Biological Insights into the Expression of Translation Initiation Factors from Recombinant CHOK1SV Cell Lines and their Relationship to Enhanced Productivity

Translation initiation is on the critical pathway for the production of monoclonal antibodies (mAb) by mammalian cells. Formation of a closed loop structure comprised of mRNA, a number of eukaryotic initiation factors and ribosomal proteins has been proposed to aid re-initiation of translation and therefore increase global translational efficiency. We have determined mRNA and protein levels of the key components of the closed loop; eukaryotic initiation factors (eIF3a, eIF3b, eIF3c, eIF3h, eIF3i and eIF4G1), poly(A) binding protein (PABP) 1 and PABP interacting protein 1 (PAIP1) across a panel of 30 recombinant mAb-producing GS-CHOK1SV cell lines with a broad range of growth characteristics and production levels of a model recombinant mAb. We have used a multi-level statistical approach to investigate the relationship between key performance indicators (cell growth and recombinant antibody productivity) and the intracellular amounts of target translation initiation factor proteins and the mRNAs encoding them. We show that high-producing cell lines maintain amounts of the translation initiation factors involved in the formation of the closed loop mRNA, maintaining these proteins at appropriate levels to deliver enhanced recombinant protein production. We then utilise knowledge of the amounts of these factors to build predictive models for, and use cluster analysis to identify, high-producing cell lines. This study therefore defines the translation initiation factor amounts that are associated with highly productive recombinant GS-CHOK1SV cell lines that may be targets for screening highly productive cell lines or to engineer new host cell lines with the potential for enhanced recombinant antibody productivity

Sequestration of Highly Expressed mRNAs in Cytoplasmic Granules, P-Bodies, and Stress Granules Enhances Cell Viability

Transcriptome analyses indicate that a core 10%–15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance its ability to withstand stress

Building Partnerships Towards a Democratic Police Force in the Post-Revolutionary Tunisia Context

This research analyzes security sector reform in Tunisia, and focuses on Tunisia’s Community Policing (CP) program in particular. CP is identified as an effective form of policing in the context of Tunisia’s political transition and continuing security concerns. This work pinpoints a number of gaps in the existing pilot CP program and areas for improvement, and proposes additional ways to address separate but interrelated problems by means of new policing methods, based on comparative research into methods used in the United States and Germany. It proposes to expand the existing private-public partnerships, which are at the heart of CP, and it identifies some areas of cooperation between the police and society that can be generalized across nations. Finally, the argument is put forth that police reform is a multi-pronged, multi-sectoral effort, relying on the efforts of many actors other than the polic

Stress-induced granules appear only in <i>UFO1-MS2L</i> stressed cells.

<p>A. Wild type and <i>UFO1-MS2L</i> cells transformed with p<i>CP-MS2L-GFPx3</i> at <i>A</i>₆₀₀ = 0.5, were transferred to SC 2% glucose without methionine for 1 hour to induce the CP^GFP. Cells were untreated or exposed to 1 mM arsenate, 8.8 mM H₂O₂, or UV-irradiated with 40 mJ/cm². Aliquots were collected 30 minutes after each treatment. Fluorescent granules appear only in cells with the tagged <i>UFO1-MS2L</i> gene. B. <i>UFO1-MS2L</i> cells expressing p<i>CP-MS2L-GFPx3</i> grown as above, treated with 1 mM arsenate for 30 minutes (stress), and transferred to fresh SC glucose medium. Stress recovery 30′ and 60′ indicate time after transfer to fresh SC. C. <i>UFO1-MS2L</i>, <i>yap1Δ</i> or <i>UFO1-MS2L</i>, <i>pdr1Δ</i> cells grown as above and untreated (Utrd), exposed to 1 mM arsenate, 8.8 mM H₂O₂, or UV-irradiated with 40 mJ/cm².</p

Yeast strains.

<p>Yeast strains.</p

<i>UFO1-MS2L</i> and <i>MFA2-U1A</i> mRNAs are sequestered in the same PBs after arsenate stress.

<p>A. Control wild type cells at <i>A</i>₆₀₀ = 0.5 producing U1A^GFP untreated or treated with 1 mM arsenate for 30 minutes. B. wild type <i>UFO1-MS2L</i> cells at <i>A</i>₆₀₀ = 0.5 expressing p<i>MFA2-U1A</i>, with their respective RNA-binding proteins, CP^mCherry and U1A^GFP, untreated or treated with 1 mM arsenate and stained with Hoechst 33342 at a final concentration of 2.5 µg/mL for 30 minutes.</p

Induction of Hsp12 protein and mRNA, and mRNA decay after stress.

<p>A. WB of protein produced from genomic <i>HSP12-GFP</i> in response to stress. B. <i>UFO1</i> and <i>HSP12</i> mRNA levels in untreated cells analyzed by qRT-PCR. mRNA levels were normalized to <i>ACT1</i>. C. Induction of <i>HSP12</i> mRNA by stress. Wild type cells at <i>A</i>₆₀₀ = 0.5, treated with 1 mM arsenate, 8.8 mM H₂O₂, irradiated with 40 mJ/cm² UV or shifted from 30°C to 37°C for 40 minutes. Aliquots were collected at the times indicated and analyzed by qRT-PCR. D. <i>HSP12</i> mRNA decay. p<i>GAL-HSP12</i> was expressed in <i>hsp12Δ</i> mutants by overnight induction with 2% galactose. Next morning cells at <i>A</i>₆₀₀ = 0.5 were untreated, or stressed with 1 mM arsenate or 8.8 mM H₂O₂ for 30 minutes, or irradiated with 40 mJ/cm² UV. The cells were washed and transferred to SC medium with 4% glucose. Samples were collected immediately after addition of glucose and at the times indicated and analyzed by qRT-PCR. mRNA levels were normalized to <i>ACT1</i> and to time 0 (untreated log cells).</p