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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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Identification of AtTBP1 T-DNA insertion mutant. (A) Genomic map showing the position of the T-DNA insertion at the AtTBP1 locus. The arrows indicate the primers used for RT-PCR. Black rectangles represent exons and white rectangles are 5′ and 3′ UTR regions. E and H represent EcoRI and Hindlll restriction sites, respectively. A β-glucuronidase (GUS) cassette was used for DNA-gel blot. LB, left border; RB, right border. (B) Northern blot analysis of AtTBP1 transcripts in mutant and wild-type plants. Total RNA was hybridized with radiolabeled cDNA corresponding to the AtTBP1 5′ region (from exon 2 to exon 7). The lower row shows an ethidium bromide staining of rRNA. (C) Analysis of the disrupted attbp1-1 allele by semi-quantitative RT-PCR. AtTBP1 transcripts were amplified by 38 cycles of PCR with the indicated sets of primers. (D) The DNA sequence of the junction between the AtTBP1 cDNA and T-DNA insertion site obtained by RT-PCR. Intron sequences are shown in lowercase letters, and exon sequences are shown in uppercase letters. T-DNA sequences and homologous junction sequences are displayed in italic and boldface uppercase letters, respectively. A translation of the predicted protein is included.
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Figure 1: Identification of AtTBP1 T-DNA insertion mutant. (A) Genomic map showing the position of the T-DNA insertion at the AtTBP1 locus. The arrows indicate the primers used for RT-PCR. Black rectangles represent exons and white rectangles are 5′ and 3′ UTR regions. E and H represent EcoRI and Hindlll restriction sites, respectively. A β-glucuronidase (GUS) cassette was used for DNA-gel blot. LB, left border; RB, right border. (B) Northern blot analysis of AtTBP1 transcripts in mutant and wild-type plants. Total RNA was hybridized with radiolabeled cDNA corresponding to the AtTBP1 5′ region (from exon 2 to exon 7). The lower row shows an ethidium bromide staining of rRNA. (C) Analysis of the disrupted attbp1-1 allele by semi-quantitative RT-PCR. AtTBP1 transcripts were amplified by 38 cycles of PCR with the indicated sets of primers. (D) The DNA sequence of the junction between the AtTBP1 cDNA and T-DNA insertion site obtained by RT-PCR. Intron sequences are shown in lowercase letters, and exon sequences are shown in uppercase letters. T-DNA sequences and homologous junction sequences are displayed in italic and boldface uppercase letters, respectively. A translation of the predicted protein is included.

Mentions: To study the biological function of AtTBP1 in planta, we screened the INRAV Arabidopsis T-DNA insertion collection (19) and identified a putative mutant plant (FST 072C05) with a T-DNA insertion into the AtTBP1 gene. In the INRAV database, the insertion was annotated within the 10th exon, with the left border oriented toward the 3′ end of the AtTBP1 gene. To determine the genomic organization in detail, including the exact position of insertion, the AtTBP1–T-DNA junctions were amplified and sequenced. As shown in Figure 1A, the T-DNA insertion resulted in vast deletion of DNA from the 8th to the 10th exons. We refer to this mutation, which disrupts the gene in the region coding for the conserved DNA-binding motif, as the attbp1-1 allele. On selective medium, the progeny of self-pollinated heterozygotes segregated in a ratio of ∼3:1 for the selection marker (Basta-resistant:Basta-sensitive = 467:160; χ2[1df] = 0.09). This result suggests that the T-DNA insertion is inherited in a Mendelian manner and indicates a single T-DNA insertion in the AtTBP1 gene. Southern blot analysis, using T-DNA border sequence as a probe, confirmed that the T-DNA insertion is present at a single locus (supplementary Figure S1).Figure 1.


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)

Identification of AtTBP1 T-DNA insertion mutant. (A) Genomic map showing the position of the T-DNA insertion at the AtTBP1 locus. The arrows indicate the primers used for RT-PCR. Black rectangles represent exons and white rectangles are 5′ and 3′ UTR regions. E and H represent EcoRI and Hindlll restriction sites, respectively. A β-glucuronidase (GUS) cassette was used for DNA-gel blot. LB, left border; RB, right border. (B) Northern blot analysis of AtTBP1 transcripts in mutant and wild-type plants. Total RNA was hybridized with radiolabeled cDNA corresponding to the AtTBP1 5′ region (from exon 2 to exon 7). The lower row shows an ethidium bromide staining of rRNA. (C) Analysis of the disrupted attbp1-1 allele by semi-quantitative RT-PCR. AtTBP1 transcripts were amplified by 38 cycles of PCR with the indicated sets of primers. (D) The DNA sequence of the junction between the AtTBP1 cDNA and T-DNA insertion site obtained by RT-PCR. Intron sequences are shown in lowercase letters, and exon sequences are shown in uppercase letters. T-DNA sequences and homologous junction sequences are displayed in italic and boldface uppercase letters, respectively. A translation of the predicted protein is included.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Figure 1: Identification of AtTBP1 T-DNA insertion mutant. (A) Genomic map showing the position of the T-DNA insertion at the AtTBP1 locus. The arrows indicate the primers used for RT-PCR. Black rectangles represent exons and white rectangles are 5′ and 3′ UTR regions. E and H represent EcoRI and Hindlll restriction sites, respectively. A β-glucuronidase (GUS) cassette was used for DNA-gel blot. LB, left border; RB, right border. (B) Northern blot analysis of AtTBP1 transcripts in mutant and wild-type plants. Total RNA was hybridized with radiolabeled cDNA corresponding to the AtTBP1 5′ region (from exon 2 to exon 7). The lower row shows an ethidium bromide staining of rRNA. (C) Analysis of the disrupted attbp1-1 allele by semi-quantitative RT-PCR. AtTBP1 transcripts were amplified by 38 cycles of PCR with the indicated sets of primers. (D) The DNA sequence of the junction between the AtTBP1 cDNA and T-DNA insertion site obtained by RT-PCR. Intron sequences are shown in lowercase letters, and exon sequences are shown in uppercase letters. T-DNA sequences and homologous junction sequences are displayed in italic and boldface uppercase letters, respectively. A translation of the predicted protein is included.
Mentions: To study the biological function of AtTBP1 in planta, we screened the INRAV Arabidopsis T-DNA insertion collection (19) and identified a putative mutant plant (FST 072C05) with a T-DNA insertion into the AtTBP1 gene. In the INRAV database, the insertion was annotated within the 10th exon, with the left border oriented toward the 3′ end of the AtTBP1 gene. To determine the genomic organization in detail, including the exact position of insertion, the AtTBP1–T-DNA junctions were amplified and sequenced. As shown in Figure 1A, the T-DNA insertion resulted in vast deletion of DNA from the 8th to the 10th exons. We refer to this mutation, which disrupts the gene in the region coding for the conserved DNA-binding motif, as the attbp1-1 allele. On selective medium, the progeny of self-pollinated heterozygotes segregated in a ratio of ∼3:1 for the selection marker (Basta-resistant:Basta-sensitive = 467:160; χ2[1df] = 0.09). This result suggests that the T-DNA insertion is inherited in a Mendelian manner and indicates a single T-DNA insertion in the AtTBP1 gene. Southern blot analysis, using T-DNA border sequence as a probe, confirmed that the T-DNA insertion is present at a single locus (supplementary Figure S1).Figure 1.

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