Fig. 1. Molecular analysis of the AtMND1 T-DNA insertion mutant. (A) Schematic representation of the Atmnd1 mutant allele with the T-DNA insertion. The T-DNA insertion site as well as the orientation of left borders are indicated. The boxes represent exons, with UTRs in white and cDNA sequence in grey. The positions of the primers (arrows) and HindIII restriction sites (H) are marked. Expected DNA fragments after digestion are indicated by double arrows. (B) DNA sequence of the junction between T-DNA and genomic DNA. Capital letters indicate genomic sequences adjacent to the borders of the T-DNA. Lowercase letters indicate the deleted 85-bp genomic region. (C) DNA gel blot hybridisation of HindIII-digested genomic DNA with a genomic AtMND1 probe results in the production of 0.8 kb and 3.2 kb bands for wild-type (+/+), 0.8 kb, 2.2/2.3 kb and 3.2 kb bands for heterozygous (+/-) and 2.2/2.3 kb and 3.2 kb for homozygous (-/-) mutant plants. (D) PCR assay to distinguish wild-type, heterozygous and homozygous mutant plants. Primers M2, M3 and LBa1 were used in the same PCR reaction. (E) RT-PCR experiment to analyse the expression of AtMND1. The AtMND1 transcript (M) was detected in various wild-type tissues but not in the Atmnd1 mutant line. Amplification of the ACTIN (A) transcripts was used as a control. C represents a control experiment with the corresponding RNA as template in the PCR amplification reaction, using primers directed against ACTIN. The expected band size for AtMND1 cDNA is 0.7 kb and for ACTIN cDNA is 0.4 kb.