Colored bars indicate positions of

gene-specific primers (GSPs) designed for RACEs, with those for 3′ RACE shown on top and those for 5′ RACE shown at the bottom. Table 1 Comparison of KU70 / 80 organization between fungal homologues Gene Strain GenBank accession no. CDS (nt) CDS CG (%) Intron no. Intron CG (%) Average intron length (nt) Reference KU70 N. crassa AB177394 2046 51.4 2 45.4 54 [18] A. niger EF061656 2283 50.7 5 45.4 67 [19] C. neoformans XM_573016 1683 48.0 10 46.6 117 [20] Y. lipolytica CR382129 1758 48.9 0 – - [21, 22] XM_501610 R. toruloides KF850470 2121 59.8 15 61.1 61 This study KU80 N. crassa AB177395 2764 51.2 7 48.3 111 [18] C. neoformans XM_568810 2511 47.9 13 43.4 53 [20] Y. lipolytica CR382131 2181 Angiogenesis inhibitor GDC-0994 supplier 48.6 1 37.5 48 [21, 22] XM_503443 R. toruloides KF850471 2769 62.1 10 61.1 66 This study Note: CDS: coding sequence; nt: nucleotide. The Ku70 ORF sequence was predicted to encode for a protein of 706 amino acids with a molecular weight of 79.5 kDa. Ku70 showed 25% to 30% identities to those from Homo sapiens, Neurospora crassa, Aspergillus niger and

Cryptococcus neoformans, with the N. crassa Ku70 being the closest homologue (Figure 2). Analysis of Ku70 against the SUPERFAMILY database [23] revealed a Ku70 core domain (aa 288–589) that is flanked by a N-terminal “von Willebrand” A (vWA)-like domain (aa 31–54, 82–258), and a C-terminal SAP domain (aa 631–663). The high sequence similarity 17-DMAG (Alvespimycin) HCl and presence of signature domains conserved among Ku70 homologues suggest that the characterized Ku70 would be the key component of the NHEJ pathway in R. toruloides. Figure 2 Sequence comparison of Ku70s. Multiple sequence alignment of R. toruloides Ku70 amino acid sequence (R_tor) with homologues from Homo sapiens (H_sap, P12956), A. niger (A_nig, ABN13872), N. crassa (N_cra, BAD16622) and C. neoformans (C_neo, XP_573016). The N-terminal von Williebrand

A (vWA)-like domain, a central core domain and the C-terminal SAP (SAF-A/B, Acinus and PIAS) domains are marked with arrow-lines. Targeted gene deletion in wild type R. toruloides and generation of KU70 null mutants To see whether targeted gene deletion could be achieved in wild type R. toruloides, KU70 was used as the first deletion target. A derivative of R. toruloides ATCC 10657 (Rt1CE6, named WT hereafter, our unpublished data), which contained a 17β-estradiol inducible Cre recombinase gene stably integrated into the genome and allowed the recycling of hygromycin selection marker, was used in ATMT using the KU70 deletion construct, pKOKU70 (Figure 3A). Eight candidates out of 96 transformants were screened for loss of the targeted deletion region as judged by multiplex PCR (absence of KU70 PCR product and presence of GPD1 reference PCR product, data not shown).