The copy number of any protein fluctuates among cells in a population; characterizing and understanding these fluctuations is a fundamental problem in biophysics. We show here that protein distributions measured under a broad range of biological realizations collapse to a single non-Gaussian curve under scaling by the first two moments. Moreover in all experiments the variance is found to depend quadratically on the mean, showing that a single degree of freedom determines the entire distribution. Our results imply that protein fluctuations do not reflect any specific molecular or cellular mechanism, and suggest that some buffering process masks these details and induces universality.The protein content of a cell is a primary determinant of its phenotype. However, protein copy number is subject to large cell-to-cell variation even among genetically identical cells grown under uniform conditions ([1-3] and references therein). This variation has been the subject of intensive research in recent years ([4-7] and references therein). Much of this previous work was devoted to characterizing the stochastic properties of various processes underlying gene expression, such as transcription and translation [8], or different stages of the cell cycle [9], and understanding their effect on protein variation. However, gene expression is generally coupled to all aspects of cell physiology, such as growth [10], metabolism [11], aging [12], division [13,14] and epigenetic processes [15,16], as well as gene location and function [17], all of which were shown to affect protein variation. The emerging picture is of a plethora of correlated mechanisms at different levels of organization; how they integrate to shape the total protein variation in a dividing population remains an open question [11,14].In this work we addressed this question by a phenomenological approach. We measured distributions of highly expressed proteins in proliferating clonal populations of bacteria and yeast under natural conditions, where gene expression is coupled to other cellular processes. By designing an array of different metabolic and regulatory conditions as well as growth environments, we collected a compendium of measurements which systematically covers the major processes of gene expression and 2 cell division, and compared the measured distributions in a wide range of biological realizations. More specifically, our comparisons included: (a) Two archetypical microorganisms, bacteria and yeast, with two well-studied regulatory systems of essential metabolic pathways: the LAC operon in E. coli [18] and the GAL system in S. cerevisiae [19]. Both systems were studied under environmental conditions in which expression is strongly coupled to metabolism, namely they control the utilization of an essential sugar (lactose and galactose, respectively) as the sole carbon The spectrum of our experiments spans an array of "control parameters" p which covers many of the essential processes affecting protein content in cells. The two organisms used, E. coli an...

We present a new experimental approach to build an artificial cell using the translation machinery of a cell-free expression system as the hardware and a DNA synthetic genome as the software. This approach, inspired by the self-replicating automata of von Neumann, uses cytoplasmic extracts, encapsulated in phospholipid vesicles, to assemble custom-made genetic circuits to develop the functions of a minimal cell. Although this approach can find applications, especially in biotechnology, the primary goal is to understand how a DNA algorithm can be designed to build an operating system that has some of the properties of life. We provide insights on this cell-free approach as well as new results to transform step by step a long-lived vesicle bioreactor into an artificial cell. We show how the green fluorescent protein can be anchored to the membrane and we give indications of a possible insertion mechanism of integral membrane proteins. With vesicles composed of different phospholipids, the fusion protein alpha-hemolysin-eGFP can be expressed to reveal patterns on the membrane. The specific degradation complex ClpXP from E. coli is introduced to create a sink for the synthesized proteins. Perspectives and subsequent limitations of this approach are discussed.

We observed that bacteria grown below a critical concentration, in batch-mode cultures, swim towards warm regions when subjected to a temperature gradient. Above that concentration, they swim towards colder regions. Our findings indicate that the secreted intercellular signal, glycine, mediates this switch through methylation of Tsr receptors. At high bacterial concentration, the switch is reinforced by an inversion of the Tar/Tsr expression ratio.

Gene transfer to eukaryotic cells requires the uptake of exogenous DNA into the cell nucleus. Except during mitosis, molecular access to the nuclear interior is limited to passage through the nuclear pores. Here we demonstrate the nuclear uptake of extended linear DNA molecules by a combination of fluorescence microscopy and single-molecule manipulation techniques, using the latter to follow uptake kinetics of individual molecules in real time. The assays were carried out on nuclei reconstituted in vitro from extracts of Xenopus eggs, which provide both a complete complement of biochemical factors involved in nuclear protein import, and unobstructed access to the nuclear pores. We find that uptake of DNA is independent of ATP or GTP hydrolysis, but is blocked by wheat germ agglutinin. The kinetics are much slower than would be expected from hydrodynamic considerations. A fit of the data to a simple model suggests femto-Newton forces and a large friction relevant to the uptake process.A common component in many types of viral infection (1, 2), strategies for genetic therapy (3), and direct DNA vaccination (4) is the accumulation of exogenous DNA in a host cell nucleus. While much attention has been paid to viral invasion mechanisms for cellular entry, relatively little is known about the uptake of DNA into the nucleus itself, particularly as it is not a part of normal cellular physiology. In nondividing cells, molecular exchange across the nuclear envelope normally takes place through the nuclear pore complexes (NPCs). These large channels, whose physiological role is primarily in regulating traffic of proteins and protein-RNA complexes between the nucleus and cytoplasm (5-8), exhibit distinct modes of passive exchange for small molecules and peptide signal-mediated selective transport for larger ones (9). Several pathways for the latter have been identified, based on affinity for receptors of the karyopherin ␤͞importin ␤ (10, 11) and related families (12). Less is known about the biochemistry related to DNA uptake, even whether such exists at all. Indeed, a prominent feature of the viral mechanisms is their ability to co-opt the native protein import machinery by expression of appropriate nuclear localization signals (NLS; ref. 1).The size scales of the NPC make it an unlikely transporter for DNA. The upper cutoff for diffusive passage of colloidal gold particles is Ϸ9 nm, whereas the NLS-mediated mechanism displays an apparent cutoff at Ϸ25 nm diameter (13,14). This scale easily encompasses the size of most protein transport substrates, for which the molecular weight of the cargo-receptor complex will be in the low hundreds of kDa. A recent study of transport rates finds the passage of several molecules per second in the NLS-mediated mode (15). Tightly compacted messenger ribonucleoprotein (mRNP) particles have been observed to unwind during their export through NPCs, conforming to the restricted space within the pore (16). The virulence proteins VirD2 and VirE2 of Agrobacterium tumefaciens mediate the import o...