HMA4 Quadruplication in Noccaea caerulescens Figure 3. Genomic organisation of HMA4 in N. caerulescens. Five overlapping genomic fosmids (horizontal blue lines) represented a 101,480 bp single locus of N. caerulescens. Genes and their transcriptional direction are represented by arrows and given A. thaliana Genome Identifier appellations (brown arrows, genes syntenic with A. thaliana HMA4 flanking regions; orange, quadruplicated NcHMA4 genes). Restriction endonuclease site locations used for fosmid fingerprinting are indicated in red; HindIII, green; BamHI, and teal; EcoRI. Numbers in brackets refer to genomic locations in base pairs. doi:10.1371/journal.pone.0017814.g003 Lines bearing the AtHMA4p::GUS construct showed expression in root and stem tissue, although no staining was observed in leaf tissues (Figure 5). For both NcHMA4-2p::GUS and AhHMA4- 3p::GUS constructs, transformed lines showed expression in most plant tissue including roots, shoots and stems (Figure 5). The GUS gene appeared to be similarly and more intensely expressed Figure 4. Genomic illustration of all four NcHMA4 tandem repeats. Exons are represented by orange squares flanked by introns (blue lines). The NcHMA4 library probe (yellow box) is illustrated at its site of hybridisation for each copy. Numbers above exons and below introns represent percentage sequence identities for each copy to a consensus NcHMA4 sequence using Dot Matrix (Vector NTI 11). doi:10.1371/journal.pone.0017814.g004 PLoS ONE | www.plosone.org 4 March 2011 | Volume 6 | Issue 3 | e17814