Important pseudoknots are conserved in the filamentous fungi.

<p><i>A. oryzae/A. flavus</i> and <i>A. nidulans</i> are shown in comparison to yeast and human sequences<b>.</b> Red nucleotides indicate U-A·U base triples in addition to nucleotides fitting the conserved proposed pseudoknot consensus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661-Gunisova1" target="_blank">[16]</a>. Numbered nucleotides for the <i>A. oryzae/A. flavus</i> pseudoknot are specific to <i>A. oryzae</i>. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661.s003" target="_blank">Figure S3</a> for additional Aspergilli pseudoknots.</p

The 3′ end of the Aspergilli TERs contain conserved elements that function in TER processing.

<p>The Sm site is slightly variable, while the 5′ splice site and branch point are much more conserved within the Aspergilli. Numbers within the sequence indicate the linker nucleotides between the 5′ splice site and the branch point. Asterisks indicate conserved nucleotides in all 13 species.</p

Strongly syntenous regions surround nine of the filamentous fungi TERs.

<p>The region to the right of TER shows more conservation of genes than the region to the left of TER. Chromosome VIII of A. nidulans contains TER, but no synteny is exhibited with these nine fungi, although five of the syntenous proteins were found dispersed across chromosome VIII of A. nidulans: vacuolar protein sorting protein, 3-oxoacyl-acyl carrier protein reductase, peptidyl-prolyl cis-trans isomerase, arrestin.</p

Phylogenetic-based secondary structure prediction revealed organization and functional elements conserved with other TER sequences.

<p>Boxed regions indicate similarity of the <i>A. oryzae</i> TER to other TERs: blue-yeast; red-vertebrates, green-similarity to both yeast and vertebrate. Red nucleotides indicate conservation across all 13 of the Aspergillus TERs.</p

A single transcribed sequence with a putative telomerase RNA template is identified in <i>A. oryzae</i>.

<p>A. RNA was reverse transcribed using one forward primer to amplify from the template towards the 5′ end (green). Different reverse primers were used to determine roughly where the transcript ended (green indicates RT-PCR product was present, red indicates RT-PCR product does not extend that far). Likewise, a reverse primer was used to amplify from the template to the 3′ end (purple). B. Sets of three products were analyzed by gel electrophoresis: RT-PCR reactions without RNase (−), reactions with RNase (+), and PCR reactions where genomic DNA was used instead of RNA (D). The set of three reactions is labeled by the genomic sequence (e.g. “H”), the strand orientation of the first primer (e.g. “3″) and the designation of the opposite primer of the pair (e.g. “J”). Alpha-tubulin primers on either side of an intron were used as a control, where an excised intron results in smaller products in the (−) lane than in the (D) lane. C. RLM-RACE results for the 3′ end (lanes 1 and 2) and the 5′ end (lanes 5 and 6). D. The sequence in green is the anticipated template. The sequences in blue were the primers used for the RT-PCR reaction that yielded products. Asterisks indicate ends that were determined by sequence analysis, some of which were redundant at the 3′ end.</p

Conservation of the three-way junction.

<p><i>A. oryzae</i>/<i>A. flavus</i>/<i>A. soaje</i> and <i>A. nidulans</i> are shown in comparison to yeast and human sequences. For the yeast and human sequences the same coloring scheme is used from Gunisova et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661-Gunisova1" target="_blank">[16]</a>. For the Aspergilli TERs nucleotides in blue are conserved for 12 of 13 Aspergilli. Combining information from the TERs examined by Gunisova et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661-Gunisova1" target="_blank">[16]</a>, the nucleotides in red are conserved in 63 of 68 TERs examined. Numbered nucleotides for the <i>A. oryzae/A. flavus/A. sojae</i> three-way junction are specific to <i>A. oryzae</i>. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661.s004" target="_blank">Figure S4</a> for additional Aspergilli three-way junctions.</p

Templates and template boundaries are conserved in the Aspergilli TERs.

<p>The TER template must contain a short repeated sequence at its 5′ and 3′ ends for alignment with the telomere end. This information allowed us to identify or confirm what telomeric repeats the template sequences of these organisms could synthesize, depending on whether the telomeric repeat sequence was already identified. For example, in <i>A. carbonarius</i> the template sequence begins with UAA at the 3′ end and ends with UAA at the 5′ end of the template, which would synthesize TTAGGG. It is unknown whether A. fumigatus, N. fischeri, A. nidulans, A. niger, and A. kawachii incorporate the C before the template boundary into their templates. Superscripts to the right of the telomeric sequences indicate the manner in which the telomeric repeats were determined or predicted: ¹Proposed by our lab based on template sequence; ²Proposed by researchers based on telomeric sequence (<i>A. clavatus</i> from the fungal genome database at Broad Institute, <a href="http://www.broadinstitute.org/science/data#" target="_blank">http://www.broadinstitute.org/science/data#</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661-Chang1" target="_blank">[56]</a>, <i>A. fumigatus </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661-Nierman1" target="_blank">[40]</a>, and <i>A. flavus </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661-Chang2" target="_blank">[65]</a>); ³Identified by Bal31digestion and southern blot (<i>A. nidulans </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661-Bhattacharyya1" target="_blank">[22]</a>, <i>A. oryzae </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661-Kusumoto1" target="_blank">[23]</a>); ⁴Proposed by our lab based on genome sequence data.</p

All permutations are present at the ultimate telomeric repeat.

<p><b>A.</b> 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 <i>nku-</i> strain. <b>B.</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. <b>C.</b> The template DNA was from strain GR5 having a wild-type <i>nkuA</i> 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.</p

Telomere-anchored PCR assay detects telomeres in <i>A. nidulans.</i>

<p><b>A.</b> 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>B.</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.</p

Alignment of an <i>A. nidulans</i> ORF to known TERT motifs.

<p>The ANID_03753.1 predicted protein sequence (AN_TERT) was aligned to the TERT sequence from mouse (MOUSE_TERT, accession number O70372) and <i>S. pombe</i> (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.</p