Chromosomal regions can adopt stable and heritable alternative states resulting in bistable gene expression without changes to the DNA sequence. Such epigenetic control is often associated with alternative covalent modifications of histones. The stability and heritability of the states are thought to involve positive feedback where modified nucleosomes recruit enzymes that similarly modify nearby nucleosomes. We developed a simplified stochastic model for dynamic nucleosome modification based on the silent mating-type region of the yeast Schizosaccharomyces pombe. We show that the mechanism can give strong bistability that is resistant both to high noise due to random gain or loss of nucleosome modifications and to random partitioning upon DNA replication. However, robust bistability required: (1) cooperativity, the activity of more than one modified nucleosome, in the modification reactions and (2) that nucleosomes occasionally stimulate modification beyond their neighbor nucleosomes, arguing against a simple continuous spreading of nucleosome modification.

We present an update of our method for systematic detection and evaluation of potential helix-turn-helix DNA-binding motifs in protein sequences [Dodd, I. and Egan, J. B. (1987) J. Mol. Biol. 194, 557-564]. The new method is considerably more powerful, detecting approximately 50% more likely helix-turn-helix sequences without an increase in false predictions. This improvement is due almost entirely to the use of a much larger reference set of 91 presumed helix-turn-helix sequences. The scoring matrix derived from this reference set has been calibrated against a large protein sequence database so that the score obtained by a sequence can be used to give a practical estimation of the probability that the sequence is a helix-turn-helix motif.

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The CI repressor of bacteriophage is a model for the role of cooperativity in the efficient functioning of genetic switches. Pairs of CI dimers interact to cooperatively occupy adjacent operator sites at O R and at O L . These CI tetramers repress the lytic promoters and activate transcription of the cI gene from P RM . CI is also able to octamerize, forming a large DNA loop between O R and O L , but the physiological role of this is unclear. Another puzzle is that, although a dimer of CI is able to repress P RM by binding to the third operator at O R , O R 3, this binding seems too weak to affect CI production in the lysogenic state. Here we show that repression of P RM at lysogenic CI concentrations is absolutely dependent on O L , in this case 3.8 kb away. A mutant defective in this CI negative autoregulation forms a lysogen with elevated CI levels that cannot efficiently switch from lysogeny to lytic development. Our results invalidate previous evidence that Cro binding to O R 3 is important in prophage induction. We propose the octameric CI:O R -O L complex increases the affinity of CI for O R 3 by allowing a CI tetramer to link O R 3 and the third operator at O L , O L 3.

We describe "clonetegration", a method for integrating DNA into prokaryotic chromosomes that approaches the simplicity of cloning DNA within extrachromosomal vectors. Compared to existing techniques, clonetegration drastically decreases the time and effort needed for integration of single or multiple DNA fragments. Additionally, clonetegration facilitates cloning and expression of genetic elements that are impossible to propagate within typical multicopy plasmids.