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Supplementary Information
A. Figure descriptions:
Supplementary Figure 1. Differential hybridisation to identify clones
corresponding to methylated genomic regions.
Supplementary Figure 2. Survey of DNA methylation in Oak Ridge (OR),
Mauriceville (M) and dim strains of N. crassa (see also Fig. 3).
Supplementary Figure 3. DNA-type transposon relics in Neurospora.
Supplementary Figure 4. Induction of DNA methylation by sequences with RIP
indices not predicted to induce methylation.
B. Figure legends:
Supplementary Figure 1. Differential hybridisation to identify clones
corresponding to methylated genomic regions. Example of replica blots of
colonies from independent clones generated with DNA from late fractions
(enriched in methylated DNA) from the MBD column that were hybridised with
probes from early (predominantly unmethylated) or late fractions (corresponding
to lanes 4 and lanes 9-11 in Fig. 1, respectively). Clones that hybridised strongly
to the late column fractions were analysed further.
Supplementary Figure 2. Further survey of DNA methylation in Oak Ridge
(OR), Mauriceville (M) and dim strains of N. crassa (see also Fig. 3 of primary
paper). Approximately 1µg of DNA from OR (FGSC 2489), M (FGSC 2225), dim-
1 (N2517), dim-2 (N1104), or dim-3 (N1089) were digested with DpnII (D) or
Sau3AI (S) and used for Southern hybridizations with probes from clones
generated in this study. The OR strain was grown in the presence (+) or absence
(-) of 1µg/ml Trichostatin A (TSA) as previously described1.
Supplementary Figure 3. DNA-type transposon relics in Neurospora. a, Three
new Tc1 Neurospora transposon relics, three new pogo relics and one hAT relic
were identified (boxed); no mariner-like transposon relics were found in
Neurospora. Transposons from Caenorhabditis elegans (Ce), human (Hs),
Drosophila simulans (Ds), Anopheles albimanus (Aa), Drosophila hydei (Dh),
Fusarium oxysporum (Fo), Aspergillus niger (An), Magnaporthe grisea (Mg),
Botryotinia fuckeliana (Bf), Drosophila melanogaster (Dm), Escherichia coli (Ec),
Antirrhinum majus (Am), Tolypocladium inflatum (Ti), and Schizosaccharomyces
pombe (Sp) centromere-binding protein Cbhp were aligned2 with predicted
translations of Neurospora crassa (Nc) methylated fragments (see Table 1;"X"
was substituted for predicted nonsense codons caused by RIP). b,Two groups of
Neurospora Tc1/mariner-type transposon relics. a. Predicted amino acid
sequence and alignment of a Nc nogo relic with Sp Cbhp, Dm pogo and human
Tigger13. c, Conserved DDE motifs of three novel Nc Tc1 relics. Alignments of a
dodo-1 relic with Ce Tc1, Aa Quetzal, Dh Minos4, a dodo-2 relic with Aa Ant15
and dodo-3 with Fo impala 6 are shown. All Neurospora DNA-type transposon
relics had extensive RIP mutations (predicted nonsense mutations are indicated
as *) but DDE/DDD motifs are conserved; in some cases, the single helix-turnhelix motif, paired-like and homeo-like domains involved in DNA binding are
conserved (data not shown). Numbers in parentheses indicate amino acids not
shown in the alignment. Numbering of the Neurospora elements refers to the
beginning of homology between the conceptually translated repeats.
Supplementary Figure 4. Induction of DNA methylation by sequences with RIP
indices not predicted to induce methylation. The 736 bp Neurospora DNA
segment of clone 5:A7, and two subfragments (dodo2 451 bp segment a and
non-dodo2 447 bp segment b) with EcoRI and BglII linkers, were cloned between
the EcoRI and BamHI sites [destroying the BglII site, (B)] in the his-3 gene
replacement vector pBM607. Transformants of Neurospora strain FGSC 6103
with the fragments integrated at his-3 were identified by Southern hybridizations
(not shown) and tested for their capacity to trigger DNA methylation by digestion
with HindIII (H) and BglII (B). All three induced methylation, as indicated by the
appearance of the 7 kb fragment (*). The 2.7 kb band results from absence of
methylation at the HindIII site; the 3.1 kb fragment in transformant b, which
represents the host his-3 locus, is evident because this strain is a heterokaryon,
with both transformed and untransformed nuclei.
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