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Most eukaryotic telomeres contain a repeating motif with stretches of guanine residues that form a 3'-terminal overhang extending beyond the telomeric duplex region. The telomeric repeat of hypotrichous ciliates, d(T(4)G(4)), forms a 16-nucleotide 3'-overhang. Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex conformations in vitro. Although it has been proposed that guanine-quadruplex conformations may have important cellular roles including telomere function, recombination, and transcription, evidence for the existence of this DNA structure in vivo has been elusive to date. We have generated high-affinity single-chain antibody fragment (scFv) probes for the guanine-quadruplex formed by the Stylonychia telomeric repeat, by ribosome display from the Human Combinatorial Antibody Library. Of the scFvs selected, one (Sty3) had an affinity of K(d) = 125 pM for the parallel-stranded guanine-quadruplex and could discriminate with at least 1,000-fold specificity between parallel or antiparallel quadruplex conformations formed by the same sequence motif. A second scFv (Sty49) bound both the parallel and antiparallel quadruplex with similar (K(d) = 3--5 nM) affinity. Indirect immunofluorescence studies show that Sty49 reacts specifically with the macronucleus but not the micronucleus of Stylonychia lemnae. The replication band, the region where replication and telomere elongation take place, was also not stained, suggesting that the guanine-quadruplex is resolved during replication. Our results provide experimental evidence that the telomeres of Stylonychia macronuclei adopt in vivo a guanine-quadruplex structure, indicating that this structure may have an important role for telomere functioning.

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.

Background: Programmed DNA-reorganization and DNA-elimination events take place frequently during cellular differentiation. An extreme form of such processes, involving DNA reorganization, DNA elimination and DNA fragmentation, is found during macronuclear differentiation in hypotrichous ciliates. Ciliated protozoa can therefore serve as a model system to analyze the molecular basis of these processes during cellular differentiation in eukaryotic cells., Results: Using a biological approach to identify cis-acting sequences involved in DNA fragmentation, we show that in the hypotrichous ciliate Stylonychia lemnae sequences required for specific DNA processing are localized in the 3'- and the 5'-subtelomeric regions of the macronuclear precursor sequence. They can be present at various positions in the two subtelomeric regions, and an interaction between the two regions seems to occur. Sequence comparison revealed a consensus inverted repeat in both subtelomeric regions that is almost identical to the putative Euplotes chromosome breakage sequence (E-Cbs), also identified by sequence comparison. When this sequence was mutagenized, a processed product could no longer be detected, demonstrating that the sequence plays a crucial role in DNA processing. By injecting a construct into the developing macronucleus, which exclusively contains the subtelomeric regions of the Stylonychia alphal-tubulin gene, we show that subtelomeric regions are not only required but are also sufficient for DNA processing in Stylonychia., Conclusions: Our results indicate that an inverted repeat with the core sequence 5'-TGAA present in both subtelomeric regions acts as a Cbs in Stylonychia. The results allow us to propose a mechanistic model for DNA processing in this ciliate.

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.

A repetitive element from the hypotrichous ciliate Stylonychia lemnae was characterized by restriction and hybridization analysis. This repetitive element is present in about 5,000-7,000 copies per haploid genome in the micronucleus and the macronuclear anlagen. Its DNA sequence is very conserved, but the length of the repetitive sequence blocs is variable. In some cases, it is associated with telomeric sequences and macronucleus-homologous sequences. Restriction analysis of genomic micronuclear and macronuclear anlagen DNA and in situ hybridization showed that the repetitive sequences are amplified during the formation of polytene chromosomes. They are localized in many bands of the polytene chromosomes and are eliminated during the degradation of the polytene chromosomes. Possible functions of the repetitive sequences during macronuclear differentiation are discussed.

A nucleotide (nt) sequence of a DNA molecule from Stylonychia lemnae with an open reading frame encoding a protein showing homology to cyclin B has been determined. The DNA molecule is 3791-nt long and the deduced 444-amino-acid (aa) sequence shares about 30% identity with the sequences of two yeast cyclin-B homologs over a length of about 210 aa.

A macronuclear DNA molecule from Stylonychia lemnae with a size of 1891 nucleotides was cloned and sequenced. The 507-amino-acid computer-predicted sequence shares 41.9% identity with the mouse t-complex polypeptide 1 (TCP1).