Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 7

1.
Figure 2.

Figure 2. From: The common ancestral core of vertebrate and fungal telomerase RNAs.

Identification of NcrTER by a deep-sequencing approach. (A) The multi-step strategy for N. crassa telomerase purification and NcrTER identification. Telomerase was purified from nuclear extract by size-exclusion chromatography and anti-FLAG immunoprecipitation (IP). Telomerase activity was followed by TRAP assay through the purification steps. The RNA extracted from purified telomerase was sequenced using next-generation sequencing, followed by bioinformatics screening for putative template sequences. (B) The average read coverage, length, putative template sequence and gene annotation for the five top-ranking TER candidates. The candidates are ranked by the average read coverage.

Xiaodong Qi, et al. Nucleic Acids Res. 2013 January;41(1):450-462.
2.
Figure 1.

Figure 1. From: The common ancestral core of vertebrate and fungal telomerase RNAs.

Evolutionary relationships and telomere repeat sequence of major fungal subphyla. The evolutionary relationships of the Ascomycota subphyla (Pezizomycotina, Saccharomycotina and Taphrinomycotina) and the Basidiomycota subphyla (Pucciniomycotina, Ustilaginomycotina and Agaricomycotina) are depicted based on recent phylogenomic studies of fungal species (17,24,25). Branch lengths are not proportional to evolutionary time. The names of subkingdom Dikarya (D), subphylum Ascomycota (A) and subphylum Basidiomycota (B) are indicated at nodes in the tree. Telomere repeat sequences of representative species from each subphylum are shown. The vertebrate-type sequence TTAGGG is shown in red. The subphyla Pezizomycotina and Taphrinomycotina studied in this work are highlighted in blue.

Xiaodong Qi, et al. Nucleic Acids Res. 2013 January;41(1):450-462.
3.
Figure 3.

Figure 3. From: The common ancestral core of vertebrate and fungal telomerase RNAs.

Validation of the NcrTER gene. (A) Schematic for generating NcrTER template mutants. The parental N. crassa strain NC1 had a loss-of-function mutation (red cross) in the his-3 allele. Mutant NcrTER genes driven by the ccg-1 promoter were integrated into the genome at the his-3 locus via homologous recombination. The template sequences of the three NcrTER mutants are presented, with the inserted nucleotides shown in red. (B) Expression of the NcrTER template mutants promotes synthesis of mutant telomeric repeats. Sequences of mutant telomeric DNA repeats (blue) are shown, with the inserted nucleotides (red) highlighted.

Xiaodong Qi, et al. Nucleic Acids Res. 2013 January;41(1):450-462.
4.
Figure 4.

Figure 4. From: The common ancestral core of vertebrate and fungal telomerase RNAs.

Secondary structure model of the NcrTER core domains. (A) Number of fungal TER sequences identified. In this study, 73 new TER sequences were identified from six Pezizomycotina classes (red) and one Taphrinomycotina TER sequence from S. complicata. The number of TER sequences identified in each class is indicated. Branch length is not proportional to evolutionary distance. (B) Secondary structure model of the NcrTER core domains: the template–pseudoknot and the TWJ that includes the P5, P6 and P6.1 stems. Invariant nucleotides (red) or nucleotides with ≥80% identity (blue) as well as base pairings supported by co-variations (solid gray boxes) are indicated. Other major structural features shown include the core-enclosing helices 1 and 2, template boundary element, template and the pseudoknot helices PK1, PK2 and PK2.1. The regions without secondary structure determined are indicated by dotted lines, with the number of omitted residues indicated.

Xiaodong Qi, et al. Nucleic Acids Res. 2013 January;41(1):450-462.
5.
Figure 7.

Figure 7. From: The common ancestral core of vertebrate and fungal telomerase RNAs.

Conservation and diversification of the common ancestral core of vertebrate and fungal TERs. Left, a minimal consensus structure of TER depicts the core domains common to both vertebrate and fungal TERs. Right, secondary structure models of TER pseudoknot and TWJ cores from human, N. crassa, S. cerevisiae, S. pombe and S. complicata are shown, with their evolutionary relationships depicted. Nucleotides shown in red indicate ≥80% identity within vertebrates, Saccharomyces and filamentous ascomycete, or identical between S. pombe and S. complicata. Sequences omitted are denoted as dashed lines. The helical regions in the pseudoknot structure and the highly conserved loop L6.1 in the TWJ domain are shaded gray. The predicted and experimentally determined base triples in the pseudoknot are indicated by gray and green dashed lines, respectively. The RNA core domains required for reconstituting telomerase activity in vitro are indicated for individual species.

Xiaodong Qi, et al. Nucleic Acids Res. 2013 January;41(1):450-462.
6.
Figure 6.

Figure 6. From: The common ancestral core of vertebrate and fungal telomerase RNAs.

Reconstitution of S. pombe telomerase activity from two separate RNA fragments. (A) The secondary structure model of S. pombe TER1 core domains. The template–pseudoknot (T-PK) is composed of nt 83–957 and the TWJ is from nt 1029–1090. (B) Left, direct primer-extension assay of in vitro-reconstituted S. pombe telomerase. Telomerase was reconstituted in RRL from recombinant S. pombe TERT protein and 1 µM T7-transcribed TER1 RNA (Full-length: 1–1207; T-PK: 83–957 or TWJ: 1029–1090). The addition of ddATP and ddTTP in place of dATP and dTTP, respectively, in the reactions is indicated. RNase A (RNase) was added to the reaction in lane 2. A DNA size marker (M) was generated by labeling the 3′ end of the DNA oligonucleotide (5′-GTTACGGTTACAGGTTACG-3′) with α-32P-dGTP using TdT. A 15-mer 32P-end-labeled oligonucleotide was included as loading control (l.c.). The expected sequence of nucleotides incorporated is shown on the right of the gel. Right, Alignment of the DNA primer sequence with the template sequence of S. pombe TER1. The previously determined template region is denoted by an open box (46). Nucleotides added to the DNA primer are shown in bold, with the radioactive 32P-dGTP underlined.

Xiaodong Qi, et al. Nucleic Acids Res. 2013 January;41(1):450-462.
7.
Figure 5.

Figure 5. From: The common ancestral core of vertebrate and fungal telomerase RNAs.

Functional characterization of N. crassa telomerase activity. (A) Direct primer-extension assay of in vitro-reconstituted N. crassa telomerase. Recombinant NcrTERT protein was synthesized in RRL and assembled with 0.1 µM full-length 2049-nt NcrTER. The reconstituted N. crassa telomerase was analyzed by direct primer-extension assay using the telomeric primer (TTAGGG)3. The major bands (+9, +15, +21 and +27) are denoted on the right of lane 2, indicating the number of nucleotides added to the primer. Addition of RNase A (lane 3), the absence of NcrTER (lane 4) and the absence of NcrTERT (lane 5) is indicated above the gel. Human telomerase (H) served as a positive control (lane 1). A 32P-end-labeled 15-mer oligonucleotide was included as loading control (l.c.) before purification and precipitation of telomerase-extended products. (B) Secondary structure of the minimal 295-nt NcrTER template–pseudoknot fragment T-PK3. The T-PK3 fragment contains nucleotides 225–1515, with two internal deletions (nt 256–433 and 463–1288) replaced with tetraloops (bold, lower case), GAAA and GGAC, respectively. The template region is denoted by an open box. (C) The 39-nt NcrTER P6/6.1 RNA fragments with L6.1 mutations. Point mutations, U1854A (m1), G1856C (m2) and U1854A/G1856C (m3), in the L6.1 loop are indicated by solid circles (D). Mutations in loop L6.1 severely reduced telomerase activity. N. crassa telomerase was reconstituted in RRL from the recombinant NcrTERT protein and two separate RNA fragments, T-PK3 and P6/6.1, and were analyzed by direct primer-extension assay. The RNA fragments assayed within each reaction are denoted above the gel. Relative telomerase activity is shown under the gel (lane 4–6) and was determined by normalizing the total intensities of all bands within each lane to wild-type activity in lane 3. The relative activity was not determined (n.d.) for lanes 1 and 2. A 32P-end-labeled 15-mer oligonucleotide was included as the loading control (l.c.). (E) Pulse-chase time course analysis of N. crassa telomerase. In vitro-reconstituted telomerase was incubated with telomeric primer (TTAGGG)3 for 20 s at room temperature in the presence of radioactive α-32P-dGTP, dTTP and dATP. An aliquot of the reaction was removed and terminated after 20 s to determine the initial length of the product (lane 1). Chase reaction was initiated by adding 50 folds of non-radioactive dGTP and 10 folds of competitive DNA oligonucletide 5′-(TTAGGG)3-3′-amine. Aliquots of the chase reaction was removed and terminated after 2, 4, 6 or 8 min from the start of the chase reaction (lane 2–5). A 15-mer 32P-end-labeled oligonucleotide was added as loading control (l.c.). (F) Effect of template length on repeat addition processivity. Full-length NcrTER (1–2049) with either the wild-type 9-nt template (wt), 10-nt template (t1) or 11-nt template (t2) was reconstituted with NcrTERT protein in RRL and analyzed by direct primer-extension assay. The NcrTER template sequence is denoted by an open box, with the alignment region shaded. Relative processivity was determined by normalization to wild-type processivity and is shown below the gel. A 15-mer 32P-end-labeled oligonucleotide was included as loading control (l.c.).

Xiaodong Qi, et al. Nucleic Acids Res. 2013 January;41(1):450-462.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk