The macronuclear DNA of the ciliated protozoa Oxytricha consists of a collection of DNA molecules ranging in length from 24 to .4kb. Each molecule is equipped with the same sequence of nucleotides at each end. A typical macronuclear DNA molecule can be depicted as follows: 5' C4A4C4A4C4 G4T4G4T4G4T4G4T4G4 3' G4T4G4T4G4T4G4T4G4 2.4 kb C4A4C4A4C4 Remarkably, when this DNA is incubated in 1.0 M NaCl for 20-40 hours at room temperature, these terminals will associate with one another to produce long concatemeric structures that are more than 40kb in length. Nuclease experiments have demonstrated that the ability of these terminals to cohere with each other is dependent upon the presence of a free 16-base pair G4T4G4T4 single chain. The complementary synthetic oligomer C4A4C4A4 will completely prevent association, while oligomers of other sequence will not. A synthetic oligomer containing 3' G4T4G4T4 will prevent the association of the terminals provided it is present at a 100-fold excess to the concentration of DNA terminals. This means that the natural terminals have a greater affinity for each other than they do for the 3' G4T4G4T4 oligomers. Thus, coherence is not simply the result of the interaction of G4T4 single chains. The first objective of this work is to discover what kind of DNA:DNA structure is formed that holds these terminals together. These 3'-ended single chains of special sequences represent, or are part of, the telomeres of these macronuclear chromosomes. The work proposed here represents a first step in learning how these gene-sized chromosomes are replicated. These terminals also may be involved in the expression of the genes adjacent to them. To approach these questions, we plan to ligate synthetic G4T4 single chains to the terminals of linearized plasmids that are able to replicate in yeast. If these single chains can function as telomeres by themselves, the plasmids should replicate as linear molecules rather than circles.
| Oka, Y; Thomas Jr, C A (1987) The cohering telomeres of Oxytricha. Nucleic Acids Res 15:8877-98 |
