Rational design of a bacterial transcriptional cascade for amplifying gene expression capacity

Cascade regulatory circuits have been described that control numerous cell processes, and may provide models for the design of artificial circuits with novel properties. Here we describe the design of a transcriptional regulatory cascade to amplify the cell response to a given signal. We used the salicylate-responsive activators of Pseudomonas putida NahR of the naphthalene degradation plasmid NAH7 and XylS2, a mutant regulator of the TOL plasmid for catabolism of m-xylene and their respective cognate promoters Psal and Pm. Control of the expression of xylS2 with the nahR/Psal system permitted either their selective activation with specific effectors for each protein or the simultaneous activation of both of them with salicylate. When cells face the common effector of the two regulators, both the increase in XylS2 concentration and the stimulation of its activity act synergistically on the Pm promoter, amplifying the gene expression capacity by at least one order of magnitude with respect to the individual systems. By changing the hierarchy of regulators, we showed that the specific features of the downstream regulator were crucial for the amplification effect. Directed changes in the effector profile of the regulators allowed the extension of the amplifying system to other molecular signals.

Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification

Rolling circle amplification (RCA) is a surface-anchored DNA replication reaction that can be exploited to visualize single molecular recognition events. Here we report the use of RCA to visualize target DNA sequences as small as 50 nts in peripheral blood lymphocytes or in stretched DNA fibers. Three unique target sequences within the cystic fibrosis transmembrane conductance regulator gene could be detected simultaneously in interphase nuclei, and could be ordered in a linear map in stretched DNA. Allele-discriminating oligonucleotide probes in conjunction with RCA also were used to discriminate wild-type and mutant alleles in the cystic fibrosis transmembrane conductance regulator, p53, BRCA-1, and Gorlin syndrome genes in the nuclei of cultured cells or in DNA fibers. These observations demonstrate that signal amplification by RCA can be coupled to nucleic acid hybridization and multicolor fluorescence imaging to detect single nucleotide changes in DNA within a cytological context or in single DNA molecules. This provides a means for direct physical haplotyping and the analysis of somatic mutations on a cell-by-cell basis.

Putting ion channels to work: Mechanoelectrical transduction, adaptation, and amplification by hair cells

As in other excitable cells, the ion channels of sensory receptors produce electrical signals that constitute the cellular response to stimulation. In photoreceptors, olfactory neurons, and some gustatory receptors, these channels essentially report the results of antecedent events in a cascade of chemical reactions. The mechanoelectrical transduction channels of hair cells, by contrast, are coupled directly to the stimulus. As a consequence, the mechanical properties of these channels shape our hearing process from the outset of transduction. Channel gating introduces nonlinearities prominent enough to be measured and even heard. Channels provide a feedback signal that controls the transducer's adaptation to large stimuli. Finally, transduction channels participate in an amplificatory process that sensitizes and sharpens hearing.