We present a method for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed Escherichia coli cells. A set of fluorescently-labeled oligonucleotide probes are hybridized to the target mRNA, so that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The number of target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete “spots” as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy-number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 days, with another 2-3 days typically required for image and data analysis.

Transcription is a highly stochastic process. To infer transcription kinetics for a gene-of-interest, researchers commonly compare the distribution of mRNA copy-number to the prediction of a theoretical model. However, the reliability of this procedure is limited because the measured mRNA numbers represent integration over the mRNA lifetime, contribution from multiple gene copies, and mixing of cells from different cell-cycle phases. We address these limitations by simultaneously quantifying nascent and mature mRNA in individual cells, and incorporating cell-cycle effects in the analysis of mRNA statistics. We demonstrate our approach on Oct4 and Nanog in mouse embryonic stem cells. Both genes follow similar two-state kinetics. However, Nanog exhibits slower ON/OFF switching, resulting in increased cell-to-cell variability in mRNA levels. Early in the cell cycle, the two copies of each gene exhibit independent activity. After gene replication, the probability of each gene copy to be active diminishes, resulting in dosage compensation.DOI: http://dx.doi.org/10.7554/eLife.12175.001

We combine immunofluorescence and single-molecule fluorescence in situ hybridization (smFISH), followed by automated image analysis, to quantify the concentration of nuclear transcription factors, number of transcription factors bound, and number of nascent mRNAs synthesized at individual gene loci. A theoretical model is used to decipher how transcription-factor binding modulates the stochastic kinetics of mRNA production. We demonstrate this approach by examining the regulation of hunchback in the early Drosophila embryo.

The stochastic kinetics of transcription is typically inferred from the distribution of RNA numbers in individual cells. However, cellular RNA reflects additional processes downstream of transcription, hampering this analysis. In contrast, nascent (actively transcribed) RNA closely reflects the kinetics of transcription. We present a theoretical model for the stochastic kinetics of nascent RNA, which we solve to obtain the probability distribution of nascent RNA per gene. The model allows us to evaluate the kinetic parameters of transcription from single-cell measurements of nascent RNA. The model also predicts surprising discontinuities in the distribution of nascent RNA, a feature which we verify experimentally.

The optic tectum is central for transforming incoming visual input into orienting behavior. Yet it is not well understood how this behavior is organized early in development and how it relates to the response properties of the developing visual system. We designed a novel behavioral assay to study the development of visually guided behavior in Xenopus laevis tadpoles. We found that, during early development, visual avoidance-an innate, tectally mediated behavior-is tuned to a specific stimulus size and is sensitive to changes in contrast. Using in vivo recordings we found that developmental changes in the spatial tuning of visual avoidance are mirrored by changes in tectal receptive field sharpness and the temporal properties of subthreshold visual responses, whereas contrast sensitivity is affected by the gain of the visual response. We also show that long- and short-term perturbations of visual response properties predictably alter behavioral output. We conclude that our assay for visual avoidance is a useful functional measure of the developmental state of the tectal circuitry. We use this assay to show that the developing visual system is tuned to facilitate behavioral output and that the system can be modulated by neural activity, allowing it to adapt to environmental changes it encounters during development.

In vivo mapping of transcription-factor binding to the transcriptional output of the regulated gene is hindered by probabilistic promoter occupancy, the presence of multiple gene copies, and cell-to-cell variability. We demonstrate how to overcome these obstacles in the lysogeny maintenance promoter of bacteriophage lambda, PRM. We simultaneously measured the concentration of the lambda repressor CI and the number of mRNAs from PRM in individual E. coli cells, and used a theoretical model to identify the stochastic activity corresponding to different CI binding configurations. We found that switching between promoter configurations is faster than mRNA lifetime, and that individual gene copies within the same cell act independently. The simultaneous quantification of transcription factor and promoter activity, followed by stochastic theoretical analysis, provides a tool that can be applied to other genetic circuits.

The formation of a complex between DNA polymerase ␦ (pol ␦) and its sliding clamp, proliferating cell nuclear antigen (PCNA), is responsible for the maintenance of processive DNA synthesis at the leading strand of the replication fork. In this study, the ability of the p125 catalytic subunit of DNA polymerase ␦ to engage in protein-protein interactions with PCNA was established by biochemical and genetic methods. p125 and PCNA were shown to co-immunoprecipitate from either calf thymus or HeLa extracts, or when they were ectopically co-expressed in Cos 7 cells. Because pol ␦ is a multimeric protein, this interaction could be indirect. Thus, rigorous evidence was sought for a direct interaction of the p125 catalytic subunit and PCNA. To do this, the ability of recombinant p125 to interact with PCNA was established by biochemical means. p125 co-expressed with PCNA in Sf9 cells was shown to form a physical complex that can be detected on gel filtration and that can be cross-linked with the bifunctional cross-linking agent Sulfo-EGS (ethylene glycol bis (sulfosuccinimidylsuccinate)). An interaction between p125 and PCNA could also be demonstrated in the yeast two hybrid system. Overlay experiments using biotinylated PCNA showed that the free p125 subunit interacts with PCNA. The PCNA overlay blotting method was also used to demonstrate the binding of synthetic peptides corresponding to the N2 region of pol ␦ and provides evidence for a site on pol ␦ that is involved in the protein-protein interactions between PCNA and pol ␦. This region contains a sequence that is a potential member of the PCNA binding motif found in other PCNA-binding proteins. These studies provide an unequivocal demonstration that the p125 subunit of pol ␦ interacts with PCNA.Proliferating cell nuclear antigen (PCNA) 1 was originally discovered as an antigen in autoimmune sera from patients with systemic lupus erythematosus and was reported to be found only in actively proliferating cells (1). It was later shown to be a factor that enhanced the processivity of DNA polymerase ␦ (pol ␦) and to have key roles in both DNA replication and repair (2, 3). There have been striking recent advances in our understanding of the structure and functions of PCNA (4). Purification and expression of human recombinant PCNA and its physiochemical characterization established that it was a trimeric protein (5). The crystal structures of both yeast and human PCNA have been determined (6, 7). Like the T4 gene 45 protein and the ␤ subunit of Escherichia coli DNA polymerase III holoenzyme, PCNA functions as a sliding DNA clamp that forms a closed ring around duplex DNA (8). The binding of pol ␦ to PCNA provides an elegant micromechanical solution to the biological need to maintain an extraordinarily high level of processivity during the synthesis of chromosomal DNA (8 -10). Recently, it has also been found that PCNA has a number of protein partners with which it interacts (4, 9, 11). Pol ␦ has been shown to be involved not only in DNA replication but also in DNA repair a...

Single-cell measurements of mRNA copy-number inform our understanding of stochastic gene expression [1-3], but these measurements coarse-grain over the individual copies of the gene, where transcription and its regulation stochastically take plasce [4, 5]. Here we combine singlemolecule quantification of mRNA and gene loci to measure the transcriptional activity of an endogenous gene in individual Escherichia coli bacteria. Interpreted using a theoretical model for mRNA dynamics, the single-cell data allows us to obtain the probabilistic rates of promoter switching, transcription initiation and elongation, mRNA release and degradation. Unexpectedly, we find that gene activity can be strongly coupled to the transcriptional state of another copy of the same gene present in the cell, and to the event of gene replication during the bacterial cell cycle. These gene-copy and cell-cycle correlations demonstrate the limits of mapping whole-cell mRNA numbers to the underlying stochastic gene activity, and instead highlight the contribution of previously hidden variables to the observed population heterogeneity. Counting RNA in individual cells revealed the bursty nature of transcription in bacteria [2] and eukaryotes [6], and showed how gene expression noise is modulated by physiological Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: