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MATE transposable elements in Aspergillus nidulans: evidence of repeat-induced point mutation
Fungal Genetics and Biology 41 (2004) 308–316 www.elsevier.com/locate/yfgbi
MATE transposable elements in Aspergillus nidulans: evidence of repeat-induced point mutation A. John Clutterbuck* Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 6NU, Scotland, UK Received 5 September 2003; accepted 12 November 2003
Abstract The sequences of five MATE transposable elements were retrieved from the Aspergillus nidulans genome sequence. These elements are 6.1 kb in length and are characterized by 9–10 bp target site duplications, paired 40 bp palindromes close to each end, and in the unmutated elements, 57 clustered Spe-motifs (RWCTAGWY) scattered through their length. Short open reading frames have no known homology. Two of the MATE elements have numerous C ! T transitions on both DNA strands relative to the remaining three elements. These mutations have all the characteristics of repeat-induced point mutation (RIP) previously described in Neurospora crassa, but not experimentally demonstrated in A. nidulans. Ninety-eight percent of mutated cytosines are in CpG and CpA doublets, the former mutating at higher frequency. Ó 2003 Elsevier Inc. All rights reserved. Index descriptors: MATE element; Transposon; Transposable element; RIP; Repeat-induced point mutation; Aspergillus nidulans
1. Introduction Aspergillus nidulans mobile Aspergillus transformation enhancer (MATE) elements were discovered during investigation of the ARp1 autonomously replicating plasmid (Gems et al., 1991, reviewed by Aleksenko and Clutterbuck, 1997). The AMA1 sequence responsible for plasmid replication is a genomic insert derived from a segment of chromosome IV containing two back-toback MATE elements. Evidence of similar elements (MATEs 4, 5, and 9) was found at three other locations in Glasgow laboratory strains, and cross-hybridizing sequences are present in varied numbers and positions in wild isolates of A. nidulans, leading to the conclusion that MATE elements are, or have at some time been, transposable (Aleksenko and Clutterbuck, 1996). Only the portion of one element present in AMA1 has previously been sequenced (Aleksenko and Clutterbuck, 1996), and nothing is known about mode of transposition. *
Fax: +44-141-330-4878. E-mail address: [email protected]. URL: http://www.gla.ac.uk:443/ibls/staff/staff.php?who=H000155. 1087-1845/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2003.11.004
I here describe the sequences of the five MATE elements in the Glasgow laboratory strains of A. nidulans, two of which are confirmed to be heavily mutated throughout in a pattern typical of the repeat-induced point mutation (RIP) process first demonstrated in Neurospora crassa (reviewed by Selker, 1990).
2. Materials and methods Fragmentary evidence for multiple MATE sequences was initially obtained by interrogation of the Cereon– Monsanto Microbial Database for sequences that matched and extended the published AMA1 sequence (GenBank Acccession No. X78051, Aleksenko and Clutterbuck, 1996). The WICGR A. nidulans genome sequence (Aspergillus Sequencing Project, Whitehead Institute/MIT Center for Genome Research, URL: http://www-genome.wi.mit.edu/annotation/fungi/aspergillus/) substantiated the existence in the Glasgow wildtype strain (G00 ¼ FGSC-A4) of five closely related 6.1 kb MATE elements corresponding in flanking restriction patterns with those previously described by Aleksenko and Clutterbuck (1996) as MATE-1 (two
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Fig. 1. (A) Diagram of MATE elements aligned with the AMA1 sequence and WICGR contigs. Unmutated elements are shaded, those suffering RIP are hatched. (B) Summary of differences between MATE-9 and other MATEs; see Section 2 for details. For MATE-1b the differences shown refer to contig 1.122, rather than those reported for sequenced portions of the right arm of AMA1 (Aleksenko and Clutterbuck, 1996).
adjacent copies in back-to-back orientation, here designated -1a and -1b), and MATEs -4, -5, and -9. The genome also contains five scattered fragments of MATE sequence, of respectively, 100, 45, 41, 40, and 32 bp. DNA sequences were aligned using GeneJockey II, and doublet and tetrad frequencies were counted with Microsoft Word. WICGR contig 1.178 (GenBank Accession No. AACD01000178) includes, at co-ordinates 2111–8203, the full sequence of MATE-9 (Figs. 1 and 2), but is not incorporated into a supercontig nor aligned with the linkage map. However it overlaps contig 1.166 by 800 bases without disagreement, and contig 1.165 by 2919 bases, with five single base differences, all insertions into 1.178. If these differences are assumed to be sequencing/ compilation errors, then 1.178 forms a link between contigs 1.165 and 1.166, and places MATE-9 in supercontig 14, aligned with the left arm of linkage group VII. The contig 1.178 sequence of MATE-9 differs from the version in the last 796 bases of contig 1.165 (GenBank Accession No. AACD01000165) by one cytosine insertion after MATE-9 base 109. Since this insertion is not found in other MATE elements, the 1.165 version is shown in Figs. 1 and 2. WICGR contig 1.121 (supercontig 9, aligned with linkage group IV, left arm; GenBank Accession No. AACD01000121) contains at co-ordinates 10678–13642, a sequence of 2965 bases identifiable by the presence of an identical target site duplication (Fig. 3) as the left end of MATE-1a, whose right end is the sequenced arm of AMA1 (Aleksenko and Clutterbuck, 1996). The remainder of MATE-1a and the majority of the 375 bp
central sequence between the two arms of AMA1 (Aleksenko and Clutterbuck, 1996) are missing from the WICGR database, lying between contigs 1.121 and 1.122. Contig 1.121 differs from MATE-9 (Fig. 1) at two sites: MATE-9 AA161-2 are exchanged for TT in contig 1.121, and contig 1.121 adds an A after MATE-9 1393. The 2801 bases of MATE-1a in AMA1 (Aleksenko and Clutterbuck, 1996) differ from MATE-9 as follows: deletions: G3712*, A5894*, G5983*; addition: C after 4841; substitutions A5154T, T5388A, TC5749-50CT*; tandem duplication of ATCGTT5720-5. The adjacent WICGR contig 1.122 (GenBank Accession No. AACD01000122) contains, at co-ordinates 27-6120, ()ve orientation) a full copy of MATE-1b, identified by 10 bases of the unique central sequence as the partially sequenced right arm of AMA1 (Aleksenko and Clutterbuck, 1996). Nine of these bases are mirrored at the other end of MATE-1b as a target site duplication (Fig. 3). MATE-1b differs from MATE-9 (Fig. 1) by two guanine additions, after MATE-9 bases 2588 and 4291, and tandem duplication of the first 14 bases (Fig. 3). It also differs from the segments reported by Aleksenko and Clutterbuck (1996) to be identical to the AMA1 left arm, at the sites asterisked in the paragraph above. MATE-4 is represented by WICGR contig 1.45 (supercontig 3, linkage group VI; GenBank Accession No. AACD01000045), bases 43,715–51,173 ()ve orientation). It differs from MATE-9 by 264 transitions (see Section 3), and four single base insertions: G after MATE-9 1613, T after 2101, A after 3989, and G after 5949. In addition, MATE-4 contains a 1363-base
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Fig. 2. Nucleotide sequence of MATE-9, as derived from WICGR contigs 1.178 and 1.165: see Section 2 for details. Potential stem–loop structures (Fig. 4) at each end are underlined, and Spe-motifs (6/8 match to the RWCTAGWY consensus) are shown in red.
Fig. 3. Ends of MATE elements aligned to show target site duplications of 9 or 10 bases (in bold). MATE sequences are underlined; a tandem duplication of the first 14 bases of MATE-1b is italicized.
insertion after base 5962, identified as a Mariner/Tc1like transposable element (AJC unpublished). MATE-5 is represented by WICGR contig 1.108 (supercontig 7, linkage group I; GenBank Accession No. AACD01000108) bases 311,969–318,267. It differs from MATE-9 by 306 transitions (see Section 3), addition of a cytosine after base 1395 and GTAT after 1442, deletion of A3990, and substitution T4036G. MATE-5 also contains an insertion of 203 bases after 1616. This in-
sertion is identifiable as the footprint of an long terminal repeat (LTR) retrotransposon plus its target site duplication (AATAT), putatively resulting from excision of the body of the transposon by recombination between LTRs (AJC, unpublished). MATE element sequence data are available in the Third Party Annotation Section of the DDBJ/EMBL/ GenBank databases under the Accession Nos. TPA: BK001592 (MATE-9), BK001593 (MATE-1a),
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BK0015924 (MATE-1b), BK001595 (MATE-4), and BK001596 (MATE-5).
3. Results 3.1. Characteristics of MATE elements Five MATE sequences were retrieved from the WICGR A. nidulans genome assembly. The genome assembly also contains a few scattered MATE fragments of no more than 100 bases. Fig. 1 diagrams the relationship of the five MATE elements to the genome sequence contigs and summarizes their differences from MATE-9, which can be regarded as a basic, possibly ancestral, sequence. The DNA sequence of MATE-9 is shown in Fig. 2, and its doublet composition is in Table 1. The overall DNA composition (given by Table 1A, ‘‘total’’ column and row, bearing in mind that the first base is a guanine and the last a cytosine) is 42.6% G + C, compared to the overall WICGR genome composition of 50.3% G + C, and 47.2% G + C for non-coding regions (www-genome.wi.mit.edu/annotation/fungi/aspergillus/). In comparison with random base distribution, MATE-9 has a significant deficiency of CpG and ApT doublets, while ApG and CpT doublets are in excess. MATE elements are characterized by the following features: individual MATE elements have target site duplications (Fig. 3) of 9 bases (MATEs 1a, 1b, 4, and 5), or 10 bases (MATE-9). Secondly, as observed for AMA1 (Aleksenko and Clutterbuck, 1996), and predicted for extended MATE sequences (Aleksenko et al., 2001), MATE elements are characterized throughout their length by a high frequency of ‘‘Spe motif’’ clusters (shown in red in Fig. 2), with the consensus (RWCTAGWYNNN)1–4 (where R ¼ purine; Y ¼ pyrimidine; W ¼ A or T; N ¼ any base). The AMA1 se-
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quence has a palindromic potential stem–loop structure close to the right hand end of the sequenced MATE-1a fragment (Aleksenko and Clutterbuck, 1996). This is present in all MATE elements and is mirrored by similar, although not identical, structures at their left ends (Fig. 4). A number of short open reading frames (ORFs) can be identified in MATE-9, the longest spanning nucleotides 3184–3648 and encoding 154 amino acids. However none show significant similarity with any GenBank sequence. 3.2. Evidence of repeat-induced point mutation Aleksenko and Clutterbuck (1996) noted that the sequenced fragments of MATE-4 and -5 differ from MATE-1 and -9 by unidirectional transitions C ! T and G ! A. This observation is substantiated for the complete MATE elements: relative to MATE-9, MATE-4 shows 172 C ! T, 123 G ! A transitions, and one T ! C transition, while MATE-5 shows 144 C ! T and 188 G ! A transitions (Fig. 5). This contrasts with the relative scarcity of other differences (see Fig. 1 and Section 2), and strongly suggests the operation of a RIPlike process specifically generating C ! T substitutions (Selker, 1990), where G ! A substitutions are assumed to be the result of C ! T transitions on the lower strand. Other features of these transitions are typical of RIP products: overall frequencies are not equal (Figs. 1 and 5), either between elements or between strands of the same element; implying that mutation events affect a strand, or substantial portions of a strand, as a unit rather than a random scattering of bases throughout the affected sequences. Secondly, mutations are most frequent at the centre of the element and rarer at both ends (Fig. 5) as in N. crassa (Grayburn and Selker, 1989), which again implies a mechanism sensing a stretch of susceptible DNA, which for MATE elements, as in N.
Table 1 MATE-9 base composition A. Doublet composition of MATE-9
First base
A C G T Total
Second base A
C
G
T
Total
463 391 389 523
384 270 276 386
490 162 286 341
429 492 329 480
1766 1315 1280 1730
1766
1316
1279
1730
6091
B. Doublet observed/those expected on the basis of nucleotide frequency Second base A C First base A 0.90 1.01 C 1.03 0.95 G 1.05 1.00 T 1.04 1.03 *
p < 0:05 (v21 ). p < 0:001 (v21 ).
***
G 1.32 0.59 1.06 0.94
T 0.86 1.32 0.90 0.98
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wider sequence context has a role in mutation-site preference. Totaling C ! T substitutions for both strands of MATEs -4 and -5, with adjacent mutations included, gives 318 CpA ! TpA substitutions and 183 CpG ! TpG substitutions. However, because CpG doublets are relatively rare in the unaffected sequence (Table 1) the mutation frequency for CpG doublets is 50% higher than for CpA doublets. As a result of this doublet preference, RIP indices applied to N. crassa (Margolin et al., 1998), are ineffective indicators of RIP in A. nidulans. More significant measures of RIP in A. nidulans depend on comparisons with the unaffected sequence: % (C + G) for MATE-9 is 42.6, while in both MATEs -4 and -5 it is 38.3 and 37.8 respectively. Similarly, the CpG/GpC ratio in MATE-9 is 0.59, while in MATEs -4 and -5 it declines to 0.27 and 0.22, respectively. The number of substitutions coinciding in MATEs -4 and -5, predicted on the basis of random mutation of CpA and CpG doublets, would be 62.4, while the observed number is 137, an excess of 120%. This may indicate that these two copies were derived from a common ancestor that had suffered limited RIP before duplication. An alternative explanation is that coinciding mutations are due to hot-spots determined by sequence context, as outlined above.
4. Discussion 4.1. MATE transposons
Fig. 4. Imperfect palindromes at each end of MATE elements, drawn as potential stem-loop structures of the top strand. Dashes between the two structures indicate identity.
crassa, is a repeated sequence (Selker, 1990). While frequencies are fairly uniform along each strand, there are signs of local concentrations of mutations, suggesting that mutation-generating scans are shorter than the whole length of the element. Table 2 shows the sequence context of RIP in MATEs -4 and -5. With few exceptions (Table 2C) only cytosines in CpA (Table 2A) and CpG (Table 2B) doublets are subject to RIP. Adjacent mutation pairs (19 CpG ! TpA and 7 TpGpCpA/G ! TpApTpA/G) are omitted from Table 2 since each mutation will change the context of the other. It is evident from these data that guanines, and to a lesser extent, adenines, in the +2 (‘‘Y’’) position reduce RIP, especially for CpA doublets, and that a similar but less marked effect, applies in the -1 (‘‘X’’) position. There are signs of interactions between )1 and +2 sites, and it is also probable that the
The mechanism of MATE transposition is unknown. Target site duplications imply insertion of a DNA element into a staggered chromosome break, but this does not distinguish between type II DNA transposons and retrotransposons inserted as DNA after reverse transcription. The repeats at each end of the elements are ambiguous in that they could equally well be described as imperfect direct or inverted repeats; however, they do not extend to the ends of the elements and their palindromic form suggests that they could be recognition sites for a DNA transposase. Since no significant homology of the short open reading frames to any database sequences has been found, we do not know whether they encode any transposition functions. The remarkable ability of MATE fragments to facilitate plasmid replication in A. nidulans and its relatives (Gems et al., 1991), tempts speculation that replication may play a role in transposition. Aleksenko and Clutterbuck (1997) suggested that the Spe-motifs might represent double topoisomerase I recognition sites that would allow efficient unwinding during plasmid transcription and replication. It is therefore possible that accumulation of these motifs has allowed multiplication of free MATE
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Fig. 5. Histograms showing frequency of C to T and G to A transitions (vertical axis) per 100 bp in MATEs -4 and -5 (horizontal axes) relative to MATE-9.
elements during transposition. If this is the case, MATE elements must be assumed to circularize during transposition or heterologous transfer, since AMA1 does not facilitate replication of linear plasmids (Aleksenko and Ivanova, 1998). Such a mode of replication suggests a parallel with helitrons (Kapitov and Jurka, 2001), but these typically have palindromic sequences only at the 30 end, insert without target site duplications, and in active copies, encode a helicase. MATE-9, which can be considered as representing a possible ancestral sequence for the other MATE elements in the Glasgow strains (Fig. 1), shows a nonrandom doublet composition (Table 1), but there is no excess of TpA or deficiency of CpA doublets, and therefore no evidence of previous encounter with a RIP mechanism working on either the A. nidulans or N. crassa doublet preferences. 4.2. Repeat-induced point mutation Active RIP has not been demonstrated in Aspergillus, although indications that it has acted at some time in the
past have been reported in A. nidulans (Aleksenko and Clutterbuck, 1997, Nielsen et al., 2001) and in A. fumigatus (Neuveglise et al., 1996). It is also becoming clear that this process is widespread in fungi, although with differing site specificities (Table 3). Among these fungi, A. fumigatus and Fusarium oxysporum have no known sexual phase, which prompts the question whether RIP occurs at non-sexual stages of the life cycle of these fungi or is an ancient phenomenon relating to a sexual past. Although inserted into a heavily mutated region, the 203-base transposon relic interrupting MATE-5 is not itself mutated relative to the many other elements of the same type in the A. nidulans genome (data not shown). In N. crassa only repeats longer than 400 bp are susceptible to RIP, so this repeat may in itself be too short to be mutated by this means, and while mutation can extend beyond the repeat region in N. crassa, such mutation is very limited in extent (Irelan et al., 1994). However that fact that this insertion has no apparent effect on the pattern of mutation of the MATE element may mean that insertion occurred after the RIP process
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Table 2 Percentages of available sites mutated by C to T transitions relative to MATE-9, summed for both strands of MATE-4 and MATE-5, and shown according to sequence context A. C–T transitions for cytosines in context XCAY Base Y A Base X
G
T
Overall
A C G T
16.1 7.6 9.5 20.5
40.6 40.0 18.0 34.1
7.6 15.9 2.1 7.3
38.1 36.2 27.1 41.2
21.9 24.3 12.5 25.2
Overall
14.1
32.7
7.9
36.3
21.1
B. C–T transitions for cytosines in context XCGY Base Y A Base X
C
C
G
T
Overall
A C G T
26.8 31.7 10.9 26.3
32.1 54.5 21.9 34.8
18.4 37.5 13.6 12.2
39.5 52.6 25.0 56.1
29.0 42.3 17.2 32.5
Overall
23.5
34.4
22.1
44.8
30.8
C. Cytosines (in bold) mutated in other contexts MATE-4 MATE-5 ACCC ACCT ACTC CCTA TCCT
ACCA ACCT CCTA (2) GCCA TCTA
Adjacent pairs of substitutions, where the flanking bases will be altered by mutation, are omitted.
Table 3 Susceptibility of fungal cytosines to RIP with respect to adjacent basesa Fungus
30 preference
50 preference
Reference
A. nidulans A. fumigatus Leptosphaeria maculans Mycosphaerella graminicola: F. oxysporum: N. crassa P. anserina: Magnaporthe grisea
G>ACT G>AC GA A>G A>G A>T>G>C A>CGT T>AC>G
A, C or T C or T C or T A, C or T A, C or T A T A or T
This work Neuveglise et al. (1996) Idnurm and Howlett (2003) Goodwin and Tian (2002) Hua-Van et al. (1998) Grayburn and Selker (1989) Gra€ıa et al. (2001) Nakayashiki et al. (1999)
a
Relative frequencies of C to T transitions as a proportion of available sites of each type.
was complete. The Tc1/Mariner transposon inserted into MATE-4 (Figs. 1 and 5), also shows no clear evidence of RIP, but is close to the end of the MATE element where mutation is sparse. In N. crassa RIP invariably affects both copies of experimentally produced duplications (Fincham et al., 1989); it is therefore striking that A. nidulans MATEs -4 and 5 are heavily affected while MATEs -1a, -1b, and -9 have avoided RIP. Moreover, RIP in N. crassa is most effective on tandem duplications, but has not been applied to the adjacent A. nidulans MATE-1a/b pair. There are two plausible explanations for this: RIP may be an occasional event or may have declined in frequency, so that elements that have recently arrived from an exog-
enous source have not yet encountered the process. Aleksenko and Clutterbuck (1996) compared six wild isolates of A. nidulans and concluded that none possessed the MATE-1 pair found in the Glasgow laboratory strains, suggesting that these may be relatively recent insertions into the Glasgow wild-type. Other repeated elements in the A. nidulans genome show varied degrees of RIP, including in some cases either C ! T or G ! A transitions, but not both, indicating mutation on one strand only (AJC unpublished). This is in agreement with RIP in N. crassa and Podospora anserina where mildly mutated sequences show evidence of limited RIP on one DNA strand at a time. The heavily but unequally mutated DNA strands of
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MATEs -4 and -5 are therefore likely to be the products of multiple rounds of RIP at successive meioses, making an explanation in terms of rarity of RIP unlikely unless it depends on a historical change in occurrence of the process. The second possibility is that RIP depends on chromosomal location: MATE-1a and 1b are central on the left arm of chromosome IV and MATE-9 is in the middle of the short left arm of chromosome VII, at least 265 kb from either end. Telomeric repeats have not yet been identified on any of the chromosomes relevant to this study, but the RIP-affected MATE-5 is 41 kb from the centromere-distal left end of the known sequence of chromosome I, and MATE-4 is 62 kb from the centromere-distal right end of the chromosome VI sequence. RIP is believed to be initiated in N. crassa by pairing of ectopic repeats at the premeiotic dikaryon stage (Selker, 1990), and it is plausible that telomeric sequences in A. nidulans are particularly susceptible to ectopic pairing and recombination, as they are in yeast (Goldman and Lichten, 1996). 4.3. Cytosine methylation It is postulated that RIP C ! T transitions are generated by deamination of 5-methylcytosine, and Freitag et al. (2002) have demonstrated that a cytosine methyl transferase gene, rid, is essential for RIP inN. crassa and has homologues in the genomes of both A. nidulans and A. fumigatus. Vegetative methylation in N. crassa, mainly applied to sequences previously suffering RIP, is dependent on a separate enzyme, encoded by the dim-2 gene (Kouzminova and Selker, 2001). It is not yet clear whether free methylcytosine during the sexual phase of N. crassa is too rare or ephemeral to be detected, or whether the rid-encoded enzyme also deaminates cytosines directly. No methylcytosine was detected in A. nidulans by Antequera et al. (1984), but has more recently been found at very low levels in Aspergillus oryzae (Gowher et al., 2001).
Acknowledgments I am grateful to Cereon (now Monsanto) for initial use of their Microbial Sequence Database, and subsequently to the Aspergillus Sequencing Project, Whitehead Institute/MIT Center for Genome Research, for access to the A. nidulans genome database.
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MATE transposable elements in Aspergillus nidulans: evidence of repeat-induced point mutation A. John Clutterbuck* Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 6NU, Scotland, UK Received 5 September 2003; accepted 12 November 2003
Abstract The sequences of five MATE transposable elements were retrieved from the Aspergillus nidulans genome sequence. These elements are 6.1 kb in length and are characterized by 9–10 bp target site duplications, paired 40 bp palindromes close to each end, and in the unmutated elements, 57 clustered Spe-motifs (RWCTAGWY) scattered through their length. Short open reading frames have no known homology. Two of the MATE elements have numerous C ! T transitions on both DNA strands relative to the remaining three elements. These mutations have all the characteristics of repeat-induced point mutation (RIP) previously described in Neurospora crassa, but not experimentally demonstrated in A. nidulans. Ninety-eight percent of mutated cytosines are in CpG and CpA doublets, the former mutating at higher frequency. Ó 2003 Elsevier Inc. All rights reserved. Index descriptors: MATE element; Transposon; Transposable element; RIP; Repeat-induced point mutation; Aspergillus nidulans
1. Introduction Aspergillus nidulans mobile Aspergillus transformation enhancer (MATE) elements were discovered during investigation of the ARp1 autonomously replicating plasmid (Gems et al., 1991, reviewed by Aleksenko and Clutterbuck, 1997). The AMA1 sequence responsible for plasmid replication is a genomic insert derived from a segment of chromosome IV containing two back-toback MATE elements. Evidence of similar elements (MATEs 4, 5, and 9) was found at three other locations in Glasgow laboratory strains, and cross-hybridizing sequences are present in varied numbers and positions in wild isolates of A. nidulans, leading to the conclusion that MATE elements are, or have at some time been, transposable (Aleksenko and Clutterbuck, 1996). Only the portion of one element present in AMA1 has previously been sequenced (Aleksenko and Clutterbuck, 1996), and nothing is known about mode of transposition. *
Fax: +44-141-330-4878. E-mail address: [email protected]. URL: http://www.gla.ac.uk:443/ibls/staff/staff.php?who=H000155. 1087-1845/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2003.11.004
I here describe the sequences of the five MATE elements in the Glasgow laboratory strains of A. nidulans, two of which are confirmed to be heavily mutated throughout in a pattern typical of the repeat-induced point mutation (RIP) process first demonstrated in Neurospora crassa (reviewed by Selker, 1990).
2. Materials and methods Fragmentary evidence for multiple MATE sequences was initially obtained by interrogation of the Cereon– Monsanto Microbial Database for sequences that matched and extended the published AMA1 sequence (GenBank Acccession No. X78051, Aleksenko and Clutterbuck, 1996). The WICGR A. nidulans genome sequence (Aspergillus Sequencing Project, Whitehead Institute/MIT Center for Genome Research, URL: http://www-genome.wi.mit.edu/annotation/fungi/aspergillus/) substantiated the existence in the Glasgow wildtype strain (G00 ¼ FGSC-A4) of five closely related 6.1 kb MATE elements corresponding in flanking restriction patterns with those previously described by Aleksenko and Clutterbuck (1996) as MATE-1 (two
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Fig. 1. (A) Diagram of MATE elements aligned with the AMA1 sequence and WICGR contigs. Unmutated elements are shaded, those suffering RIP are hatched. (B) Summary of differences between MATE-9 and other MATEs; see Section 2 for details. For MATE-1b the differences shown refer to contig 1.122, rather than those reported for sequenced portions of the right arm of AMA1 (Aleksenko and Clutterbuck, 1996).
adjacent copies in back-to-back orientation, here designated -1a and -1b), and MATEs -4, -5, and -9. The genome also contains five scattered fragments of MATE sequence, of respectively, 100, 45, 41, 40, and 32 bp. DNA sequences were aligned using GeneJockey II, and doublet and tetrad frequencies were counted with Microsoft Word. WICGR contig 1.178 (GenBank Accession No. AACD01000178) includes, at co-ordinates 2111–8203, the full sequence of MATE-9 (Figs. 1 and 2), but is not incorporated into a supercontig nor aligned with the linkage map. However it overlaps contig 1.166 by 800 bases without disagreement, and contig 1.165 by 2919 bases, with five single base differences, all insertions into 1.178. If these differences are assumed to be sequencing/ compilation errors, then 1.178 forms a link between contigs 1.165 and 1.166, and places MATE-9 in supercontig 14, aligned with the left arm of linkage group VII. The contig 1.178 sequence of MATE-9 differs from the version in the last 796 bases of contig 1.165 (GenBank Accession No. AACD01000165) by one cytosine insertion after MATE-9 base 109. Since this insertion is not found in other MATE elements, the 1.165 version is shown in Figs. 1 and 2. WICGR contig 1.121 (supercontig 9, aligned with linkage group IV, left arm; GenBank Accession No. AACD01000121) contains at co-ordinates 10678–13642, a sequence of 2965 bases identifiable by the presence of an identical target site duplication (Fig. 3) as the left end of MATE-1a, whose right end is the sequenced arm of AMA1 (Aleksenko and Clutterbuck, 1996). The remainder of MATE-1a and the majority of the 375 bp
central sequence between the two arms of AMA1 (Aleksenko and Clutterbuck, 1996) are missing from the WICGR database, lying between contigs 1.121 and 1.122. Contig 1.121 differs from MATE-9 (Fig. 1) at two sites: MATE-9 AA161-2 are exchanged for TT in contig 1.121, and contig 1.121 adds an A after MATE-9 1393. The 2801 bases of MATE-1a in AMA1 (Aleksenko and Clutterbuck, 1996) differ from MATE-9 as follows: deletions: G3712*, A5894*, G5983*; addition: C after 4841; substitutions A5154T, T5388A, TC5749-50CT*; tandem duplication of ATCGTT5720-5. The adjacent WICGR contig 1.122 (GenBank Accession No. AACD01000122) contains, at co-ordinates 27-6120, ()ve orientation) a full copy of MATE-1b, identified by 10 bases of the unique central sequence as the partially sequenced right arm of AMA1 (Aleksenko and Clutterbuck, 1996). Nine of these bases are mirrored at the other end of MATE-1b as a target site duplication (Fig. 3). MATE-1b differs from MATE-9 (Fig. 1) by two guanine additions, after MATE-9 bases 2588 and 4291, and tandem duplication of the first 14 bases (Fig. 3). It also differs from the segments reported by Aleksenko and Clutterbuck (1996) to be identical to the AMA1 left arm, at the sites asterisked in the paragraph above. MATE-4 is represented by WICGR contig 1.45 (supercontig 3, linkage group VI; GenBank Accession No. AACD01000045), bases 43,715–51,173 ()ve orientation). It differs from MATE-9 by 264 transitions (see Section 3), and four single base insertions: G after MATE-9 1613, T after 2101, A after 3989, and G after 5949. In addition, MATE-4 contains a 1363-base
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Fig. 2. Nucleotide sequence of MATE-9, as derived from WICGR contigs 1.178 and 1.165: see Section 2 for details. Potential stem–loop structures (Fig. 4) at each end are underlined, and Spe-motifs (6/8 match to the RWCTAGWY consensus) are shown in red.
Fig. 3. Ends of MATE elements aligned to show target site duplications of 9 or 10 bases (in bold). MATE sequences are underlined; a tandem duplication of the first 14 bases of MATE-1b is italicized.
insertion after base 5962, identified as a Mariner/Tc1like transposable element (AJC unpublished). MATE-5 is represented by WICGR contig 1.108 (supercontig 7, linkage group I; GenBank Accession No. AACD01000108) bases 311,969–318,267. It differs from MATE-9 by 306 transitions (see Section 3), addition of a cytosine after base 1395 and GTAT after 1442, deletion of A3990, and substitution T4036G. MATE-5 also contains an insertion of 203 bases after 1616. This in-
sertion is identifiable as the footprint of an long terminal repeat (LTR) retrotransposon plus its target site duplication (AATAT), putatively resulting from excision of the body of the transposon by recombination between LTRs (AJC, unpublished). MATE element sequence data are available in the Third Party Annotation Section of the DDBJ/EMBL/ GenBank databases under the Accession Nos. TPA: BK001592 (MATE-9), BK001593 (MATE-1a),
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BK0015924 (MATE-1b), BK001595 (MATE-4), and BK001596 (MATE-5).
3. Results 3.1. Characteristics of MATE elements Five MATE sequences were retrieved from the WICGR A. nidulans genome assembly. The genome assembly also contains a few scattered MATE fragments of no more than 100 bases. Fig. 1 diagrams the relationship of the five MATE elements to the genome sequence contigs and summarizes their differences from MATE-9, which can be regarded as a basic, possibly ancestral, sequence. The DNA sequence of MATE-9 is shown in Fig. 2, and its doublet composition is in Table 1. The overall DNA composition (given by Table 1A, ‘‘total’’ column and row, bearing in mind that the first base is a guanine and the last a cytosine) is 42.6% G + C, compared to the overall WICGR genome composition of 50.3% G + C, and 47.2% G + C for non-coding regions (www-genome.wi.mit.edu/annotation/fungi/aspergillus/). In comparison with random base distribution, MATE-9 has a significant deficiency of CpG and ApT doublets, while ApG and CpT doublets are in excess. MATE elements are characterized by the following features: individual MATE elements have target site duplications (Fig. 3) of 9 bases (MATEs 1a, 1b, 4, and 5), or 10 bases (MATE-9). Secondly, as observed for AMA1 (Aleksenko and Clutterbuck, 1996), and predicted for extended MATE sequences (Aleksenko et al., 2001), MATE elements are characterized throughout their length by a high frequency of ‘‘Spe motif’’ clusters (shown in red in Fig. 2), with the consensus (RWCTAGWYNNN)1–4 (where R ¼ purine; Y ¼ pyrimidine; W ¼ A or T; N ¼ any base). The AMA1 se-
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quence has a palindromic potential stem–loop structure close to the right hand end of the sequenced MATE-1a fragment (Aleksenko and Clutterbuck, 1996). This is present in all MATE elements and is mirrored by similar, although not identical, structures at their left ends (Fig. 4). A number of short open reading frames (ORFs) can be identified in MATE-9, the longest spanning nucleotides 3184–3648 and encoding 154 amino acids. However none show significant similarity with any GenBank sequence. 3.2. Evidence of repeat-induced point mutation Aleksenko and Clutterbuck (1996) noted that the sequenced fragments of MATE-4 and -5 differ from MATE-1 and -9 by unidirectional transitions C ! T and G ! A. This observation is substantiated for the complete MATE elements: relative to MATE-9, MATE-4 shows 172 C ! T, 123 G ! A transitions, and one T ! C transition, while MATE-5 shows 144 C ! T and 188 G ! A transitions (Fig. 5). This contrasts with the relative scarcity of other differences (see Fig. 1 and Section 2), and strongly suggests the operation of a RIPlike process specifically generating C ! T substitutions (Selker, 1990), where G ! A substitutions are assumed to be the result of C ! T transitions on the lower strand. Other features of these transitions are typical of RIP products: overall frequencies are not equal (Figs. 1 and 5), either between elements or between strands of the same element; implying that mutation events affect a strand, or substantial portions of a strand, as a unit rather than a random scattering of bases throughout the affected sequences. Secondly, mutations are most frequent at the centre of the element and rarer at both ends (Fig. 5) as in N. crassa (Grayburn and Selker, 1989), which again implies a mechanism sensing a stretch of susceptible DNA, which for MATE elements, as in N.
Table 1 MATE-9 base composition A. Doublet composition of MATE-9
First base
A C G T Total
Second base A
C
G
T
Total
463 391 389 523
384 270 276 386
490 162 286 341
429 492 329 480
1766 1315 1280 1730
1766
1316
1279
1730
6091
B. Doublet observed/those expected on the basis of nucleotide frequency Second base A C First base A 0.90 1.01 C 1.03 0.95 G 1.05 1.00 T 1.04 1.03 *
p < 0:05 (v21 ). p < 0:001 (v21 ).
***
G 1.32 0.59 1.06 0.94
T 0.86 1.32 0.90 0.98
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wider sequence context has a role in mutation-site preference. Totaling C ! T substitutions for both strands of MATEs -4 and -5, with adjacent mutations included, gives 318 CpA ! TpA substitutions and 183 CpG ! TpG substitutions. However, because CpG doublets are relatively rare in the unaffected sequence (Table 1) the mutation frequency for CpG doublets is 50% higher than for CpA doublets. As a result of this doublet preference, RIP indices applied to N. crassa (Margolin et al., 1998), are ineffective indicators of RIP in A. nidulans. More significant measures of RIP in A. nidulans depend on comparisons with the unaffected sequence: % (C + G) for MATE-9 is 42.6, while in both MATEs -4 and -5 it is 38.3 and 37.8 respectively. Similarly, the CpG/GpC ratio in MATE-9 is 0.59, while in MATEs -4 and -5 it declines to 0.27 and 0.22, respectively. The number of substitutions coinciding in MATEs -4 and -5, predicted on the basis of random mutation of CpA and CpG doublets, would be 62.4, while the observed number is 137, an excess of 120%. This may indicate that these two copies were derived from a common ancestor that had suffered limited RIP before duplication. An alternative explanation is that coinciding mutations are due to hot-spots determined by sequence context, as outlined above.
4. Discussion 4.1. MATE transposons
Fig. 4. Imperfect palindromes at each end of MATE elements, drawn as potential stem-loop structures of the top strand. Dashes between the two structures indicate identity.
crassa, is a repeated sequence (Selker, 1990). While frequencies are fairly uniform along each strand, there are signs of local concentrations of mutations, suggesting that mutation-generating scans are shorter than the whole length of the element. Table 2 shows the sequence context of RIP in MATEs -4 and -5. With few exceptions (Table 2C) only cytosines in CpA (Table 2A) and CpG (Table 2B) doublets are subject to RIP. Adjacent mutation pairs (19 CpG ! TpA and 7 TpGpCpA/G ! TpApTpA/G) are omitted from Table 2 since each mutation will change the context of the other. It is evident from these data that guanines, and to a lesser extent, adenines, in the +2 (‘‘Y’’) position reduce RIP, especially for CpA doublets, and that a similar but less marked effect, applies in the -1 (‘‘X’’) position. There are signs of interactions between )1 and +2 sites, and it is also probable that the
The mechanism of MATE transposition is unknown. Target site duplications imply insertion of a DNA element into a staggered chromosome break, but this does not distinguish between type II DNA transposons and retrotransposons inserted as DNA after reverse transcription. The repeats at each end of the elements are ambiguous in that they could equally well be described as imperfect direct or inverted repeats; however, they do not extend to the ends of the elements and their palindromic form suggests that they could be recognition sites for a DNA transposase. Since no significant homology of the short open reading frames to any database sequences has been found, we do not know whether they encode any transposition functions. The remarkable ability of MATE fragments to facilitate plasmid replication in A. nidulans and its relatives (Gems et al., 1991), tempts speculation that replication may play a role in transposition. Aleksenko and Clutterbuck (1997) suggested that the Spe-motifs might represent double topoisomerase I recognition sites that would allow efficient unwinding during plasmid transcription and replication. It is therefore possible that accumulation of these motifs has allowed multiplication of free MATE
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Fig. 5. Histograms showing frequency of C to T and G to A transitions (vertical axis) per 100 bp in MATEs -4 and -5 (horizontal axes) relative to MATE-9.
elements during transposition. If this is the case, MATE elements must be assumed to circularize during transposition or heterologous transfer, since AMA1 does not facilitate replication of linear plasmids (Aleksenko and Ivanova, 1998). Such a mode of replication suggests a parallel with helitrons (Kapitov and Jurka, 2001), but these typically have palindromic sequences only at the 30 end, insert without target site duplications, and in active copies, encode a helicase. MATE-9, which can be considered as representing a possible ancestral sequence for the other MATE elements in the Glasgow strains (Fig. 1), shows a nonrandom doublet composition (Table 1), but there is no excess of TpA or deficiency of CpA doublets, and therefore no evidence of previous encounter with a RIP mechanism working on either the A. nidulans or N. crassa doublet preferences. 4.2. Repeat-induced point mutation Active RIP has not been demonstrated in Aspergillus, although indications that it has acted at some time in the
past have been reported in A. nidulans (Aleksenko and Clutterbuck, 1997, Nielsen et al., 2001) and in A. fumigatus (Neuveglise et al., 1996). It is also becoming clear that this process is widespread in fungi, although with differing site specificities (Table 3). Among these fungi, A. fumigatus and Fusarium oxysporum have no known sexual phase, which prompts the question whether RIP occurs at non-sexual stages of the life cycle of these fungi or is an ancient phenomenon relating to a sexual past. Although inserted into a heavily mutated region, the 203-base transposon relic interrupting MATE-5 is not itself mutated relative to the many other elements of the same type in the A. nidulans genome (data not shown). In N. crassa only repeats longer than 400 bp are susceptible to RIP, so this repeat may in itself be too short to be mutated by this means, and while mutation can extend beyond the repeat region in N. crassa, such mutation is very limited in extent (Irelan et al., 1994). However that fact that this insertion has no apparent effect on the pattern of mutation of the MATE element may mean that insertion occurred after the RIP process
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Table 2 Percentages of available sites mutated by C to T transitions relative to MATE-9, summed for both strands of MATE-4 and MATE-5, and shown according to sequence context A. C–T transitions for cytosines in context XCAY Base Y A Base X
G
T
Overall
A C G T
16.1 7.6 9.5 20.5
40.6 40.0 18.0 34.1
7.6 15.9 2.1 7.3
38.1 36.2 27.1 41.2
21.9 24.3 12.5 25.2
Overall
14.1
32.7
7.9
36.3
21.1
B. C–T transitions for cytosines in context XCGY Base Y A Base X
C
C
G
T
Overall
A C G T
26.8 31.7 10.9 26.3
32.1 54.5 21.9 34.8
18.4 37.5 13.6 12.2
39.5 52.6 25.0 56.1
29.0 42.3 17.2 32.5
Overall
23.5
34.4
22.1
44.8
30.8
C. Cytosines (in bold) mutated in other contexts MATE-4 MATE-5 ACCC ACCT ACTC CCTA TCCT
ACCA ACCT CCTA (2) GCCA TCTA
Adjacent pairs of substitutions, where the flanking bases will be altered by mutation, are omitted.
Table 3 Susceptibility of fungal cytosines to RIP with respect to adjacent basesa Fungus
30 preference
50 preference
Reference
A. nidulans A. fumigatus Leptosphaeria maculans Mycosphaerella graminicola: F. oxysporum: N. crassa P. anserina: Magnaporthe grisea
G>ACT G>AC GA A>G A>G A>T>G>C A>CGT T>AC>G
A, C or T C or T C or T A, C or T A, C or T A T A or T
This work Neuveglise et al. (1996) Idnurm and Howlett (2003) Goodwin and Tian (2002) Hua-Van et al. (1998) Grayburn and Selker (1989) Gra€ıa et al. (2001) Nakayashiki et al. (1999)
a
Relative frequencies of C to T transitions as a proportion of available sites of each type.
was complete. The Tc1/Mariner transposon inserted into MATE-4 (Figs. 1 and 5), also shows no clear evidence of RIP, but is close to the end of the MATE element where mutation is sparse. In N. crassa RIP invariably affects both copies of experimentally produced duplications (Fincham et al., 1989); it is therefore striking that A. nidulans MATEs -4 and 5 are heavily affected while MATEs -1a, -1b, and -9 have avoided RIP. Moreover, RIP in N. crassa is most effective on tandem duplications, but has not been applied to the adjacent A. nidulans MATE-1a/b pair. There are two plausible explanations for this: RIP may be an occasional event or may have declined in frequency, so that elements that have recently arrived from an exog-
enous source have not yet encountered the process. Aleksenko and Clutterbuck (1996) compared six wild isolates of A. nidulans and concluded that none possessed the MATE-1 pair found in the Glasgow laboratory strains, suggesting that these may be relatively recent insertions into the Glasgow wild-type. Other repeated elements in the A. nidulans genome show varied degrees of RIP, including in some cases either C ! T or G ! A transitions, but not both, indicating mutation on one strand only (AJC unpublished). This is in agreement with RIP in N. crassa and Podospora anserina where mildly mutated sequences show evidence of limited RIP on one DNA strand at a time. The heavily but unequally mutated DNA strands of
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MATEs -4 and -5 are therefore likely to be the products of multiple rounds of RIP at successive meioses, making an explanation in terms of rarity of RIP unlikely unless it depends on a historical change in occurrence of the process. The second possibility is that RIP depends on chromosomal location: MATE-1a and 1b are central on the left arm of chromosome IV and MATE-9 is in the middle of the short left arm of chromosome VII, at least 265 kb from either end. Telomeric repeats have not yet been identified on any of the chromosomes relevant to this study, but the RIP-affected MATE-5 is 41 kb from the centromere-distal left end of the known sequence of chromosome I, and MATE-4 is 62 kb from the centromere-distal right end of the chromosome VI sequence. RIP is believed to be initiated in N. crassa by pairing of ectopic repeats at the premeiotic dikaryon stage (Selker, 1990), and it is plausible that telomeric sequences in A. nidulans are particularly susceptible to ectopic pairing and recombination, as they are in yeast (Goldman and Lichten, 1996). 4.3. Cytosine methylation It is postulated that RIP C ! T transitions are generated by deamination of 5-methylcytosine, and Freitag et al. (2002) have demonstrated that a cytosine methyl transferase gene, rid, is essential for RIP inN. crassa and has homologues in the genomes of both A. nidulans and A. fumigatus. Vegetative methylation in N. crassa, mainly applied to sequences previously suffering RIP, is dependent on a separate enzyme, encoded by the dim-2 gene (Kouzminova and Selker, 2001). It is not yet clear whether free methylcytosine during the sexual phase of N. crassa is too rare or ephemeral to be detected, or whether the rid-encoded enzyme also deaminates cytosines directly. No methylcytosine was detected in A. nidulans by Antequera et al. (1984), but has more recently been found at very low levels in Aspergillus oryzae (Gowher et al., 2001).
Acknowledgments I am grateful to Cereon (now Monsanto) for initial use of their Microbial Sequence Database, and subsequently to the Aspergillus Sequencing Project, Whitehead Institute/MIT Center for Genome Research, for access to the A. nidulans genome database.
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