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Increasing amounts of data support a role for guanine quadruplex (G4) DNA and RNA structures in various cellular processes. We stained different organisms with monoclonal antibody 1H6 specific for G4 DNA. Strikingly, immuno-electron microscopy showed exquisite specificity for heterochromatin. Polytene chromosomes from Drosophila salivary glands showed bands that co-localized with heterochromatin proteins HP1 and the SNF2 domain-containing protein SUUR. Staining was retained in SUUR knock-out mutants but lost upon overexpression of SUUR. Somatic cells in Macrostomum lignano were strongly labeled, but pluripotent stem cells labeled weakly. Similarly, germline stem cells in Drosophila ovaries were weakly labeled compared to most other cells. The unexpected presence of G4 structures in heterochromatin and the difference in G4 staining between somatic cells and stem cells with germline DNA in ciliates, flatworms, flies and mammals point to a conserved role for G4 structures in nuclear organization and cellular differentiation., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

During macronuclear differentiation in ciliated protozoa a series of programed DNA reorganization processes occur. These include the elimination of micronuclear-specific DNA sequences, the specific fragmentation of the genome into small gene-sized DNA molecules, the de novo addition of telomeric sequences to these DNA molecules and the specific amplification of the remaining DNA molecules. Recently we constructed a vector containing the modified micronuclear version of macronuclear destined DNA sequences that was correctly fragmented and telomeres were added de novo after injection into the developing macronucleus. It therefore must contain all the cis- acting sequences required for these processes. We made a series of vectors deleting different sequences from the original vector. It could be shown that at least in the case studied here no micronuclear-specific sequences are required for specific fragmentation of the genome and telomere addition. However, a short subtelomeric sequence at the 3[prime]-end is essential for these processes, whereas no specific cut seems to occur at the 5[prime]-end. In addition, we can show that the processing activity is restricted to a short period of time during macronuclear differentiation and that a preceding transcription is required for correct processing of macronuclear-destined DNA sequences. Possible mechanisms of these processes will be discussed.

We have developed an episomal replicating expression vector in which the SV40 gene coding for the large T-antigen was replaced by chromosomal scaffold/matrix attached regions. Southern analysis as well as vector rescue experiments in CHO cells and in Escherichia coli demonstrate that the vector replicates episomally in CHO cells. It occurs in a very low copy number in the cells and is stably maintained over more than 100 generations without selection pressure.

Elimination of internal eliminated sequences (IES) during macronuclear development of the hypotrichous ciliate Stylonychia lemnae was analyzed in one cluster of macronuclear precursor DNA sequences. The results indicate that IES elimination is a highly ordered process, it starts very early during macronuclear development and has only finished immediately before DNA fragmentation takes place. It occurs in distinct steps and the IES are eliminated in a specific order, where a defined IES is only removed after complete elimination of other IES. Transfection experiments clearly demonstrate that the structure of the IES itself is not sufficient for its correct excision but other cis-acting sequences or additional structural requirements are needed for IES elimination.

We describe the construction of a vector carrying the micronuclear versions of two macronuclear DNA molecules, one of which was modified by the insertion of a polylinker sequence. This vector was injected into the polytene chromosomes of the developing macronucleus of Stylonychia and its processing during further macronuclear development and its fate in the mature macronucleus were analyzed. In up to 30% of injected cells the modified macronuclear DNA sequence could be detected. While the internal eliminated sequences (IES) present in the macronuclear precursor DNA sequence are still retained in the mature macronucleus, the modified macronuclear DNA sequence is correctly cut out from the vector, telomeres are added de novo and it is stably retained in the macronucleus during vegetative growth of the cells. This vector system represents an experimental system that allows the identification of DNA sequences involved in the processing of macronuclear DNA sequences during macronuclear development.

There exist about 1000-1500 internal telomeric sequences per haploid genome in the polytene chromosomes of the hypotrichous ciliate Stylonychia lemnae. All these telomeric repeats are contained in a very conserved element. This element consists of two 2 kb direct repeats flanking a 2.6 kb sequence. Immediately adjacent to one of the repeats a 18mer C4A4C4A4C2 telomeric sequence is localized. Sequences homologous to macronuclear DNA follow 180 bp downstream of the C4A4-bloc. These macronuclear homologous sequences are flanked by the second direct repeat. The possible origin and function of these telomere containing elements is discussed.

Evidence is presented that macronuclear chromatin in the hypotrichous ciliate Stylonychia mytilus occurs in discrete fragments, each representing at least single genes. The size of these fragments varies between 3 and more than 70 nucleosomes with an average length of about 18 nucleosomes. This observation is discussed with respect to macronuclear structure of hypotrichous ciliates.