Figure 1.
Telomere-anchored PCR assay detects telomeres in A. nidulans.
A. The design of telomere-anchored PCR of C-tailed DNA using chromosome II-L. Forward primers were constructed at three different positions, labeled A, B, and C. Reverse primers were made to either a “G-only” sequence or to a G sequence that contained all six possible terminal sequences at the 3′end. B. PCR yielded products of expected sizes when telomere-anchored PCR primer 1 or 4 (TAP) were used with primer C (lanes 2 and 3, respectively) or primer B (lanes 4 and 5, respectively), and when telomere-anchored PCR primer 4 was used with primer A (lane 8) or primer B (lane 9). No PCR product was detected on non-tailed template without terminal transferase (TdT), using telomere-anchored PCR primer 4 and primer A (lane 11) or primer B (lane 12). The positive controls in lanes 6, 10, and 13 used primer A and a reverse primer internal to the telomere.
Figure 2.
All permutations are present at the ultimate telomeric repeat.
A. PCR products were obtained for all six telomere anchored PCR primers with no significant variation in intensity, indicating all six permutations at the ultimate sequence were amplified equally. The positive control used primers E and F. The template DNA was from an nku- strain. B. DNA sequence analysis performed on the PCR products of telomere-anchored primers 1 and 4 from panel A showed the presence of a G tract (highlighted in yellow) and the first telomere repeat (bracketed in red) as expected. The first synthesized telomeric repeat is shown in the second red bracket. The number of Gs varied from one cloned sequence to the next, and is indicated as “Gs” to the left. The number of telomeric repeats is shown at right. C. The template DNA was from strain GR5 having a wild-type nkuA gene. The telomere-anchored PCR was run using primer A coupled with the six different telomere-anchored permutation primers. PCR products were seen for all the telomere-anchored primers.
Table 1.
Cloned telomere length in wild-type and trtA deletion.
Figure 3.
Determining the optimal conditions and sensitivity of the telomere-anchored PCR assay.
A. PCR products were amplified for 30 cycles (lanes 2–5) or 45 cycles (lanes 7–10). Though the two telomere-associated sequences were visible at both 30 and 45 cycles (lanes 4, 5, 9, 10), the telomeric sequences were clearly seen at 45 cycles (lanes 2, 3, 7, 8). PCR was performed using either forward primer A (lanes 2 and 7) or B (lanes 3 and 8) coupled with telomere-anchored PCR primer 4. Telomere-associated sequences were used as controls in lanes 4, 5, 9, and 10. B. Diluted genomic DNA, ranging from 100 ng to 0.1 ng (lanes 2–8), was tailed and subjected to PCR analysis, and the intensity of PCR products was observed at a DNA concentration as low as 0.1 ng. PCR was performed using forward primer B coupled with telomere-anchored PCR primer 4.
Figure 4.
Comparable telomere lengths in A. nidulans hyphae and ascospores.
A. Color microscopy of intact sexual ascospores (arrowhead) determined to be >99% of total cell concentration. The conidium (arrow) was thus relatively rare after cleaning the cleistothecium, and one is shown here for comparison. B. Broken ascospore lysate obtained by using a FastPrep Homogenizer. C. No difference was observed between A. nidulans hyphal and ascospore telomere length (lanes 2 and 5). Telomere PCR products for both cell types were observed at ∼350 bp. Diagram to the left corresponds to regions used for densitometry; solid black rectangle indicates majority of products and white rectangle indicates larger telomeric products. Forward and reverse primers were used to amplify telomere associated sequences (TAS, lanes 3 and 6) and the Pot1 gene (lanes 4 and 7), both located chromosome internally as controls.
Figure 5.
Alignment of an A. nidulans ORF to known TERT motifs.
The ANID_03753.1 predicted protein sequence (AN_TERT) was aligned to the TERT sequence from mouse (MOUSE_TERT, accession number O70372) and S. pombe (SP_TERT, accession number O13339) using the Align program in Vector NTI (Invitrogen). The region of the alignment including AN_TERT residues 601 through 1120 were selected and are shown. The conserved telomerase reverse transcriptase motifs (Motif T, 1, 2, Motif A, Motif B, Motif C, Motif D and Motif E) are indicated above the aligned sequences. Residues identical in all three sequences are in shown with dark shading, whereas sequences that are similar or identical in only two sequences are shown in lighter shading.
Figure 6.
Telomerase mutants showed dramatic shortening of telomeres compared to wild-type as assayed by telomere-anchored PCR.
A. Genomic DNA was isolated from wild-type strains (GR5 in lane 1, TN02A7 in lane 2) and two putative trtA deletion mutants (TDP3-2 in lane 3, TDP3-32 in lane 4), digested with EcoRI and analyzed by Southern blot using PCR product A as probe. B. Telomere-anchored PCR was performed on genomic DNA isolated from wild-type and trtA deletion mutants with either forward primer A or B coupled with telomere-anchored PCR primer 4. At both eight hours and sixteen hours after transformation, trtA deletion mutants show significantly shorter telomeres (lanes 7-10, red dot in lane 8) than wild-type (lanes 1-2) and nkuA deletion mutant (lanes 3-6, blue dot in lane 4).