Table 1. Fission yeast strains used in this study. Name Genotype/description S. japonicus NIG2021 a single colony isolate from type strain IFO 1609 DSMZ70573 Windisch isolate CBS11777 type strain S. octosporus S. cryophilus S. pombe h2 ade6-M210 leu1-32 ura4-D18 h2 ade6-M210 leu1-32 h+ (Leupold standard strain L975) h2 (Leupold standard strain L972) h2 h2 erh1::kanMX6 h2 ade6-M210 leu1-32 erh1::kanMX6 h2 ade6-M210 leu1-32 [pREP1/SpErh1p-yEGFP] h2 ade6-M210 leu1-32 [pREP1/SpErh1p-3HA] h2 ade6-M210 leu1-32 [pREP1] h2 ade6-M210 leu1-32 erh1::kanMX6 [pREP1] FY7266 FY7269 FY7519 FY12697 ZBM1004 ZBM1005 ZBM1020 ZBM1021 ZBM1022 ZBM1023 ZBM1024 ZBM1025 ZBM1026 ZBM1027 ZBM1028 ZBM1030 h2 ade6-M210 leu1-32 erh1::kanMX6 [pREP1/SpErh1p] This study h2 ade6-M210 leu1-32 erh1::kanMX6 [pREP1/SjErh1p] This study h2 ade6-M210 leu1-32 erh1::kanMX6 [pREP1/HsERH] This study h2 ade6-M210 leu1-32 erh1-yEGFP::kanMX6 This study h2 ade6-M210 leu1-32 ura4-D18 erh1::kanMX6 This study 1The National Institute of Genetics, Japan. 2Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany. 3Centraal Bureau voor Schimmelcultures, the Netherlands. 4The Yeast Genetics Resource Center, Japan. doi:10.1371/journal.pone.0049059.t001 for 3 hours against the same buffer supplemented with 10% glycerol. Lysates were incubated with anti-HA epitope monoclonal antibody (F-7, Santa Cruz) followed by incubation with protein A- coated beads (Santa Cruz). After washing four times with the lysis buffer, immunoprecipitates were resolved on a 15% SDS/ polyacrylamide gel and stained with silver as described previously [9]. Cloning of the ERH Genes and Plasmid Constructs All amplifications by PCR were performed with a high-fidelity PfuTurbo hotstart DNA polymerase (Stratagene) and all constructs were verified by automated DNA sequencing. Cloning was performed according to standard procedures [46]. All genomic copies and cDNAs of the fission yeast ERH genes were amplified employing as templates total DNA or total cDNA isolated from wild-type strains (the first four strains in Table 1) and the following pairs of primers: forward (59-GCGGAATTCCCTTGCTCGA- TAAAAACAG-39) and reverse (59-GCGCTGCAGATTTAAAG- CATAGCAATGAG-39) for S. pombe, forward (59- CGCGAATTCTTCGTTCCATCATTCCTC-39) and reverse (59-GCGCTGCAGAATAATAGTATTTCAACGAA-39) for S. octosporus, forward (59-GCGGAATTCCCTTTC- CATTGCTCCTCTGTC-39) and reverse (59-GCGCTGCAG- CAGTAGGCGACATTAGAAGAT-39) for S. cryophilus and for- ward (59-GCGGAATTCCGTGAAAACACGTACGCG-39) and reverse (59-GCGCTGCAGTGAGACATACAAGAAAATAAG- Source NIG1 [51] DSMZ2 CBS3 [23] YGRC4 YGRC YGRC YGRC This study This study This study This study This study This study This study ERH Genes from Fission Yeasts 39) for S. japonicus; restriction sites introduced for cloning are underlined. Amplified DNA fragments were ligated into pUC18 (Amersham) at the EcoRI and PstI sites yielding plasmids with ERH genomic copies (pUC18/Sperh1, pUC18/Soerh1, pUC18/ Scerh1 and pUC18/Sjerh1; GenBank accession numbers: JN194210, JN194212, JN194213 and JN194211, respectively) and plasmids with ERH cDNAs (pUC18/cSperh1, pUC18/ cSoerh1, pUC18/cScerh1 and pUC18/cSjerh1; GenBank acces- sion numbers: JN194206, JN194208, JN194209 and JN194207, respectively). pFA6a-59arm(Sperh1)-Kan-39arm(Sperh1). The region of 271 bp immediately downstream from the stop codon of Sperh1+ was amplified from total DNA from S. pombe with forward (59- GCGAATTCAAATATCGAACACCATATTATG-39) and re- verse (59-GCACTAGTAGACAAATGCCATTTATTAGAAC- 39) primers and ligated into pFA6a-link-yEGFP-Kan [47] at the EcoRI and BcuI sites yielding pFA6a-link-yEGFP-Kan-39arm(- Sperh1). Similarly, the region of 201 bp immediately upstream from the start codon of Sperh1+ was amplified with forward (59- GCGTCGACTCGCTACTCAATAACTGTGCTAC-39) and re- verse (59-GCAGATCTTACGAGCCAAGTATTTTGAAG-39) primers and used to replace the sequence coding for link-yEGFP in pFA6a-link-yEGFP-Kan-39arm(Sperh1) by employing SalI and BglII. pFA6a-39ORF(Sperh1)-link-yEGFP-Kan-39arm(Sperh1). The region of 219 bp immediately upstream from the stop codon of Sperh1+ was amplified from total DNA from S. pombe with forward (59-GCGCTCGAGGAATCTATTGATGTCTCAA-39) and re- verse (59-GCGCTCGAGAAGGGATCTGTCTAGCAGC-39) primers and ligated into pFA6a-link-yEGFP-Kan-39arm(Sperh1) at the SalI site. pREP1/SpErh1p-link-yEGFP. The Sperh1+ ORF was amplified from pUC18/cSperh1 with forward (59-GATGTCGACCG- TAATGTCGCCTCCACCAGC-39) and reverse (59- GATCCCGGGAGGGATCTGTCTAGCAG-39) primers and ligated into pREP1 [48] at the SalI and Cfr9I sites yielding pREP1/SpErh1-nsc. The sequence coding for link-yEGFP was amplified from pFA6a-link-yEGFP-Kan with forward (59- GCGCCCGGGGTGATCGGTGACGGTGCTGGTTTA-39) and reverse (59-GCGCCCGGGTTATTTGTACAATTCATC- CAT-39) primers and ligated into pREP1/SpErh1-nsc at the Cfr9I site. pREP1/SpErh1p-3HA. The sequence coding for 3HA was amplified from pFA6a-3HA-His3MX6 [49] with forward (59- CAGCCCGGGAACATCTTTTACCCATAC-39) and reverse (59-GAGCCCGGGTCAGCACTGAGCAGCG-39) primers and ligated into pREP1/SpErh1-nsc at the Cfr9I site. pREP1/SpErh1p. The Sperh1+ CDS was amplified from pUC18/cSperh1 with forward (59-GATGTCGACCG- TAATGTCGCCTCCACCAGC-39) and reverse (59- GATCCCGGGTTAAGGGATCTGTCTAGC-39) primers and ligated into pREP1 at the SalI and Cfr9I sites. pREP1/SjErh1p. The Sjerh1+ CDS was amplified from pUC18/cSjerh1 with forward (59-GCGGTCGACTATGT- CATTTGCATCCATGG-39) and reverse (59-GATCCCGGGT- TATGCAGATACTTCCTCAT-39) primers and ligated into pREP1 at the SalI and Cfr9I sites. pREP1/HsERH. The human ERH CDS was amplified from pCR2.1/ER [9] with forward (59-GATGTCGACCG- TAATGTCTCACACCATT-39) and reverse (59- GATCCCGGGTTATTTCCCAGCCTG-39) primers and ligated into pREP1 at the SalI and Cfr9I sites. pEGFP-N1/SpErh1p. The Sperh1+ ORF was amplified from pUC18/cSperh1 with forward (59- GAGCTCGAGCAC- PLOS ONE | www.plosone.org 11 November 2012 | Volume 7 | Issue 11 | e49059