Research Paper Volume 1, Issue 3 pp 328—334

The S-adenosylmethionine dependent O-methyltransferase PaMTH1: a longevity assurance factor protecting Podospora anserina against oxidative stress

Figure 1. Construction and validation of a PaMth1 deletion strain of P. anserina. (A) Physical maps and sizes of the restriction products for the genomic region bearing PaMth1 and the recombined version with the hygromycin B resistance cassette. Reading frames of the two genes are indicated in grey. The genomic sequences flanking PaMth1 are indicated by punctuation. Restriction sites of EcoRV are indicated. (B) Southern blot analysis of Eco RV digested wild-type strain s genomic DNA and genomic DNA of two secondary transformants isolated from a primary deletion strain. The PaMth1 gene-specific probe (left panel) detects the 3.55 kbp fragment only in the sample of the wild-type s (wt) but not in the samples of the deletions strains (ΔPaMth1). The hygromycin B resistance gene-specific probe (right panel) detects the 3.26 kbp fragment only in the sample of the deletions strains. (C) Western blot analysis verifying the successful construction of a PaMth1 deletion strain using total- and mitochondrial protein samples of juvenile and senescent P. anserina wild-type strain s and the secondary transformants of the deletion strain, respectively. The PaMTH1 specific antibody detects PaMTH1 in the samples of the wild-type s strains but not in samples of the deletion strains. As loading controls an actin specific antibody for total proteins and a porin specific antibody for the mitochondrial proteins were used.