Branched-chain amino acids are transported into Escherichia coli by a low-affinity system, LIV-II, and by two high-affinity systems, 18). The latter two high-affinity systems are the focus of this paper. The LIV-I system transports leucine, isoleucine, and valine, whereas the LS system transports only leucine. The two systems have a common set of membrane components and are distinguished by the specificities of their periplasmic binding proteins (9). The LIV-I binding protein binds L-leucine, L-isoleucine, and L-valine with approximately equal affinity and is coded for by the livJ gene, whereas the LS binding protein binds D-and L-leucine but neither isoleucine nor valine and is coded for by livK, the first gene in an operon that also codes for the common membrane components. The genes are closely linked near min 76 on the E. coli chromosome within a region referred to as the LIV-I locus.The LIV-I locus is regulated by leucine, as determined by assaying the transport and leucine binding activities of periplasmic shock fluids (1,19 scribed and termed lrp (leucine-responsive regulatory protein) (11, 16). Mutations in lrp affect the expression of a number of operons, including ilvIH (16), serA (11,20), sdaA (11), tdh (11,20), and oppABCDF (5). The expression of each of these operons, like those composing the LIV-I locus, is altered by growing cells in the presence of leucine. Given the similarity in map location and the common connection to leucine, it seemed possible that livR and lrp are the same locus. Rex et al. raised this possibility recently in connection with studies showing that a livR allele had a dramatic effect upon the regulation of the tdh and serA operons (20). Here we show that livR and lrp are indeed the same gene. A livRJ-containing strain had mutations in lrp, and these mutations were shown to affect the expression of ilvIH, an operon known to be regulated by lrp. Moreover, lrp mutations that arose under conditions having nothing to do with amino acid transport were shown to affect the expression of livJ and livK.An important finding that came out of these studies is the amino acid transport phenotype of a strain with a null mutation in lrp. Such a strain shows high, constitutive transport of branched-chain amino acids. This result reflects the fact that expression from the livJ and livK promoters is similarly high and constitutive. These results suggest that Lrp, the product of lrp, negatively regulates livJ and livK expression. As we point out in the Discussion, a negative effect of lrp coupled with leucine-mediated repression is a pattern that is unique for operons known to be regulated by lrp.

Heterogeneity is a fundamental property of biological systems at all scales that must be addressed in a wide range of biomedical applications including basic biomedical research, drug discovery, diagnostics and the implementation of precision medicine. There are a number of published approaches to characterizing heterogeneity in cells in vitro and in tissue sections. However, there are no generally accepted approaches for the detection and quantitation of heterogeneity that can be applied in a relatively high throughput workflow. This review and perspective emphasizes the experimental methods that capture multiplexed cell level data, as well as the need for standard metrics of the spatial, temporal and population components of heterogeneity. A recommendation is made for the adoption of a set of three heterogeneity indices that can be implemented in any high throughput workflow to optimize the decision-making process. In addition, a pairwise mutual information method is suggested as an approach to characterizing the spatial features of heterogeneity, especially in tissue-based imaging. Furthermore, metrics for temporal heterogeneity are in the early stages of development. Example studies indicate that the analysis of functional phenotypic heterogeneity can be exploited to guide decisions in the interpretation of biomedical experiments, drug discovery, diagnostics and the design of optimal therapeutic strategies for individual patients.

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Small interfering RNAs (siRNAs) are routinely used to reduce mRNA levels for a specific gene with the goal of studying its function. Several studies have demonstrated that siRNAs are not always specific and can have many off-target effects. The 3′ UTRs of off-target mRNAs are often enriched in sequences that are complementary to the seed-region of the siRNA. We demonstrate that siRNA off-targets can be significantly reduced when cells are treated with a dose of siRNA that is relatively low (e.g. 1 nM), but sufficient to effectively silence the intended target. The reduction in off-targets was demonstrated for both modified and unmodified siRNAs that targeted either STAT3 or hexokinase II. Low concentrations reduced silencing of transcripts with complementarity to the seed region of the siRNA. Similarly, off-targets that were not complementary to the siRNA were reduced at lower doses, including up-regulated genes that are involved in immune response. Importantly, the unintended induction of caspase activity following treatment with a siRNA that targeted hexokinase II was also shown to be a concentration-dependent off-target effect. We conclude that off-targets and their related phenotypic effects can be reduced for certain siRNA that potently silence their intended target at low concentrations.

Cac3p/Msi1p, the Saccharomyces cerevisiae homolog of retinoblastoma-associated protein 48 (RbAp48), is a component of chromatin assembly factor I (CAF-I), a complex that assembles histones H3 and H4 onto replicated DNA. CAC3 overexpression also suppresses the RAS/cyclic AMP (cAMP) signal transduction pathway by an unknown mechanism. We investigated this mechanism and found that CAC3 suppression of RAS/cAMP signal transduction was independent of either CAC1 or CAC2, subunits required for CAF-I function. CAC3 suppression was also independent of other chromatin-modifying activities, indicating that Cac3p has at least two distinct, separable functions, one in chromatin assembly and one in regulating RAS function. Unlike Cac1p, which localizes primarily to the nucleus, Cac3p localizes to both the nucleus and the cytoplasm. In addition, Cac3p associates with Npr1p, a cytoplasmic kinase that stablizes several nutrient transporters by antagonizing a ubiquitin-mediated protein degradation pathway. Deletion of NPR1, like overexpression of Cac3p, suppressed the RAS/cAMP pathway. Furthermore, NPR1 overexpression interfered with the ability of CAC3 to suppress the RAS/cAMP pathway, indicating that extra Cac3p suppresses the RAS/cAMP pathway by sequestering Npr1p. Deletion of NPR1 did not affect the quantity, phosphorylation state, or localization of Ras2p. Consistent with the idea that Npr1p exerts its effect on the RAS/cAMP pathway by antagonizing a ubiquitin-mediated process, excess ubiquitin suppressed both the heat shock sensitivity and the sporulation defects caused by constitutive activation of the RAS/cAMP pathway. Thus, CAC3/MSI1 regulates the RAS/cAMP pathway via a chromatin-independent mechanism that involves the sequestration of Npr1p and may be due to the increased ubiquitination of an Npr1p substrate.