In Escherichia coli , tetracycline prevents translation. When subject to tetracycline, E. coli express TetA to pump it out by a mechanism that is sensitive, while fairly independent of cellular metabolism. We constructed a target gene, P tetA -mRFP1-96BS, with a 96 MS2-GFP binding site array in a single-copy BAC vector, whose expression is controlled by the tetA promoter. We measured the in vivo kinetics of production of individual RNA molecules of the target gene as a function of inducer concentration and temperature. From the distributions of intervals between transcription events, we find that RNA production by P tetA is a sub-Poissonian process. Next, we infer the number and duration of the prominent sequential steps in transcription initiation by maximum likelihood estimation. Under full induction and at optimal temperature, we observe three major steps. We find that the kinetics of RNA production under the control of P tetA , including number and duration of the steps, varies with induction strength and temperature. The results are supported by a set of logical pairwise Kolmogorov-Smirnov tests. We conclude that the expression of TetA is controlled by a sequential mechanism that is robust, whereas sensitive to external signals.
BackgroundSeveral algorithms have been proposed for detecting fluorescently labeled subcellular objects in microscope images. Many of these algorithms have been designed for specific tasks and validated with limited image data. But despite the potential of using extensive comparisons between algorithms to provide useful information to guide method selection and thus more accurate results, relatively few studies have been performed.ResultsTo better understand algorithm performance under different conditions, we have carried out a comparative study including eleven spot detection or segmentation algorithms from various application fields. We used microscope images from well plate experiments with a human osteosarcoma cell line and frames from image stacks of yeast cells in different focal planes. These experimentally derived images permit a comparison of method performance in realistic situations where the number of objects varies within image set. We also used simulated microscope images in order to compare the methods and validate them against a ground truth reference result. Our study finds major differences in the performance of different algorithms, in terms of both object counts and segmentation accuracies.ConclusionsThese results suggest that the selection of detection algorithms for image based screens should be done carefully and take into account different conditions, such as the possibility of acquiring empty images or images with very few spots. Our inclusion of methods that have not been used before in this context broadens the set of available detection methods and compares them against the current state-of-the-art methods for subcellular particle detection.
bEscherichia coli cells employ an asymmetric strategy at division, segregating unwanted substances to older poles, which has been associated with aging in these organisms. The kinetics of this process is still poorly understood. Using the MS2 coat protein fused to green fluorescent protein (GFP) and a reporter construct with multiple MS2 binding sites, we tracked individual RNA-MS2-GFP complexes in E. coli cells from the time when they were produced. Analyses of the kinetics and brightness of the spots showed that these spots appear in the midcell region, are composed of a single RNA-MS2-GFP complex, and reach a pole before another target RNA is formed, typically remaining there thereafter. The choice of pole is probabilistic and heavily biased toward one pole, similar to what was observed by previous studies regarding protein aggregates. Additionally, this mechanism was found to act independently on each disposed molecule. Finally, while the RNA-MS2-GFP complexes were disposed of, the MS2-GFP tagging molecules alone were not. We conclude that this asymmetric mechanism to segregate damage at the expense of aging individuals acts probabilistically on individual molecules and is capable of the accurate classification of molecules for disposal. Escherichia coli is a well-established and simple model in aging studies. Stewart and colleagues demonstrated that two apparently identical sister cells resulting from cell division are functionally asymmetric (14). This asymmetry is representative of the aging of the cells, since unwanted protein aggregates tend to concentrate at the older pole of the mother cell. This can be observed as an accumulation of cell constituents with limited diffusion and a long half-life at the old pole of the mother cell, resulting in larger old poles and, consequently, a cumulatively slower growth of the daughter cells receiving these substances. Additional evidence of this phenomenon of asymmetric segregation of protein aggregates at older poles and its association with cellular aging in E. coli was presented previously (9), where it was shown that this pattern of segregation of unwanted substances appears to occur by a common mechanism irrespective of the unwanted substance segregated. The kinetics of this process is yet poorly understood.To better understand the mechanism of the preferential accumulation of unwanted protein aggregates at older poles in E. coli, it is essential to detect and track these aggregates individually. Although the detection of individual molecules in bacteria has been elusive and difficult, a recent technique has been developed that, provided the proper automated image segmentation algorithms, allows the tracking of single RNA molecules in E. coli by tagging them with multiple fluorescent proteins. This technique may be used for the purpose of tracking unwanted aggregates in E. coli. The technique uses the RNA bacteriophage MS2 coat protein fused to green fluorescent protein (GFP) and a reporter construct with multiple MS2 binding sites (5). The RNA-MS2-GFP comple...
BackgroundA gene network's capacity to process information, so as to bind past events to future actions, depends on its structure and logic. From previous and new microarray measurements in Saccharomyces cerevisiae following gene deletions and overexpressions, we identify a core gene regulatory network (GRN) of functional interactions between 328 genes and the transfer functions of each gene. Inferred connections are verified by gene enrichment.ResultsWe find that this core network has a generalized clustering coefficient that is much higher than chance. The inferred Boolean transfer functions have a mean p-bias of 0.41, and thus similar amounts of activation and repression interactions. However, the distribution of p-biases differs significantly from what is expected by chance that, along with the high mean connectivity, is found to cause the core GRN of S. cerevisiae's to have an overall sensitivity similar to critical Boolean networks. In agreement, we find that the amount of information propagated between nodes in finite time series is much higher in the inferred core GRN of S. cerevisiae than what is expected by chance.ConclusionsWe suggest that S. cerevisiae is likely to have evolved a core GRN with enhanced information propagation among its genes.
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