 Kb human RNP UCOE performed better in expressing BDD-FVIII than their corresponding 3.2 Kb mouse RPS3 UCOE. Surprisingly, in certain scenarios described here, expression from a number of promoters was equivalent or higher than the commonly used and industry standard human CMV promoter.ConclusionThis study indicates that certain UCOE-promoter combinations are better than others in expressing the BDD-FVIII protein in a stable manner in BHK21 cells. An empirical study such as this is required to determine the best combination of UCOE-promoter in a vector for a particular production cell line.

Compounds routinely used to increase the quality of life and combat disease undergo stringent potency and biosafety tests before approval. However, based on the outcome of ongoing research, new norms need to be effected to ensure that the compounds conform to biosafety at all target levels of activity. Whereas in vitro tests used to assess biosafety lack the potency and the translational attribute of a whole animal, mammalian preclinical models are expensive and time exhaustive. Zebrafish (Danio rerio) has emerged as an attractive alternative for biosafety studies due to its small size, genetics, breeding capabilities, and most importantly, similarity at the molecular and physiological levels with humans. It has been used extensively for testing various forms of toxicity, including developmental toxicity, cardiotoxicity, nephrotoxicity, and hepatotoxicity. We review here the utility of zebrafish as a powerful, sensitive, quantitative, noninvasive, and high-throughput whole-animal assay to screen for toxicity. Different forms of toxicity will be discussed briefly before we highlight the present state of genotoxicity study in zebrafish. This review, a first in this research area, will serve as a comprehensive introduction to the field of genotoxicity assay using zebrafish, a nascent but promising field that assays compounds for DNA damage. We also discuss possible approaches that could potentially be pursued to overcome some of the shortcomings in current genotoxic studies.

No abstract

Recombinant proteins can be targeted to the Escherichia coli periplasm by fusing them to signal peptides. The popular pET vectors facilitate fusion of target proteins to the PelB signal. A systematic comparison of the PelB signal with native E. coli signal peptides for recombinant protein expression and periplasmic localization is not reported. We chose the Bacillus stearothermophilus maltogenic amylase (MA), an industrial enzyme widely used in the baking and brewing industry, as a model protein and analyzed the competence of seven, codon-optimized, E. coli signal sequences to translocate MA to the E. coli periplasm compared to PelB. MA fusions to three of the signals facilitated enhanced periplasmic localization of MA compared to the PelB fusion. Interestingly, these three fusions showed greatly improved MA yields and between 18- and 50-fold improved amylase activities compared to the PelB fusion. Previously, non-optimal codon usage in native E. coli signal peptide sequences has been reported to be important for protein stability and activity. Our results suggest that E. coli signal peptides with optimal codon usage could also be beneficial for heterologous protein secretion to the periplasm. Moreover, such fusions could even enhance activity rather than diminish it. This effect, to our knowledge has not been previously documented. In addition, the seven vector platform reported here could also be used as a screen to identify the best signal peptide partner for other recombinant targets of interest.