111109_paszkowski

We cloned a genomic fragment anking the left border

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Unformatted text preview: of the met1 mutant. We extracted genomic DNA from the TSIexpressing T-DNA line using Nucleon Phytopure Plant and Fungal DNA Extraction Kits according to the manufacturer’s instruction (Amersham Pharmacia Biotech). We cloned a genomic fragment flanking the left border of the inserted T-DNA by thermal asymmetric interlaced PCR. We cloned PCR products into the T/A vector pCR2.1 (Invitrogen) and sequenced them. We compared the flanking genomic DNA with the A. thaliana genome database using The Arabidopsis Information Resource website. We carried out Southern hybridization analysis and DNA sequencing as previously described16. A second insertion in MET1 was identified in and supplied by the Syngenta Arabidopsis Insertion Library. We determined the T-DNA insertions in MET1 and heterozygous or homozygous MET1 mutant genotypes either by Southern blotting or by PCR with primers differentiating between wildtype and mutated loci. We obtained RT–PCR products for MET1 transcripts 68 with poly-T primer for cDNA synthesis, MEF-1 and MER-1 primers (Fig. 1a) for PCR. We used the gene encoding actin 2 (ACT2) as an RT–PCR control. Primer sequences used for genotyping MET1 mutants and for RT–PCR are available on request. Segregation tests using the dominant marker gene residing on the TDNA showed that the frequency of plants with the mutated allele was significantly lower than the expected 75% (see Supplementary Table 2 online). Frequent seed abortion observed in siliques of heterozygous plants corroborates gametophytic or early embryonic lethality of strains carrying the met1 mutated allele. The transmission frequency of the met1 mutated allele varied significantly between individual lines (see Supplementary Table 2 online), suggesting secondary effects of DNA demethylation in met1 strains. The loss of seeds was not restricted to homozygous mutant plants, as T-DNA transmission in most cases was significantly lower than the 66% expected for survival of all heterozygotes. Indeed, genotyping in T2 segregating populations identified 46 wild-type, 63 heterozygous and 3 homozygous mutant plants. Considering the expectation based on the number of wild-types, the number of heterozygotes was 32% lower and of homozygotes was 94% lower. To determine whether the distortion in heterozygotes occurred during maternal or paternal transmission, we did reciprocal outcrosses of plants heterozygous with respect to the mutations. Maternal transmission of the mutated allele, which resulted in viable seeds, was roughly equal to the wild-type but paternal transmission was 20% lower (see Supplementary Table 2 online). This may reflect a role for MET1 in transcriptional inactivation of the paternal genome during gametogenesis or early embryogenesis. DNA methylation analysis. To analyze the methylation status at 180-bp pericentromeric repeats, we carried out genomic bisulfite sequencing. We chose a sequence from BAC clone F9M8 on chromosome I (nucleotides 61,536–62,127) because a unique sequence flanks...
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This document was uploaded on 03/17/2014.

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