Both subtelomeric regions are required and sufficient for specific DNA fragmentation during macronuclear development in Stylonychia lemnae
© BioMed Central Ltd 2001
Received: 21 August 2000
Accepted: 5 December 2000
Published: 25 January 2001
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.
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 αl-tubulin gene, we show that subtelomeric regions are not only required but are also sufficient for DNA processing in Stylonychia.
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.
Programmed DNA-reorganization and DNA-elimination processes are frequently observed in differentiating eukaryotic cells. Examples include the mating type switch in yeast , the antigen variation in trypanosomes and other parasitic flagellates [2,3], the specific DNA elimination observed during embryogensis of nematodes, Cyclops and Sciara [4,5,6], and the processing of mammalian immunoglobulin and T-cell receptor genes [7,8,9]. However, these processes are most extreme in hypotrichous ciliates, which therefore serve as model systems to study the molecular basis of programmed DNA reorganization, DNA elimination and specific DNA fragmentation during cellular differentiation .
The molecular mechanisms of these processes are still not well understood, but in the holotrichous ciliate Tetrahymena, and lately in the hypotrichous ciliate Euplotes, conserved cis-acting sequences have been identified that are involved in directing the process of specific fragmentation. In Tetrahymena thermophila, a 15 base pair (bp) sequence, the chromosome breakage sequence (Cbs), is located in the eliminated sequences flanking the macronuclear precursor [18,19,20]. Sequence comparisons in Euplotes crassus have identified a 5 bp sequence, the proposed E-Cbs, that either resides inside the macronuclear precursor at position 18, or is located in the flanking micronuclear-specific DNA 12 bp from the macronuclear precursor [21,22]. A model for chromosome fragmentation in E. crassus was proposed on the basis of the positions of the E-Cbs and the finding of overlapping sequences, involving a 6 bp staggered cut on both sides of the macronuclear precursor [22,23]. To date, no consensus sequence has been found at defined positions near the fragmentation sites in Stylonychia lemnae . We therefore decided to identify cis-acting sequences involved in DNA fragmentation and telomere addition by injecting modified macronuclear precursor sequences into the developing macronuclei. Recently, we demonstrated that injection of such a construct (pCE5) into the developing macronucleus resulted in correct fragmentation and de novo telomere addition . This construct contained two macronuclear precursor sequences homologous to a 1.1 kb and a 1.3 kb macronuclear DNA molecule (; GenBank accession numbers X72955 and X72956). They are separated by an 11 bp spacer and are flanked by micronuclear-specific sequences. To distinguish between the injected precursor sequence and the endogenous macronuclear DNA molecule, the 1.3 kb precursor sequence was modified by inserting a 500 bp polylinker sequence. Moreover, we showed that neither sequences of the neighboring 1.1 kb macronuclear precursor sequence nor flanking micronuclear-specific sequences are required for specific fragmentation and telomere addition. Deletion of 70 bp of the 3' end of the 1.3 kb precursor sequence resulted in no detectable processing, however, indicating that a sequence located in this subtelomeric region is indeed required for fragmentation and/or telomere addition. In contrast, deletion of 69 bp of the 5' end still led to a processed product. Surprisingly, this processed product contained the subtelomeric sequences that were deleted in the construct, suggesting the presence of a so far uncharacterized proofreading mechanism during macronuclear development. Only after deletion of 520 bp of the 5' end was a processed product no longer observed .
Here, we show that both subtelomeric regions are required for correct DNA fragmentation but, at least in the case of the 1.3 kb precursor sequence, the distance of cis-acting sequences from the fragmentation sites are different in the 3' and 5' region. In addition, we show that the subtelomeric regions of the α1-tubulin gene are sufficient for correct DNA processing. Sequence analysis of all these regions revealed the presence of an inverted repeat with a sequence almost identical to the core E-Cbs described in Euplotes crassus.
It is still noteworthy that whenever processing was obtained from a 5'-deletion construct the processed product again contained the previously deleted sequences, suggesting the presence of a correction mechanism during macronuclear development as postulated before . To rule out that these results are due to PCR artefacts by template switching, we performed control experiments in which various amounts of construct DNA were mixed with total cellular DNA from uninjected cells. Under our experimental conditions, the ratio of injected construct DNA to total cellular DNA is below 10-6. In our control experiments, however, we obtained non-specific PCR amplification only by increasing the ratio of construct DNA to cellular DNA to at least 1:10 and at DNA concentrations above 50 μg/ml, as described previously . These results therefore make a PCR artefact by template switching very unlikely, although homologous recombination events can not be completely ruled out with these control elements.
We analyzed whether the inverted repeats found in the subtelomeric region of the 1.3 kb DNA molecule are also present in the subtelomeric regions of the α1-tubulin macronuclear DNA molecule. In both subtelomeric regions, the core inverted sequence 5'-TGAA could be identified and was found to be 5'-TTGAA at position 62-66 in the 5'-subtelomeric region and 5'-TGAAA at position 45-49 in the 3'-subtelomeric region. Again, these repeats were only found once in the subtelomeric regions of this DNA molecule.
Here we describe an experimental approach to characterize cis-acting DNA sequences involved in DNA processing during macronuclear development in Stylonychia lemnae. By sequence comparison, a putative fragmentation sequence (E-Cbs) has previously been located at defined positions either in the subtelomeric regions of macronuclear DNA molecules or in flanking micronuclear-specific sequences of Euplotes crassus . In contrast, by sequence analysis of a large number of Stylonychia macronuclear DNA molecules no such sequences could be detected at defined positons in the subtelomeric regions of these molecules . We therefore decided to use a biological approach to identify such sequences by injecting various constructs carrying a modified macronuclear precursor sequence into the macronuclear anlagen [25,27].
For the 1.3 kb macronuclear precursor sequence, we had already demonstrated that no micronuclear specific sequences are required for correct fragmentation and we localized a Cbs within the first 70 bp of the 3'-subtelomeric region. Deletion of the same number of base pairs from the 5' end still led to a processed product, however. Only after deletion of more than 500 bp from the 5' end could no product be detected. These results suggested that either the 3' sequence is the only Cbs required for fragmentation at both ends or that a second Cbs resides further downstream in the 5'-subtelomeric region. In fact, we now show that the 500 bp 5'-deletion construct was not fragmented, and the failure to detect a processed product from this construct was not just due to deletion of sequences required for amplification and replication later during macronuclear development. To characterize this putative Cbs in the 5'-subtelomeric region, we analyzed further deletion constructs for their ability to become processed. From these results we localize a 5'-Cbs to a position between 230 and 350. Interestingly, sequences absent from deletion constructs were restored in the processed product. Control experiments from an earlier study , and those made within this study, make a PCR artefact by template switching very unlikely. Homologous recombination does not seem sufficient to produce the products with the restored sequences because they were not obtained from all deletion constructs, but only from those containing the putative Cbs. Perhaps some as yet uncharacterized proofreading mechanism occurs during macronuclear development, possibly using transcripts from the old macronucleus as templates .
All experiments described so far were performed with only one macronuclear precursor sequence. To determine whether subtelomeric regions are not only required but are also sufficient for DNA processing in Stylonychia, a vector was constructed carrying only the subtelomeric regions of the DNA molecule encoding the α1-tubulin gene; the tubulin ORF was replaced by enhanced GFP. Correct processing of this construct could be demonstrated by PCR analysis, showing that in this case subtelomeric regions are required and indeed are sufficient for correct fragmentation and telomere addition. No or only weak GFP-expression could be detected by fluorescence microscopy. This again may be explained by the low copy number of the macronuclear molecule derived from this vector as indicated by the PCR analysis (Figure 6a,b; lane3) and would imply that sequences required for copy number control also reside within the subtelomeric regions.
A sequence comparison of the subtelomeric regions of the 1.3 kb precursor sequence containing the putative Cbs revealed the presence of a six-base inverted repeat resembling the core of the E-Cbs described in Euplotes crassus . A subsequent search showed that a similar sequence is also present in the subtelomeric regions of the α1-tubulin macronuclear DNA-molecule and in the subtelomeric region of many other (over 80%) macronuclear DNA molecules from Stylonychia lemnae (data not shown). As in the case of the 1.3 kb macronuclear molecule, this sequence could only be found once in each subtelomeric region. However, in contrast to Euplotes it never occurred at the same position in both subtelomeric regions. Although it was always found within the first 60 bp of the 3' end, the position at the 5' end was more variable and in all cases examined it is localized further downstream than at the 3' end. To analyze whether this sequence is indeed involved in DNA processing we mutagenized this sequence in the 3'-subtelomeric region of the 1.3 kb precursor sequence. The fact that a processed product was never observed after injection of this construct is a strong indication that this sequence is required for specific DNA processing during macronuclear development; we therefore now call it St-Cbs.
Our data indicate that no micronuclear specific DNA sequences are required for specific DNA fragmentation during macronuclear development of the hypotrichous ciliate Stylonychia. Instead they are found in both subtelomeric regions of macronuclear precursor sequences, although they do not have to be localized at identical positions with respect to the breakage site. We show that these sequences are not only required but also sufficient for DNA fragmentation. Moreover, a functional analysis of an inverted repeat found in this region revealed that it functions as a St-Cbs.
Materials and methods
Stylonychia lemnae were grown in neutral Pringsheim solution and fed daily with the alga Chlorogonium elongatum . To achieve conjugation, cells of two different mating types were mixed and the stages of macronuclear development were determined by phase microscopy. DNA at concentrations between 5-20 μg/ml of the various constructs was injected into the developing macronucleus of the polytene chromosome stage using the procedure described earlier [25,27]. Cells were allowed to finish macronuclear development and, after 15-20 cell divisions, DNA was isolated from vegetative macronuclei. Sometimes DNA was isolated from exconjugant cells only 6-10 h after injection. To characterize the DNA of injected cells, PCR analyses were performed. For these analyses, total cellular DNA isolated from 30-40 cells  was dissolved in 40 μl and aliquots of 10 μl were used for each PCR reaction carried out as described by Saiki et al. . The PCR program used was described in Wen et al. . Following PCR, the samples were separated on 1% agarose gels. The gels were blotted onto nylon membranes (Amersham Pharmacia Biotech) and hybridized with random primed probes  labeled with DIG-oxigenin-dUTP (Boehringer Mannheim).
Primers used for construction of the deletion vectors and for PCR analysis
Different deletion constructs
For mutagenesis of the sequence 5'-TTGAAA present in the 3'-subtelomeric region of the 1.3 kb macronuclear precursor sequence, a megaprimer was generated in a PCR reaction using the primer combination P20/PMUT2 (Table 1 and Figure 2b). This PCR product was then used as megaprimer for amplifying the whole mutagenized pCE5mut using pCE5 as a template. By simultaneously inserting a second mutation in pCE5mut, it was possible to identify the mutagenized product by restriction analysis with BcuI. To ensure correct amplification the PCR reactions were performed using Pfu-Polymerase (Stratagene).
This work was supported by the Deutsche Forschungsgemeinschaft and the Alfried Krupp von Bohlen und Halbach Foundation. We thank N.K. Jakob for his work on the stGFP construct and Sabine Feiler for technical assistance.
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