Limits...
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.

Show MeSH

Related in: MedlinePlus

The effect of mutagenizing the St-Cbs on DNA-processing. Cells were injected with either pCE5 (lane 1) or pCE5mut (lane 2). Cells were allowed to finish macronuclear development and a PCR reaction was performed using the primer combination P9/P548 for specific detection of the modified precursor sequence. M, molecular weight marker (1 kb ladder, Gibco BRL).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC25775&req=5

Figure 5: The effect of mutagenizing the St-Cbs on DNA-processing. Cells were injected with either pCE5 (lane 1) or pCE5mut (lane 2). Cells were allowed to finish macronuclear development and a PCR reaction was performed using the primer combination P9/P548 for specific detection of the modified precursor sequence. M, molecular weight marker (1 kb ladder, Gibco BRL).

Mentions: We examined the 70 bp of the 3'-subtelomeric region and the 5'-subtelomeric regions from position 230-350 for the presence of palindromic sequences, direct and inverted repeats, and found a six-base inverted repeat of the sequence 5'-TTGAAA at position 267-272 in the 5'-subtelomeric region and at position 20-25 in the 3'-subtelomeric region. This sequence is almost identical to the core of the E-Cbs, 5'-TTGAA, described in Euplotes [21] and can be found only once in each subtelomeric region. In order to demonstrate that this sequence is indeed required for specific DNA processing, we mutagenized the 5'-TTGAAA present in the 3'-subtelomeric region into 5'-TTCAGA. Thirty exconjugant cells were injected with this construct, and the same number of cells was injected with pCE5 as a control. In 23 cells injected with pCE5, a processed product could be obtained using the primer combination P9/P548. This number corresponds to the normal success rate of our injection procedure [25,27]. However, a processed product could not be detected in any of the 30 cells injected with the mutagenized construct pCE5mut (Figure 5). The failure to detect such a product cannot be explained by a variation in the success rate of injection but rather provides significant evidence that this inverted repeat acts as a Cbs.


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)

The effect of mutagenizing the St-Cbs on DNA-processing. Cells were injected with either pCE5 (lane 1) or pCE5mut (lane 2). Cells were allowed to finish macronuclear development and a PCR reaction was performed using the primer combination P9/P548 for specific detection of the modified precursor sequence. M, molecular weight marker (1 kb ladder, Gibco BRL).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC25775&req=5

Figure 5: The effect of mutagenizing the St-Cbs on DNA-processing. Cells were injected with either pCE5 (lane 1) or pCE5mut (lane 2). Cells were allowed to finish macronuclear development and a PCR reaction was performed using the primer combination P9/P548 for specific detection of the modified precursor sequence. M, molecular weight marker (1 kb ladder, Gibco BRL).
Mentions: We examined the 70 bp of the 3'-subtelomeric region and the 5'-subtelomeric regions from position 230-350 for the presence of palindromic sequences, direct and inverted repeats, and found a six-base inverted repeat of the sequence 5'-TTGAAA at position 267-272 in the 5'-subtelomeric region and at position 20-25 in the 3'-subtelomeric region. This sequence is almost identical to the core of the E-Cbs, 5'-TTGAA, described in Euplotes [21] and can be found only once in each subtelomeric region. In order to demonstrate that this sequence is indeed required for specific DNA processing, we mutagenized the 5'-TTGAAA present in the 3'-subtelomeric region into 5'-TTCAGA. Thirty exconjugant cells were injected with this construct, and the same number of cells was injected with pCE5 as a control. In 23 cells injected with pCE5, a processed product could be obtained using the primer combination P9/P548. This number corresponds to the normal success rate of our injection procedure [25,27]. However, a processed product could not be detected in any of the 30 cells injected with the mutagenized construct pCE5mut (Figure 5). The failure to detect such a product cannot be explained by a variation in the success rate of injection but rather provides significant evidence that this inverted repeat acts as a Cbs.

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