Higher order chromatin structure plays a vital role in many cellular processes including, gene regulation, cell type determination and chromosome inheritance. We have found that we can disrupt chromatin structure using the toxic agent, camphor. Camphor decondenses chromosomes both in vivo and in vitro. We have used camphor in both Escherichia coli and Schizosaccharomyces pombe to isolate genes, which when overproduced confer camphor resistance on cells. In E. coli, camphor resistance identified a three gene system. CrcA alters the permeability of the outer membrane to give partial camphor resistance. CspE, a cold-shock-like protein, and CrcB, a predicted hydrophobic protein, are responsible for chromosome condensation and complete camphor resistance. We have demonstrated that, both in vivo and in vitro, overproduction of the three genes results in more tightly condensed nucleoids. If we overproduce CspE alone, we can demonstrate partial camphor resistance and nucleoid condensation. CspE binds to single-stranded DNA, double-stranded DNA and messenger RNA. We have identified mutations in CspE that are capable of binding only dsDNA or only single stranded nucleic acids. Those that bind dsDNA are still capable of chromosome condensation, whereas single stranded nucleic acid binding mutants do not. We are currently working to understand how CspE condenses the chromosome through binding to dsDNA.In S.pombe, we have found that camphor resistance can result from overproduction of the histone deacetylases, Clr3 and Clr6. Both of these proteins are known to affect the higher order chromatin structure and to be required for silencing of the DNA of the mating type loci. The camphor selection has also identified several novel genes, with approximately one third of them also decreasing mating type silencing and affecting chromosome stability. We are currently investigating the nature and function of these genes.In summary, we are using camphor as a probe of chromatin structure. These studies will allow us to determine how higher-order chromatin structure is formed and maintained, thus allowing intact and fully functional chromosomes to be inherited by daughter cells. - Bacterial genetics, chromosome condensation, Escherichia coli, Fission Yeast Genetics, Higher Order Chromatin Sturcture, Schizosaccharomyces pombe, - Neither Human Subjects nor Human Tissues
