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Arabidopsis thaliana telomeric DNA-binding protein 1 is required for telomere length homeostasis and its Myb-extension domain stabilizes plant telomeric DNA binding.

Hwang MG, Cho MH - Nucleic Acids Res. (2007)

Bottom Line: Here, we demonstrated that lack of AtTBP1 results in a deregulation of telomere length control, with mutant telomeres expanding steadily by the fourth generation.DNA-binding studies with mutant AtTBP1 proteins showed that the Myb-extension domain of AtTBP1 is required for binding to plant telomeric DNA.Our results suggest that AtTBP1 is involved in the telomere length mechanism in A. thaliana and that the Myb-extension domain of AtTBP1 may stabilize plant telomeric DNA binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Yonsei University, Seoul 120-749, Republic of Korea.

ABSTRACT
Telomeres are specific protein-DNA complexes that protect the ends of eukaryotic chromosomes from fusion and degradation and are maintained by a specialized mechanism exerted by telomerase and telomere-binding proteins (TBPs), which are evolutionarily conserved. AtTBP1 is an Arabidopsis thaliana protein that binds plant telomeric DNA in vitro. Here, we demonstrated that lack of AtTBP1 results in a deregulation of telomere length control, with mutant telomeres expanding steadily by the fourth generation. DNA-binding studies with mutant AtTBP1 proteins showed that the Myb-extension domain of AtTBP1 is required for binding to plant telomeric DNA. Our results suggest that AtTBP1 is involved in the telomere length mechanism in A. thaliana and that the Myb-extension domain of AtTBP1 may stabilize plant telomeric DNA binding.

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Molecular complementation of AtTBP1 deficiency using wild-type AtTBP1 gene. (A) Analysis of the complemented plants by semi-quantitative RT-PCR. RNAs of complemented transformants were extracted from seedlings of T3 homozygous and heterozygous plants. AtTBP1 transcripts were amplified by 35 cycles of PCR with specific sets of primers (F1 and R3). The 60S RP L27A gene was used to indicate the amount of template for quantitative comparison (bottom panel). (B) TRF assay of complemented plants. Tru9I-digested DNAs from wild-type, AtTBP1 mutant plants, complemented heterozygous plants (compl.+/0) and complemented homozygous plants (compl.+/+) were analyzed.
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Figure 3: Molecular complementation of AtTBP1 deficiency using wild-type AtTBP1 gene. (A) Analysis of the complemented plants by semi-quantitative RT-PCR. RNAs of complemented transformants were extracted from seedlings of T3 homozygous and heterozygous plants. AtTBP1 transcripts were amplified by 35 cycles of PCR with specific sets of primers (F1 and R3). The 60S RP L27A gene was used to indicate the amount of template for quantitative comparison (bottom panel). (B) TRF assay of complemented plants. Tru9I-digested DNAs from wild-type, AtTBP1 mutant plants, complemented heterozygous plants (compl.+/0) and complemented homozygous plants (compl.+/+) were analyzed.

Mentions: To verify that the telomere elongation phenotype was associated with the T-DNA insertion in the AtTBP1 gene, molecular complementation was performed. A wild-type genomic fragment, containing a putative promoter sequence, was introduced into the AtTBP1 mutant background via in planta transformation. Selected T3 transformants were analyzed for AtTBP1 mRNA expression and telomere length (Figure 3). The complemented mutants expressed wild-type AtTBP1 mRNA ectopically (Figure 3A). In each case, the ectopic expression of AtTBP1 prevented the telomere extension and restored the telomere length to that of the wild-type plants (Figure 3B, lanes 4 and 5). Therefore, we conclude that attbp1−/− corresponds to a mutant allele of the AtTBP1 gene. These results also demonstrate that AtTBP1 is involved in the regulation of telomere length.Figure 3.


Arabidopsis thaliana telomeric DNA-binding protein 1 is required for telomere length homeostasis and its Myb-extension domain stabilizes plant telomeric DNA binding.

Hwang MG, Cho MH - Nucleic Acids Res. (2007)

Molecular complementation of AtTBP1 deficiency using wild-type AtTBP1 gene. (A) Analysis of the complemented plants by semi-quantitative RT-PCR. RNAs of complemented transformants were extracted from seedlings of T3 homozygous and heterozygous plants. AtTBP1 transcripts were amplified by 35 cycles of PCR with specific sets of primers (F1 and R3). The 60S RP L27A gene was used to indicate the amount of template for quantitative comparison (bottom panel). (B) TRF assay of complemented plants. Tru9I-digested DNAs from wild-type, AtTBP1 mutant plants, complemented heterozygous plants (compl.+/0) and complemented homozygous plants (compl.+/+) were analyzed.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851659&req=5

Figure 3: Molecular complementation of AtTBP1 deficiency using wild-type AtTBP1 gene. (A) Analysis of the complemented plants by semi-quantitative RT-PCR. RNAs of complemented transformants were extracted from seedlings of T3 homozygous and heterozygous plants. AtTBP1 transcripts were amplified by 35 cycles of PCR with specific sets of primers (F1 and R3). The 60S RP L27A gene was used to indicate the amount of template for quantitative comparison (bottom panel). (B) TRF assay of complemented plants. Tru9I-digested DNAs from wild-type, AtTBP1 mutant plants, complemented heterozygous plants (compl.+/0) and complemented homozygous plants (compl.+/+) were analyzed.
Mentions: To verify that the telomere elongation phenotype was associated with the T-DNA insertion in the AtTBP1 gene, molecular complementation was performed. A wild-type genomic fragment, containing a putative promoter sequence, was introduced into the AtTBP1 mutant background via in planta transformation. Selected T3 transformants were analyzed for AtTBP1 mRNA expression and telomere length (Figure 3). The complemented mutants expressed wild-type AtTBP1 mRNA ectopically (Figure 3A). In each case, the ectopic expression of AtTBP1 prevented the telomere extension and restored the telomere length to that of the wild-type plants (Figure 3B, lanes 4 and 5). Therefore, we conclude that attbp1−/− corresponds to a mutant allele of the AtTBP1 gene. These results also demonstrate that AtTBP1 is involved in the regulation of telomere length.Figure 3.

Bottom Line: Here, we demonstrated that lack of AtTBP1 results in a deregulation of telomere length control, with mutant telomeres expanding steadily by the fourth generation.DNA-binding studies with mutant AtTBP1 proteins showed that the Myb-extension domain of AtTBP1 is required for binding to plant telomeric DNA.Our results suggest that AtTBP1 is involved in the telomere length mechanism in A. thaliana and that the Myb-extension domain of AtTBP1 may stabilize plant telomeric DNA binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Yonsei University, Seoul 120-749, Republic of Korea.

ABSTRACT
Telomeres are specific protein-DNA complexes that protect the ends of eukaryotic chromosomes from fusion and degradation and are maintained by a specialized mechanism exerted by telomerase and telomere-binding proteins (TBPs), which are evolutionarily conserved. AtTBP1 is an Arabidopsis thaliana protein that binds plant telomeric DNA in vitro. Here, we demonstrated that lack of AtTBP1 results in a deregulation of telomere length control, with mutant telomeres expanding steadily by the fourth generation. DNA-binding studies with mutant AtTBP1 proteins showed that the Myb-extension domain of AtTBP1 is required for binding to plant telomeric DNA. Our results suggest that AtTBP1 is involved in the telomere length mechanism in A. thaliana and that the Myb-extension domain of AtTBP1 may stabilize plant telomeric DNA binding.

Show MeSH