The expression level and cytotoxicity of green fluorescent protein are modulated by an additional N-terminal sequence

Nucleotide and amino acid sequences at the N-terminus affect the expression level and cytotoxicity of proteins; however, their effects are not fully understood yet. Here, N-terminal 30 nucleotide/10 amino acid (N10) sequences that affect the expression level and cytotoxicity of a green fluorescent protein were systematically isolated in the budding yeast Saccharomyces cerevisiae. The expression per gene (EPG) and gene copy number limit (CNL) relationships were examined to assess the effects of the N10 sequence. The isolated N10 nucleotide sequences suggested that codon optimality is the major determinant of the protein expression level. A higher number of hydrophobic or cysteine residues in the N10 sequence seemed to increase the cytotoxicity of the protein. Therefore, a high frequency of specific amino acid residues in the outside of the main tertiary structure of proteins might not be preferable

Mathematical analysis of copy number variation of 2 μ-based plasmids in yeast cells

　Plasmids with the 2 μ plasmid origin are commonly-used in the genetic engineering of the budding yeast Saccharomyces cerevisiae. Intracellular copy numbers of 2 μ plasmids are different depending on the genes inserted into the plasmids. This difference is thought to occur from the difference in the growth efficiency （fitness） produced by the positive- and negative-selection biases of genes inserted in the plasmid. In this study, we made a mathematical model based on this assumption. Computational simulations of the model validated that copy numbers of the plasmids are rapidly settled depending on the fitness created by the gene on the plasmid. The copy number of a plasmid only contains a bias to keep the plasmid in a single copy became average 20copies per cell when the plasmid is randomly distributed, suggesting that no positive distribution mechanism is required for a plasmid to become multicopy

Exploring the Complexity of Protein-Level Dosage Compensation that Fine-Tunes Stoichiometry of Multiprotein Complexes

Proper control of gene expression levels upon various perturbations is a fundamental aspect of cellular robustness. Protein-level dosage compensation is one mechanism buffering perturbations to stoichiometry of multiprotein complexes through accelerated proteolysis of unassembled subunits. Although N-terminal acetylation- and ubiquitin-mediated proteasomal degradation by the Ac/N-end rule pathway enables selective compensation of excess subunits, it is unclear how widespread this pathway contributes to stoichiometry control. Here we report that dosage compensation depends only partially on the Ac/N-end rule pathway. Our analysis of genetic interactions between 18 subunits and 12 quality control factors in budding yeast demonstrated that multiple E3 ubiquitin ligases and N-acetyltransferases are involved in dosage compensation. We find that N-acetyltransferases-mediated compensation is not simply predictable from N-terminal sequence despite their sequence specificity for N-acetylation. We also find that the compensation of Pop3 and Bet4 is due in large part to a minor N-acetyltransferase NatD. Furthermore, canonical NatD substrates histone H2A/H4 were compensated even in its absence, suggesting N-acetylation-independent stoichiometry control. Our study reveals the complexity and robustness of the stoichiometry control system. Author summary Quality control of multiprotein complexes is important for maintaining homeostasis in cellular systems that are based on functional complexes. Proper stoichiometry of multiprotein complexes is achieved by the balance between protein synthesis and degradation. Recent studies showed that translation efficiency tends to scale with stoichiometry of their subunits. On the other hand, although protein N-terminal acetylation- and ubiquitin-mediated proteolysis pathway is involved in selective degradation of excess subunits, it is unclear how widespread this pathway contributes to stoichiometry control due to the lack of a systematic investigation using endogenous proteins. To better understand the landscape of the stoichiometry control system, we examined genetic interactions between 18 subunits and 12 quality control factors (E3 ubiquitin ligases and N-acetyltransferases), in total 114 combinations. Our data suggest that N-acetyltransferases are partially responsible for stoichiometry control and that N-acetylation-independent pathway is also involved in selective degradation of excess subunits. Therefore, this study reveals the complexity and robustness of the stoichiometry control system. Further dissection of this complexity will help to understand the mechanisms buffering gene expression perturbations and shaping proteome stoichiometry

Aneuploid proliferation defects in yeast are not driven by copy number changes of a few dosage-sensitive genes

Aneuploidy—the gain or loss of one or more whole chromosome—typically has an adverse impact on organismal fitness, manifest in conditions such as Down syndrome. A central question is whether aneuploid phenotypes are the consequence of copy number changes of a few especially harmful genes that may be present on the extra chromosome or are caused by copy number alterations of many genes that confer no observable phenotype when varied individually. We used the proliferation defect exhibited by budding yeast strains carrying single additional chromosomes (disomes) to distinguish between the “few critical genes” hypothesis and the “mass action of genes” hypothesis. Our results indicate that subtle changes in gene dosage across a chromosome can have significant phenotypic consequences. We conclude that phenotypic thresholds can be crossed by mass action of copy number changes that, on their own, are benign.National Institutes of Health (U.S.) (GM056800

Development of an experimental method of systematically estimating protein expression limits in HEK293 cells

Protein overexpression sometimes causes cellular defects, although the underlying mechanism is still unknown. A protein's expression limit, which triggers cellular defects, is a useful indication of the underlying mechanism. In this study, we developed an experimental method of estimating the expression limits of target proteins in the human embryonic kidney cell line HEK293 by measuring the proteins' expression levels in cells that survived after the high-copy introduction of plasmid DNA by which the proteins were expressed under a strong cytomegalovirus promoter. The expression limits of nonfluorescent target proteins were indirectly estimated by measuring the levels of green fluorescent protein (GFP) connected to the target proteins with the self-cleaving sequence P2A. The expression limit of a model GFP was similar to 5.0% of the total protein, and sustained GFP overexpression caused cell death. The expression limits of GFPs with mitochondria-targeting signals and endoplasmic reticulum localization signals were 1.6% and 0.38%, respectively. The expression limits of four proteins involved in vesicular trafficking were far lower compared to a red fluorescent protein. The protein expression limit estimation method developed will be valuable for defining toxic proteins and consequences of protein overexpression

Plasmid Construction Using Recombination Activity in the Fission Yeast Schizosaccharomyces pombe

BACKGROUND: Construction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC), is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date. METHODOLOGY/PRINCIPAL FINDINGS: We investigated GRC efficiency in S. pombe in this study. We first showed that GRC was feasible in S. pombe by constructing a plasmid that contained the LEU2 auxotrophic marker gene in vivo and showed sufficient efficiency with short homology sequences (>25 bp). No preference was shown for the sequence length from the cut site in the vector plasmid. We next showed that plasmids could be constructed in a proper way using 3 DNA fragments with 70% efficiency without any specific selections being made. The GRC efficiency with 3 DNA fragments was dramatically increased >95% in lig4Delta mutant cell, where non-homologous end joining is deficient. Following this approach, we successfully constructed plasmid vectors with leu1+, ade6+, his5+, and lys1+ markers with the low-copy stable plasmid pDblet as a backbone by applying GRC in S. pombe. CONCLUSIONS/SIGNIFICANCE: We concluded that GRC was sufficiently feasible in S. pombe for genome-wide gene functional analysis as well as for regular plasmid construction. Plasmids with different markers constructed in this research are available from NBRP-yeast (http://yeast.lab.nig.ac.jp/)

Sub-kHz-linewidth VECSELs for cold atom experiments

We report and characterize sub-kHz linewidth operation of an AlGaInP-based VECSEL system suitable for addressing the narrow cooling transition of neutral strontium atoms at 689 nm. When frequency-stabilized to a standard air-spaced Fabry-Perot cavity (finesse 1000) via the Pound-Drever-Hall (PDH) technique, it delivers output power >150 mW in a circularly-symmetric single transverse mode with low frequency and intensity noise. The optical field was reconstructed from the frequency noise error signal via autocorrelation and the Wiener-Khintchine theorem, leading to an estimated linewidth of (125±2) Hz. Optical beat note measurements were performed against a commercial locked laser system and a second, almost identical, VECSEL system resulting in linewidths of 200 Hz and 160 Hz FWHM, respectively. To the best of our knowledge, this is the first demonstration of a VECSEL compatible with the narrowest of lines (few hundred Hz) used for cooling and trapping atoms and ions

Monolithic VECSEL for stable kHz linewidth

Vertical-external-cavity surface-emitting semiconductor lasers (VECSELs) are of increasing interest for applications requiring ultra-coherence and/or low noise at novel wavelengths; performance that is currently achieved via high-Q, air-spaced resonators to achieve long intra-cavity photon lifetimes (for the so-called class-A low noise regime), power scaling and high beam quality. Here, we report on the development of a compact, electronically tunable, monolithic-cavity, class-A VECSEL (monolithic VECSEL) for ultra-narrow free-running linewidths. A multi-quantum-well, resonant periodic gain structure with integrated distributed Bragg reflector (DBR) was optically-bonded to an air-gap-free laser resonator created inside a right-angle fused-silica prism to suppress the influence of environmental noise on the external laser oscillation, thus achieving high stability. Mode-hop-free wavelength tuning is performed via the stabilized temperature; or electronically, and with low latency, via a shear piezo-electric transducer mounted on the top of the prism. The free-running linewidth, estimated via the frequency power spectral density (PSD), is sub-kHz over ms timescales and <1.9 kHz for time sampling as long as 1s, demonstrating at least two orders-of-magnitude improvement in noise performance compared to previously reported single frequency VECSELs. The stable, total internal reflection resonator concept is akin to the prevalent monolithic non-planar ring oscillator (NPRO), however the monolithic VECSEL has several important advantages: tailored emission wavelength (via semiconductor bandgap engineering), no relaxation oscillations, no applied magnetic field, and low requirements on the pump beam quality. This approach is power-scalable in principle and could be applied to VECSELs at any of the wavelengths from the visible to the mid-infrared at which they are already available, to create a range of robust, ultra-coherent laser systems with reduced bulkiness and complexity. This is of particular interest for remote metrology and the translation of quantum technologies, such as optical clocks, from research laboratories into real world applications

Single-frequency optical parametric oscillator intracavity-pumped by a visible VECSEL for low-noise down-conversion to 1.55 µm

We report, to the best of our knowledge, the first optical parametric oscillator (OPO) pumped by a visible AlGaInP-based vertical-external-cavity surface-emitting laser (VECSEL). Tunable emission over 1155–1300 nm in the signal and 1474–1718 nm in the idler are observed by temperature adjustment of a 40 mm-long 5%-MgO:PPLN crystal intracavity-pumped at 690 nm. When optimized for low oscillation threshold, and by implementing resonant idler output-coupling (TOC = 1.7%), extracted output powers of 26.2 mW (signal) and 5.6 mW (idler; one-way) are measured, corresponding to a total down-conversion efficiency and extraction efficiency of 70.2% and 43%, respectively. Further, a total down-conversion efficiency of 72.1% is achieved in the absence of idler output-coupling. Of particular interest for high-precision applications, including quantum optics experiments and squeezed light generation, high stability and single-frequency operation are also demonstrated. We measure RMS stabilities of 0.4%, 1.8% and 2.3% for the VECSEL fundamental, signal and idler, with (resolution-limited) frequency linewidths of 2.5 MHz (VECSEL) and 7.5 MHz (signal and idler)