TAF8 regions important for TFIID lobe B assembly or for TAF2 interactions are required for embryonic stem cell survival.

The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. TFIID is composed of three lobes, named A, B, and C. A 5TAF core complex can be assembled in vitro constituting a building block for the further assembly of either lobe A or B in TFIID. Structural studies showed that TAF8 forms a histone fold pair with TAF10 in lobe B and participates in connecting lobe B to lobe C. To better understand the role of TAF8 in TFIID, we have investigated the requirement of the different regions of TAF8 for the in vitro assembly of lobe B and C and the importance of certain TAF8 regions for mouse embryonic stem cell (ESC) viability. We have identified a region of TAF8 distinct from the histone fold domain important for assembling with the 5TAF core complex in lobe B. We also delineated four more regions of TAF8 each individually required for interacting with TAF2 in lobe C. Moreover, CRISPR/Cas9-mediated gene editing indicated that the 5TAF core-interacting TAF8 domain and the proline-rich domain of TAF8 that interacts with TAF2 are both required for mouse embryonic stem cell survival. Thus, our study defines distinct TAF8 regions involved in connecting TFIID lobe B to lobe C that appear crucial for TFIID function and consequent ESC survival.

Differentiation of the human PAX7-positive myogenic precursors/satellite cell lineage in vitro.

Satellite cells (SC) are muscle stem cells that can regenerate adult muscles upon injury. Most SC originate from PAX7+ myogenic precursors set aside during development. Although myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we report the generation of human induced pluripotent stem cell (iPSC) reporter lines in which fluorescent proteins have been introduced into the PAX7 and MYOG loci. We use single cell RNA sequencing to analyze the developmental trajectory of the iPSC-derived PAX7+ myogenic precursors. We show that the PAX7+ cells generated in culture can produce myofibers and self-renew in vitro and in vivo Together, we demonstrate that cells exhibiting characteristics of human fetal satellite cells can be produced in vitro from iPSC, opening interesting avenues for muscular dystrophy cell therapy. This work provides significant insights into the development of the human myogenic lineage.

Modelling geochemical and kinetic processes involved in lead (Pb) remobilization during resuspension events of contaminated sediments.

The objective of this paper is to present a model simulating and predicting the exchange kinetics of lead (Pb) between contaminated sediments and water during resuspension events potentially occurring in reservoirs. We developed an innovative model that combines thermodynamic speciation of particulate surfaces (oxides and Particulate Organic Carbon (POC)), thermodynamic Pb speciation in water, and kinetic modelling of exchanges between free Pb and particulate sites (i.e., dissolution of a carbonate carrier phase, adsorption/coprecipitation and desorption/dissolution to/from oxides, and adsorption and desorption/degradation to/from particulate organic particles). We used results from laboratory resuspension experiments performed on sediments from three contaminated dam reservoirs to calibrate a new chemical speciation model. Uptake and release processes to/from sediments were found to be controlled by at least two successive reactions that are associated with two particulate pools (here oxides and POC). Kinetic adsorption and desorption rates were calibrated for seven experimental conditions. Variability in kinetic rates allowed evaluation of the effect of the solid-to-liquid ratio and sediment origin on exchange kinetics at the water-particle interface. The kinetic release of dissolved Pb by desorption or dissolution from the oxides was reproduced almost identically between the experiments, regardless of the solid-to-liquid ratio or sediment origin. Long-term readsorption on POC sites is more variable, even if ranges of variation in the adsorption and desorption kinetic rates related to POC remain limited, considering that tested sediments vary significantly. CAPSULE: A kinetic model simulating the dynamics of lead (Pb) during sediment resuspension was developed and calibrated to laboratory experiments performed on three contaminated sediments.

Co-translational assembly of mammalian nuclear multisubunit complexes.

Cells dedicate significant energy to build proteins often organized in multiprotein assemblies with tightly regulated stoichiometries. As genes encoding subunits assembling in a multisubunit complex are dispersed in the genome of eukaryotes, it is unclear how these protein complexes assemble. Here, we show that mammalian nuclear transcription complexes (TFIID, TREX-2 and SAGA) composed of a large number of subunits, but lacking precise architectural details are built co-translationally. We demonstrate that dimerization domains and their positions in the interacting subunits determine the co-translational assembly pathway (simultaneous or sequential). The lack of co-translational interaction can lead to degradation of the partner protein. Thus, protein synthesis and complex assembly are linked in building mammalian multisubunit complexes, suggesting that co-translational assembly is a general principle in mammalian cells to avoid non-specific interactions and protein aggregation. These findings will also advance structural biology by defining endogenous co-translational building blocks in the architecture of multisubunit complexes.

How to assess trace elements bioavailability for benthic organisms in lowly to moderately contaminated coastal sediments?

The bioavailability of trace elements (As, Cd, Co, Cr, Cu, Hg, Ni, Pb, Zn) in lowly to moderately contaminated coastal sediments from the Berre lagoon, France, was assessed by comparing their potentially bioavailable concentrations and bioaccumulated concentrations in the polychaete Alitta succinea. No linear correlations were observed contrarily to what is generally observed in similar works in areas with highly contaminated sediment. Correlations between trace and major elements (Fe, Ca, S, Mg, P, Al) in Alitta succinea tissues and their distribution in organism tissues show that, in such lowly to moderately contaminated sediments, biological variabilities should be considered. Normalization procedures allow to take into account these variabilities and to identify that sediment contamination is partly involved in the benthic ecosystem degradation of the Berre lagoon. Alitta succinea cannot be used as relevant bioindicator for Zn and Co bioavailability in sediment, since these elements are regulated by this organism.

Homozygous TAF8 mutation in a patient with intellectual disability results in undetectable TAF8 protein, but preserved RNA polymerase II transcription.

The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. In a child with intellectual disability, mild microcephaly, corpus callosum agenesis and poor growth, we identified a homozygous splice-site mutation in TAF8 (NM_138572.2: c.781-1G > A). Our data indicate that the patient's mutation generates a frame shift and an unstable TAF8 mutant protein with an unrelated C-terminus. The mutant TAF8 protein could not be detected in extracts from the patient's fibroblasts, indicating a loss of TAF8 function and that the mutation is most likely causative. Moreover, our immunoprecipitation and proteomic analyses show that in patient cells only partial TAF complexes exist and that the formation of the canonical TFIID is impaired. In contrast, loss of TAF8 in mouse embryonic stem cells and blastocysts leads to cell death and to a global decrease in Pol II transcription. Astonishingly however, in human TAF8 patient cells, we could not detect any cellular phenotype, significant changes in genome-wide Pol II occupancy and pre-mRNA transcription. Thus, the disorganization of the essential holo-TFIID complex did not affect global Pol II transcription in the patient's fibroblasts. Our observations further suggest that partial TAF complexes, and/or an altered TFIID containing a mutated TAF8, could support human development and thus, the absence of holo-TFIID is less deleterious for transcription than originally predicted.

Recapitulating early development of mouse musculoskeletal precursors of the paraxial mesoderm in vitro.

Body skeletal muscles derive from the paraxial mesoderm, which forms in the posterior region of the embryo. Using microarrays, we characterize novel mouse presomitic mesoderm (PSM) markers and show that, unlike the abrupt transcriptome reorganization of the PSM, neural tube differentiation is accompanied by progressive transcriptome changes. The early paraxial mesoderm differentiation stages can be efficiently recapitulated in vitro using mouse and human pluripotent stem cells. While Wnt activation alone can induce posterior PSM markers, acquisition of a committed PSM fate and efficient differentiation into anterior PSM Pax3+ identity further requires BMP inhibition to prevent progenitors from drifting to a lateral plate mesoderm fate. When transplanted into injured adult muscle, these precursors generated large numbers of immature muscle fibers. Furthermore, exposing these mouse PSM-like cells to a brief FGF inhibition step followed by culture in horse serum-containing medium allows efficient recapitulation of the myogenic program to generate myotubes and associated Pax7+ cells. This protocol results in improved in vitro differentiation and maturation of mouse muscle fibers over serum-free protocols and enables the study of myogenic cell fusion and satellite cell differentiation.

The WHHERE coactivator complex is required for retinoic acid-dependent regulation of embryonic symmetry.

Bilateral symmetry is a striking feature of the vertebrate body plan organization. Vertebral precursors, called somites, provide one of the best illustrations of embryonic symmetry. Maintenance of somitogenesis symmetry requires retinoic acid (RA) and its coactivator Rere/Atrophin2. Here, using a proteomic approach we identify a protein complex, containing Wdr5, Hdac1, Hdac2 and Rere (named WHHERE), which regulates RA signaling and controls embryonic symmetry. We demonstrate that Wdr5, Hdac1, and Hdac2 are required for RA signaling in vitro and in vivo. Mouse mutants for Wdr5 and Hdac1 exhibit asymmetrical somite formation characteristic of RA-deficiency. We also identify the Rere-binding histone methyltransferase Ehmt2/G9a, as a RA coactivator controlling somite symmetry. Upon RA treatment, WHHERE and Ehmt2 become enriched at RA target genes to promote RNA polymerase II recruitment. Our work identifies a protein complex linking key epigenetic regulators acting in the molecular control of embryonic bilateral symmetry.Retinoic acid (RA) regulates the maintenance of somitogenesis symmetry. Here, the authors use a proteomic approach to identify a protein complex of Wdr5, Hdac1, Hdac2 that act together with RA and coactivator Rere/Atrophin2 and a histone methyltransferase Ehmt2 to regulate embryonic symmetry.

A no-stop mutation in MAGEB4 is a possible cause of rare X-linked azoospermia and oligozoospermia in a consanguineous Turkish family.

PURPOSE: The purpose of this study was to identify mutations that cause non-syndromic male infertility using whole exome sequencing of family cases. METHODS: We recruited a consanguineous Turkish family comprising nine siblings with male triplets; two of the triplets were infertile as well as one younger infertile brother. Whole exome sequencing (WES) performed on two azoospermic brothers identified a mutation in the melanoma antigen family B4 (MAGEB4) gene which was confirmed via Sanger sequencing and then screened for on control groups and unrelated infertile subjects. The effect of the mutation on messenger RNA (mRNA) and protein levels was tested after in vitro cell transfection. Structural features of MAGEB4 were predicted throughout the conserved MAGE domain. RESULTS: The novel single-base substitution (c.1041A>T) in the X-linked MAGEB4 gene was identified as a no-stop mutation. The mutation is predicted to add 24 amino acids to the C-terminus of MAGEB4. Our functional studies were unable to detect any effect either on mRNA stability, intracellular localization of the protein, or the ability to homodimerize/heterodimerize with other MAGE proteins. We thus hypothesize that these additional amino acids may affect the proper protein interactions with MAGEB4 partners. CONCLUSION: The whole exome analysis of a consanguineous Turkish family revealed MAGEB4 as a possible new X-linked cause of inherited male infertility. This study provides the first clue to the physiological function of a MAGE protein.

KAT2A/KAT2B-targeted acetylome reveals a role for PLK4 acetylation in preventing centrosome amplification.

Lysine acetylation is a widespread post-translational modification regulating various biological processes. To characterize cellular functions of the human lysine acetyltransferases KAT2A (GCN5) and KAT2B (PCAF), we determined their acetylome by shotgun proteomics. One of the newly identified KAT2A/2B substrate is polo-like kinase 4 (PLK4), a key regulator of centrosome duplication. We demonstrate that KAT2A/2B acetylate the PLK4 kinase domain on residues K45 and K46. Molecular dynamics modelling suggests that K45/K46 acetylation impairs kinase activity by shifting the kinase to an inactive conformation. Accordingly, PLK4 activity is reduced upon in vitro acetylation of its kinase domain. Moreover, the overexpression of the PLK4 K45R/K46R mutant in cells does not lead to centrosome overamplification, as observed with wild-type PLK4. We also find that impairing KAT2A/2B-acetyltransferase activity results in diminished phosphorylation of PLK4 and in excess centrosome numbers in cells. Overall, our study identifies the global human KAT2A/2B acetylome and uncovers that KAT2A/2B acetylation of PLK4 prevents centrosome amplification.

Convergent Signaling Pathways Controlled by LRP1 (Receptor-related Protein 1) Cytoplasmic and Extracellular Domains Limit Cellular Cholesterol Accumulation.

The low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitously expressed cell surface receptor that protects from intracellular cholesterol accumulation. However, the underlying mechanisms are unknown. Here we show that the extracellular (α) chain of LRP1 mediates TGFß-induced enhancement of Wnt5a, which limits intracellular cholesterol accumulation by inhibiting cholesterol biosynthesis and by promoting cholesterol export. Moreover, we demonstrate that the cytoplasmic (ß) chain of LRP1 suffices to limit cholesterol accumulation in LRP1(-/-) cells. Through binding of Erk2 to the second of its carboxyl-terminal NPXY motifs, LRP1 ß-chain positively regulates the expression of ATP binding cassette transporter A1 (ABCA1) and of neutral cholesterol ester hydrolase (NCEH1). These results highlight the unexpected functions of LRP1 and the canonical Wnt5a pathway and new therapeutic potential in cholesterol-associated disorders including cardiovascular diseases.

Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy.

During embryonic development, skeletal muscles arise from somites, which derive from the presomitic mesoderm (PSM). Using PSM development as a guide, we establish conditions for the differentiation of monolayer cultures of mouse embryonic stem (ES) cells into PSM-like cells without the introduction of transgenes or cell sorting. We show that primary and secondary skeletal myogenesis can be recapitulated in vitro from the PSM-like cells, providing an efficient, serum-free protocol for the generation of striated, contractile fibers from mouse and human pluripotent cells. The mouse ES cells also differentiate into Pax7(+) cells with satellite cell characteristics, including the ability to form dystrophin(+) fibers when grafted into muscles of dystrophin-deficient mdx mice, a model of Duchenne muscular dystrophy (DMD). Fibers derived from ES cells of mdx mice exhibit an abnormal branched phenotype resembling that described in vivo, thus providing an attractive model to study the origin of the pathological defects associated with DMD.

Using DET and DGT probes (ferrihydrite and titanium dioxide) to investigate arsenic concentrations in soil porewater of an arsenic-contaminated paddy field in Bangladesh.

Arsenic concentration in the pore water of paddy fields (Csoln) irrigated with arsenic-rich groundwater is a key parameter in arsenic uptake by rice. Pore water extracts from cores and in situ deployment of DET and DGT probes were used to measure the arsenic concentration in the pore water. Ferrihydrite (Fe) and titanium dioxide (Ti) were used as DGT binding agents. Six sampling events during different growing stages of the rice, inducing different biogeochemical conditions, were performed in one rice field. A time series of DGT experiments allow the determination of an in situ arsenic diffusion coefficient in the diffusive gel (3.34×10(-6) cm(2) s(-1)) needed to calculate the so-called CDGT(Fe) and CDGT(Ti) concentrations. Over 3 days of a given sampling event and for cores sampled at intervals smaller than 50 cm, great variability in arsenic Csoln concentrations between vertical profiles was observed, with maxima of concentrations varying from 690 to 2800 µg L(-1). Comparisons between arsenic measured Csol and CDET and calculated CDGT(Fe) and CDGT(Ti) concentrations show either, in a few cases, roughly similar vertical profiles, or in other cases, significantly different profiles. An established iron oxyhydroxide precipitation in the DET gel may explain why measured arsenic CDET concentrations occasionally exceeded Csoln. The large spread in results suggests limitations to the use of DET and type of DGT probes used here for similarly representing the spatio-temporal variations of arsenic content in soil pore water in specific environmental such as paddy soils.

Bioavailability of copper in contaminated sediments assessed by a DGT approach and the uptake of copper by the aquatic plant Myriophyllum aquaticum.

The assessment of the potentially harmful effects of metals on biota depends on the speciation and bioavailability of the metals. In the present study, the authors investigated Cu accumulation and toxicity in the aquatic plant Myriophyllum aquaticum after exposure to artificial sediments varying in peat or ferric hydroxide content and spiked with Cu (5-200 mg kg(-1)). Modeling of the kinetic diffusive gradient in thin film (DGT) measurements revealed fast and slow Cu resupply from the solid phase for sediment formulated with and without peat, respectively. Myriophyllum aquaticum proved to be sensitive to Cu, as the Cu accumulation and growth differed depending on the sediment composition and Cu concentration. Comparing the Cu accumulation in M. aquaticum with total dissolved concentration, free concentration, and concentration in solution derived from DGT measurements (CDGT), Cu concentrations revealed that CDGT concentrations were a better predictor of accumulation than the others. However, the relatively weak correlation observed (r(2) = 0.6) and the fact that plant uptake does not increase proportionally to DGT fluxes suggest that Cu uptake in plants was not diffusion limited. Thus, the free Cu concentrations near the root surface were sufficient to meet the plant's demand during the experiment. Furthermore, labile complexes that continuously resupply the Cu(2+) pool may also contribute to the concentrations available for plant uptake. In the range of Cu concentrations investigated in the present study, saturation of uptake processes as well as toxicity are considered responsible for the poor DGT prediction of plant uptake.

Speciation and bioavailability of dissolved copper in different freshwaters: comparison of modelling, biological and chemical responses in aquatic mosses and gammarids.

Biological and chemical measurements were performed in mesocosms to investigate the bioavailability of copper, with a greater emphasis on the effects of competing ions and copper speciation. Measurements were achieved in three different natural waters for two aquatic species (Gammarus pulex and Fontinalis antipyretica) along a copper gradient concentration: natural concentration, spiked at 5 and 15 µg L(-1). Aquatic mosses exhibited high enrichment rates that were above the background levels compared to gammarids. The accumulation of copper in F. antipyretica is better correlated to the weakly complexed copper concentrations measured using differential pulse anodic stripping voltammetry (DPASV) and diffusive gradient in thin film (DGT) than to the free copper concentration measured using an ion selective electrode (ISE). In unspiked natural waters, the presence of dissolved organic ligands strongly controls the metal speciation and consequently largely minimised the impact of competing cations on the accumulation of Cu in mosses. Furthermore, the BioMet Biotic Ligand Model (BLM) successfully describes the site-specific copper bioaccumulation for the freshwater mosses studied. However, the comparison of the results with a previous study appears to indicate that the adsorption/desorption of Cu in mosses is impacted by seasons. This highlights a limit of the BioMet model in which the physiological state of aquatic organisms is not considered. No toxic effect of Cu exposure on lipid peroxidation was observed in the mosses and gammarids regardless of the site and the concentration considered. However, the oxidative stress measured in the mosses via their guaiacol peroxidase (GPX) activity increased in the case where internalised Cu reached maximal values, which suggests a threshold effect on the GPX activity.

DGT as surrogate of biomonitors for predicting the bioavailability of copper in freshwaters: an ex situ validation study.

The present report is the companion study of our previous study in which we investigated the impact of the dissolved organic matter, water cationic composition and pH on the bioavailability and the bioaccumulation of copper (Cu) in aquatic mosses (Fontinalis antipyretica). The impact had been assessed under laboratory controlled conditions and modelled using a two-compartment model calibrated under a wide range of water compositions (Ferreira et al., 2008, 2009). Herein are reported the validation stage of the abovementioned approach for contrasted geochemical field conditions. Experiments were performed with aquatic mosses that were exposed for 7d to two nominal Cu concentrations (5 and 15µgL(-1)) in a flow-through field microcosm supplied with four contrasting natural waters. At the end of the exposure period, a 6-fold difference in the bioaccumulated Cu contamination levels was found among the four deployment sites, suggesting a significant control of the water quality on the metal bioaccumulation by aquatic mosses. In parallel, the so-called 'labile' Cu concentration for the same four field conditions was determined using a DGT device (Diffusive Gradient in Thin film). By coupling these DGT measurements and a cation competition model involving Ca(2+), Mg(2+), Na(+) and H(+), the time-dependent Cu concentrations in aquatic mosses were predicted; these simulation results were compared to the actual bioaccumulation of Cu in mosses. We found that any bioaccumulation model that ignores water characteristics is not suitable to predict the Cu accumulation by aquatic mosses under various water quality conditions. Instead, we found that our approach integrating DGT measurements and cationic composition was able to reproduce the Cu bioaccumulation kinetics by aquatic mosses for a wide range of water quality conditions. In conclusion, the DGT approach was demonstrated to be a dynamic in situ measuring technique that can be used as a surrogate of bioindicators if the cationic correction is taken into account.

Temporally controlled targeted somatic mutagenesis in mouse eye pigment epithelium.

To generate temporally controlled site-specific somatic mutations in the mouse eye pigment epithelium, we generated a TRP1-Cre-ER(T2) transgenic mouse line that expresses the tamoxifen-dependent Cre-ER(T2) recombinase under the control of the tyrosinase-related protein 1 (TRP1) promoter. Cre-ER(T2) transcripts were readily detected in the retinal pigment epithelium (RPE), and tamoxifen treatment of adult TRP1-Cre-ER(T2) transgenic mice induced efficient excision of floxed DNA in patches of RPE cells, in numerous epithelial cells of the iris and ciliary body, and in very few cells of the neural retina. Importantly, no excision was detected in any cells in the absence of tamoxifen treatment. Thus, the TRP1-Cre-ER(T2) mouse line provides a powerful tool to study in vivo gene functions in the mouse eye pigment epithelium.

Genotoxicity of sediment extracts of the Berre lagoon (France).

To evaluate the genotoxic risk that contaminated sediment could constitute for benthic organisms, three contaminated (VA, VC and VN) and one uncontaminated (RN) sediment samples were collected in the Berre lagoon (France). Potentially bioavailable contaminants in sediments were obtained using sediment extraction with synthetic seawater adjusted to pH 4 or pH 6, simulating the range of pH prevailing in the digestive tract of benthic organisms. The genotoxic activities of these extracts were evaluated by three short-term bioassays: the Salmonella mutagenicity test using the Salmonella typhimurium strain TA102, the alkaline comet assay and the micronucleus assay on the Chinese Hamster Ovary cells CHO-K1. Results of the Salmonella mutagenicity assay detected a mutagenic response for RN extract at pH 6, and for VA extract at pH 4. Results of the comet and micronucleus assays detected low genotoxic/clastogenic activities for VA and VC extracts at pH 6 and higher activities for RN, VA and VC extracts at pH 4. To identify if metals (Al, Fe, Mn, As, Cd, Co, Cr, Cu, Hg, Ni, Pb and Zn) were involved in these genotoxic activities, their concentrations were determined in the extracts, and their speciation was assessed by thermodynamic calculations. Results showed that extracts from sites VA, VC and VN generally presented the highest trace metal contents for both extractants, while the site RN presented lower trace metal contents but the highest Fe and Mn contents. Thermodynamic calculations indicated that Fe, Mn, As and in a lower extend Co, Ni and Zn were mainly present under free forms in extracts, and were consequently, more likely able to induce a genotoxic effect. Results globally showed no correspondence between free metal contents and genotoxic activities. They suggested that these positive results could be due to uncharacterized compounds, acting as direct genotoxic agents or enhancing the genotoxic properties of analyzed metals.

RSK2 signaling in brain habenula contributes to place aversion learning.

RSK2 is a Ser/Thr kinase acting in the Ras/MAPK pathway. Rsk2 gene deficiency leads to the Coffin-Lowry Syndrome, notably characterized by cognitive deficits. We found that mrsk2 knockout mice are unable to associate an aversive stimulus with context in a lithium-induced conditioned place aversion task requiring both high-order cognition and emotional processing. Virally mediated shRNA-RSK2 knockdown in the habenula, whose involvement in cognition is receiving increasing attention, also ablated contextual conditioning. RSK2 signaling in the habenula, therefore, is essential for this task. Our study reveals a novel role for RSK2 in cognitive processes and uncovers the critical implication of an intriguing brain structure in place aversion learning.

Vectors for recombinational cloning and gene expression in mammalian cells using modified vaccinia virus Ankara.

Modified vaccinia virus Ankara (MVA) is a safe vector for high-level expression of proteins in mammalian cells. To simplify the molecular cloning procedures for shuttling genes into the MVA genome, we constructed generic destination plasmids that allow in vitro recombinational cloning (Gateway) and quick isolation of expression plasmids for any gene to be incorporated into the virus. Downstream purification steps were simplified by including N-terminal peptide tags (His, Strep, and Flag) in the generic plasmids. We demonstrate the ability to produce 10mg of beta-glucuronidase from 10(8) hamster cells and to purify tagged proteins with affinity gels.