Threshold effects in gene regulation: when some is not enough.
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Bacteriophage lambda↗
Biomedical subjects
John W Little
Publications and source records attributed to John W Little.
Regulatory circuit design and evolution using phage lambda.
Bistable gene regulatory circuits can adopt more than one stable epigenetic state. To understand how natural circuits have this and other systems properties, several groups have designed regulatory circuits de novo. Here we describe an alternative approach. We have modified an existing bistable circuit, that of phage lambda. With this approach, we used powerful genetic selections to identify functional circuits and selected for variants with altered behavior. The lambda circuit involves two antagonistic repressors, CI and Cro. We replaced lambda Cro with a module that included Lac repressor and several lac operators. Using a combinatorial approach, we isolated variants with different types of regulatory behavior. Several resembled wild-type lambda--they could grow lytically, could form highly stable lysogens, and carried out prophage induction. Another variant could form stable lysogens in the presence of a ligand for Lac repressor but switched to the lytic state when the ligand was removed. Several isolates evolved toward a desired behavior under selective pressure. These results strongly support the idea that complex circuits can arise during the course of evolution by a combination of simpler regulatory modules. They also underscore the advantages of modifying a natural circuit as an approach to understanding circuit design, systems behavior, and circuit evolution.
Bacteria↗
Sequence tolerance of the phage lambda PRM promoter: implications for evolution of gene regulatory circuitry.
Much of the gene regulatory circuitry of phage lambda centers on a complex region called the O(R) region. This approximately 100-bp region is densely packed with regulatory sites, including two promoters and three repressor-binding sites. The dense packing of this region is likely to impose severe constraints on its ability to change during evolution, raising the question of how the specific arrangement of sites and their exact sequences could evolve to their present form. Here we ask whether the sequence of a cis-acting site can be widely varied while retaining its function; if it can, evolution could proceed by a larger number of paths. To help address this question, we developed a lambda cloning vector that allowed us to clone fragments spanning the O(R) region. By using this vector, we carried out intensive mutagenesis of the P(RM) promoter, which drives expression of CI repressor and is activated by CI itself. We made a pool of fragments in which 8 of the 12 positions in the -35 and -10 regions were randomized and cloned this pool into the vector, making a pool of P(RM) variant phage. About 10% of the P(RM) variants were able to lysogenize, suggesting that the lambda regulatory circuitry is compatible with a wide range of P(RM) sequences. Analysis of several of these phages indicated a range of behaviors in prophage induction. Several isolates had induction properties similar to those of the wild type, and their promoters resembled the wild type in their responses to CI. We term this property of different sequences allowing roughly equivalent function "sequence tolerance " and discuss its role in the evolution of gene regulatory circuitry.
Bacteriophage lambda↗