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We exploit the unusual genome organization of the ciliate cell to analyze the control of specific gene amplification during a nuclear differentiation process. Ciliates contain two types of nuclei within one cell, the macronucleus and the micronucleus; and after sexual reproduction a new macronucleus is formed from a micronuclear derivative. During macronuclear differentiation, most extensive DNA reorganization, elimination, and fragmentation processes occur, resulting in a macronucleus containing short DNA molecules (nanochromosomes) representing individual genetic units and each being present in high copy number. It is believed that these processes are controlled by small nuclear RNAs but also by a template derived from the old macronucleus. We first describe the exact copy numbers of selected nanochromosomes in the macronucleus, and define the timing during nuclear differentiation at which copy number is determined. This led to the suggestion that DNA processing and copy number control may be closely related mechanisms. Degradation of an RNA template derived from the macronucleus leads to significant decrease in copy number, whereas injection of additional template molecules results in an increase in copy number and enhanced expression of the corresponding gene. These observations can be incorporated into a mechanistic model about an RNA-dependent epigenetic regulation of gene copy number during nuclear differentiation. This highlights that RNA, in addition to its well-known biological functions, can also be involved in the control of gene amplification.

Background: Programmed DNA elimination and reorganization frequently occur during cellular differentiation. Development of the somatic macronucleus in some ciliates presents an extreme case, involving excision of internal eliminated sequences (IESs) that interrupt coding DNA segments (macronuclear destined sequences, MDSs), as well as removal of transposon-like elements and extensive genome fragmentation, leading to 98% genome reduction in Stylonychia lemnae. Approximately 20-30% of the genes are estimated to be scrambled in the germline micronucleus, with coding segment order permuted and present in either orientation on micronuclear chromosomes. Massive genome rearrangements are therefore critical for development., Methodology/principal Findings: To understand the process of DNA deletion and reorganization during macronuclear development, we examined the population of DNA molecules during assembly of different scrambled genes in two related organisms in a developmental time-course by PCR. The data suggest that removal of conventional IESs usually occurs first, accompanied by a surprising level of error at this step. The complex events of inversion and translocation seem to occur after repair and excision of all conventional IESs and via multiple pathways., Conclusions/significance: This study reveals a temporal order of DNA rearrangements during the processing of a scrambled gene, with simpler events usually preceding more complex ones. The surprising observation of a hidden layer of errors, absent from the mature macronucleus but present during development, also underscores the need for repair or screening of incorrectly-assembled DNA molecules.

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.

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.