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Both subtelomeric regions are required and sufficient for specific DNA fragmentation during macronuclear development in Stylonychia lemnae.

Jönsson F, Steinbrück G, Lipps HJ - Genome Biol. (2001)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Zeilbiologie, Universität Witten/Herdecke, D-58448 Witten, Germany.

ABSTRACT

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.

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Injection of α1-GFPtel- into the developing macronucleus of the polytene chromosome stage. The presence of the injected construct in the macronuclear DNA of injected cells and its processing was analyzed by PCR. After injection, the cells were allowed to finish macronuclear development, then a PCR analysis was performed using DNA of 30-40 vegetatively growing cells as templates. Primers used were P27, derived from the endogenous α1-tubulin macronuclear molecule, and pGFPP2, derived from GFP sequences. (a) Schematic diagram of the α1-GFPtel- construct. (b) PCR products from the construct, from uninjected and injected cells were separated on an 1% agarose gel. M, molecular weight marker (1 kb ladder, Gibco, BRL). Lane 1, PCR product from construct DNA; lane 2, PCR product from uninjected cells; lane 3, PCR product from cells injected with α1-GFPtel-. The gel was hybridized with a DIG-labeled probe of the α1-GFPtel- vector DNA.
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Figure 6: Injection of α1-GFPtel- into the developing macronucleus of the polytene chromosome stage. The presence of the injected construct in the macronuclear DNA of injected cells and its processing was analyzed by PCR. After injection, the cells were allowed to finish macronuclear development, then a PCR analysis was performed using DNA of 30-40 vegetatively growing cells as templates. Primers used were P27, derived from the endogenous α1-tubulin macronuclear molecule, and pGFPP2, derived from GFP sequences. (a) Schematic diagram of the α1-GFPtel- construct. (b) PCR products from the construct, from uninjected and injected cells were separated on an 1% agarose gel. M, molecular weight marker (1 kb ladder, Gibco, BRL). Lane 1, PCR product from construct DNA; lane 2, PCR product from uninjected cells; lane 3, PCR product from cells injected with α1-GFPtel-. The gel was hybridized with a DIG-labeled probe of the α1-GFPtel- vector DNA.

Mentions: Our experiments using the 1.3 kb macronuclear precursor sequence showed that subtelomeric regions are required and are probably sufficient to direct DNA fragmentation and telomere addition. To test whether this is also true for other DNA molecules, a vector was constructed that contains only 195 bp of the leader- and 240 bp of the trailer sequences but no telomeric sequences of the Stylonychia α1-tubulin macronuclear molecule [28]. The α1-tubulin open reading frame (ORF) was replaced by the 717 bp ORF of the enhanced green fluorescent protein (GFP) [29] (Figure 6a). This construct was injected into the macronuclear anlage in the early polytene chromosome stage. Again, the injected cells were allowed to finish macronuclear development and a PCR analysis with DNA of 30-40 vegetative cells was performed. To detect the processed macronuclear molecules derived from the injected construct, a primer combination was used in which one primer (P27) hybridized to the 5'-α1-tubulin leader sequences while the second primer (GFPP2) was derived from GFP sequences (Figure 6a). As a PCR product could be obtained from cells injected with the α1-GFPtel- construct (Figure 6b,c; lanes 3), sequences required for fragmentation of the α1-tubulin macronuclear molecule must be located in the subtelomeric regions of the precursor sequences. No amplification was observed with primer GFPP2 and one primer from the flanking pUC vector sequences as primer combination using either total cellular DNA isolated from exconjugant cells 6-10 h after injection of α1-GFPtel- or from DNA isolated from injected vegetative cells as template, demonstrating that fragmentation had occurred (data not shown). These subtelomeric sequences therefore seem to be sufficient for correct processing of the macronuclear molecule. As in the case with other injected constructs, the copy number of the processed construct was very low compared with endogenous macronuclear tubulin genes, suggesting that not only sequences required for processing but also for regulating amplification are contained in the subtelomeric regions. The copy number of this product was obviously too low to allow detection of GFP expression using fluorescence microscopy.


Both subtelomeric regions are required and sufficient for specific DNA fragmentation during macronuclear development in Stylonychia lemnae.

Jönsson F, Steinbrück G, Lipps HJ - Genome Biol. (2001)

Injection of α1-GFPtel- into the developing macronucleus of the polytene chromosome stage. The presence of the injected construct in the macronuclear DNA of injected cells and its processing was analyzed by PCR. After injection, the cells were allowed to finish macronuclear development, then a PCR analysis was performed using DNA of 30-40 vegetatively growing cells as templates. Primers used were P27, derived from the endogenous α1-tubulin macronuclear molecule, and pGFPP2, derived from GFP sequences. (a) Schematic diagram of the α1-GFPtel- construct. (b) PCR products from the construct, from uninjected and injected cells were separated on an 1% agarose gel. M, molecular weight marker (1 kb ladder, Gibco, BRL). Lane 1, PCR product from construct DNA; lane 2, PCR product from uninjected cells; lane 3, PCR product from cells injected with α1-GFPtel-. The gel was hybridized with a DIG-labeled probe of the α1-GFPtel- vector DNA.
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Related In: Results  -  Collection

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Figure 6: Injection of α1-GFPtel- into the developing macronucleus of the polytene chromosome stage. The presence of the injected construct in the macronuclear DNA of injected cells and its processing was analyzed by PCR. After injection, the cells were allowed to finish macronuclear development, then a PCR analysis was performed using DNA of 30-40 vegetatively growing cells as templates. Primers used were P27, derived from the endogenous α1-tubulin macronuclear molecule, and pGFPP2, derived from GFP sequences. (a) Schematic diagram of the α1-GFPtel- construct. (b) PCR products from the construct, from uninjected and injected cells were separated on an 1% agarose gel. M, molecular weight marker (1 kb ladder, Gibco, BRL). Lane 1, PCR product from construct DNA; lane 2, PCR product from uninjected cells; lane 3, PCR product from cells injected with α1-GFPtel-. The gel was hybridized with a DIG-labeled probe of the α1-GFPtel- vector DNA.
Mentions: Our experiments using the 1.3 kb macronuclear precursor sequence showed that subtelomeric regions are required and are probably sufficient to direct DNA fragmentation and telomere addition. To test whether this is also true for other DNA molecules, a vector was constructed that contains only 195 bp of the leader- and 240 bp of the trailer sequences but no telomeric sequences of the Stylonychia α1-tubulin macronuclear molecule [28]. The α1-tubulin open reading frame (ORF) was replaced by the 717 bp ORF of the enhanced green fluorescent protein (GFP) [29] (Figure 6a). This construct was injected into the macronuclear anlage in the early polytene chromosome stage. Again, the injected cells were allowed to finish macronuclear development and a PCR analysis with DNA of 30-40 vegetative cells was performed. To detect the processed macronuclear molecules derived from the injected construct, a primer combination was used in which one primer (P27) hybridized to the 5'-α1-tubulin leader sequences while the second primer (GFPP2) was derived from GFP sequences (Figure 6a). As a PCR product could be obtained from cells injected with the α1-GFPtel- construct (Figure 6b,c; lanes 3), sequences required for fragmentation of the α1-tubulin macronuclear molecule must be located in the subtelomeric regions of the precursor sequences. No amplification was observed with primer GFPP2 and one primer from the flanking pUC vector sequences as primer combination using either total cellular DNA isolated from exconjugant cells 6-10 h after injection of α1-GFPtel- or from DNA isolated from injected vegetative cells as template, demonstrating that fragmentation had occurred (data not shown). These subtelomeric sequences therefore seem to be sufficient for correct processing of the macronuclear molecule. As in the case with other injected constructs, the copy number of the processed construct was very low compared with endogenous macronuclear tubulin genes, suggesting that not only sequences required for processing but also for regulating amplification are contained in the subtelomeric regions. The copy number of this product was obviously too low to allow detection of GFP expression using fluorescence microscopy.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Zeilbiologie, Universität Witten/Herdecke, D-58448 Witten, Germany.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus