Association of the telomere-telomere-binding protein complex of hypotrichous ciliates with the nuclear matrix and dissociation during replication.

Telomeric interactions with the nuclear matrix have been described in a variety of eukaryotic cells and seem to be essential for specific nuclear localization. Macronuclear DNA of hypotrichous ciliates occurs in small gene-sized DNA molecules, each being terminated by telomeres. Each macronucleus contains over 10(8 )individual DNA molecules. Owing to the high number of telomeres present in this nucleus it provides an excellent model to study telomere behaviour throughout the cell cycle. In this study we provide experimental evidence that the telomere-telomere-binding protein (TEBP) complex specifically interacts with components of the nuclear matrix in vivo. In the course of replication the specific interaction of the TEBP with components of the nuclear matrix is resolved and an attachment of the telomeres to the matrix no longer occurs.

A nuclear protein involved in apoptotic-like DNA degradation in Stylonychia: implications for similar mechanisms in differentiating and starved cells.

Ciliates are unicellular eukaryotic organisms containing two types of nuclei: macronuclei and micronuclei. After the sexual pathway takes place, a new macronucleus is formed from a zygote nucleus, whereas the old macronucleus is degraded and resorbed. In the course of macronuclear differentiation, polytene chromosomes are synthesized that become degraded again after some hours. Most of the DNA is eliminated, and the remaining DNA is fragmented into small DNA molecules that are amplified to a high copy number in the new macronucleus. The protein Pdd1p (programmed DNA degradation protein 1) from Tetrahymena has been shown to be present in macronuclear anlagen in the DNA degradation stage and also in the old macronuclei, which are resorbed during the formation of the new macronucleus. In this study the identification and localization of a Pdd1p homologous protein in Stylonychia (Spdd1p) is described. Spdd1p is localized in the precursor nuclei in the DNA elimination stage and in the old macronuclei during their degradation, but also in macronuclei and micronuclei of starved cells. In all of these nuclei, apoptotic-like DNA breakdown was detected. These data suggest that Spdd1p is a general factor involved in programmed DNA degradation in Stylonychia.

Separation of micronuclear DNA of Stylonychia lemnae by pulsed-field electrophoresis and identification of a DNA molecule with a high copy number.

DNA from the hypotrichous ciliatae Stylonychia lemnae was separated by PFGE. In addition to the separation of the macronuclear DNA molecules with a size up to approximately 40 kb, we were able to separate the micronuclear DNA with a size between approximately 90 kb and 2 Mb. One very prominent 90-kb DNA band appeared on the pulsed-field gels. We propose that this 90-kb DNA fragment represents a linear plasmid residing in the micronucleus in a very high copy number. About 10% of the micronuclear DNA consists of the 90-kb DNA molecule. It appears in the micronucleus as well as in the macronuclear anlagen during macronuclear development but not in the mature macronucleus. Thus, the multicopy DNA is eliminated during fragmentation of the macronuclear anlagen DNA in the course of macronuclear development. Therefore, this 90-kb DNA molecule might serve as an excellent tool to study the recognition and elimination of DNA during nuclear differentiation of hypotrichous ciliates.