LA LEGISLACION DEPORTIVA EN COLOMBIA PARA PcD

DNA, or type 2, transposons have TIRs and TSDs, similar to MITEs, which are derived from them (Casacuberta and Santiago, 2003). However, the autonomous DNA transposons encode the transposase required for mobilisation (Fig. 5.6) and are typically found in low copy-number, due to their mechanism of transposition via a DNA intermediate (Casacuberta and Santiago, 2003). Transposons from three superfamilies of autonomous DNA transposons, Tc1/mariner, hAT and mutator, have been identified in fungi (Daboussi and Capy, 2003).

5.3.1 Wha

Analysis of “extra” sequence between N. coenophialum dmaW-1 and the Toru MITEs, compared with dmaW-2, Neotyphodium sp. Lp1 and E. festucae E2368, revealed a putative Mutator-like element relic, Wha (four) (Fig. 5.2). Wha has 95-bp TIRs and an 8-bp putative TSD. The 2413 bp between the putative TIRs was highly degenerate (75% AT) and shared 32% identity to a 312-aa putative protein (CHGG_06856) from Chaetomium globosum, with several closely related proteins in the C. globosum genome. When submitted for BLASTX analysis itself, this sequence had 31% identity with a transposase from the Hop Mutator-like element in Fusarium oxysporum (Chalvet et al., 2003).

Figure 5.6. Schematic of DNA transposon structure. TIRs are shown as light brown arrows. Sequence coding for the transposase is shown in dark blue. TSDs are shown as arrows to each side of the element.

There are no full length Wha sequences in the E. festucae E2368 genome assembly, although the entire contig 1978 consists of an almost-full-length copy, with 38 bp of the TIRs truncated at each end. Seven other contigs contained varying length copies of one end of Wha at one end of the contig. Alignment of all of the Wha sequence fragments revealed considerable degeneration of the element, including many stop codons, SNPs and 8 deletions in one or more element of 1 – 16 bp and a larger deletion in contig 566 of 113 bp. Several of the smaller deletions were shared between two or more copies, however a shared deletion at one site did not necessarily correlate with a shared deletion at a different site, indicating that these may have been de novo deletions at the same site in different elements, rather than arising from amplification of a deleted element. Analysis of the sequence adjacent to the Wha fragments revealed that the sequences on contigs 68 and 680, and 1909 and 1609 respectively are likely to be ends of the same elements as they share the 8 bp immediately adjacent to the ends as direct repeats, which are likely to be TSDs (Fig. 5.7).

Analysis of the integration site of the elements revealed that the element found at the end of contig 566 was likely to have inserted within an ORF coding for a predicted membrane protein (Table 5.4). BLASTX analysis of the sequence adjacent to integration of Wha in other contigs revealed the remainder of this ORF on contig 803, suggesting that these were ends of the same element that had integrated within this ORF. Of all the integration sites examined for each of the different transposons in this study, this was the only evidence of an element disrupting an ORF.

Figure 5.7. TSDs are shared between some Wha end sequences. Alignment of left (A) and right (B) ends of Wha elements is shown. TSDs are underlined. Sequences are labelled with associated gene or contig.

Nc, N. coenophialum; Ef, E. festucae E2368

Table 5.4. Insertion sites of Wha elements

a_{features are FGENESH CDS predictions from the E. festucae E2368 Gbrowse. These were annotated as in either the}

5ʹ or 3ʹ region, and the top BLASTP match, along with the top match with some indication of function where available, is given.

*These Wha element ends are likely to represent each end of the same element, which has interrupted an ORF

Fg, Fusarium graminearum; Nf, Neosartorya fischerii; Ac, Aspergillus clavatus; Af, Aspergillus fumigatus

5.3.2 Waru

A fragment of a second putative autonomous type 2 element, Waru (eight) was identified by BLASTN analysis as a repeat sequence downstream of lpsA on contig 1183 of the E. festucae E2368 genome sequence assembly (Fig. 5.1). This sequence was interrupted by a Rua retroelement, however, a full-length element was identified on contig 213. Waru is 3076 bp in length and contains 34-bp TIRs and a putative 3-bp TSD (TTA for contig 213, TAA (one end only) downstream of lpsA). This putative T(T/A)A TSD suggests Waru belongs to the PIF/harbinger superfamily of transposons, transposases from which leave this signature duplication at the target site. This family of transposons had not previously been identified in fungi and this was reflected by BLASTX analysis, with the Waru sequence showing 28 – 37% identity over 115 – 184 amino acids of many transposases from various plant transposons. No fungal transposases were present in the list of BLAST matches. As for Wha, the Waru transposase sequence contained many stop codons and is thus unlikely to be functional.

A query of the E. festucae E2368 genome sequence showed 88 sequences matching Waru with an E value of 1e-05 or less, a high copy number for a type 2 transposon. Further analysis revealed that many of the copies had undergone large deletions,

Contig (size) Position Adjacent featurea _{BLAST match Distance}

1978 (2553) 1 – 2553 n/a n/a n/a

1232 (8750) 7911 – 8750 A.3.6370 (5ʹ) Fg hypothetical protein (63%, XP_390027)

Nf AAA family ATPase (47%, XP_001258398)

431

1909 (3570) 2175 – 2839 none n/a n/a

68 (14750) 14157 – 14750 A.29.8830 (5ʹ) Fg hypothetical protein (53%, XP_388120)

Ac cell wall glucanase (48%, EAW06867)

1277 680 (27703) 1 – 545 A.9.2541 (3ʹ) Fg hypothetical protein (76%,XP_388121)

Ac palmitoyl CoA transferase subunit (57%, XP_001268294)

1284

566 (29911) 1 – 897* A.9.8723 ( ORF ) Af DUF895 membrane protein (66%, EAL85419) 1 803 (35664) 34950 – 35443* unannotated Af DUF895 membrane protein (71%, EAL85419) 1

between elements. At least 13 – 25 full-length Waru copies are expected to be present in the genome based on analysis of the BLAST results. Of the internally deleted sequences containing both TIRs some appear to have been transposed as more than one copy is present in the genome. However, no evidence of a MITE derived from Waru was observed as none of the short internally deleted copies predominated and none were amplified to high-copy number.