Figure 3

<p>Sequence alignments showing the conserved motifs. (A) Conserved amino acids constituting the Alp-like (AM) motif, which was only present in the ALP family genes and neither found neither in the Enigma subfamily nor in their mutual precursor <i>eat-1/tungus.</i> (B) Conserved amino acids constituting the ZASP-like (ZM) motif found in both ALP subfamily and Enigma subfamily genes.</p

Figure 7

<p>Chromosomal locations of ALP and Enigma subfamily genes. Shown are four species were PDZ/LIM genes are co-localized on the same chromosome. Numbers indicated are distances in mega basepairs (bp). If genes were found on individual chromosomes they are not shown here. Further not shown are the results for <i>Pan troglodytes</i> and <i>Macaca mulatta</i> which show exactly the same chromosomal distribution as observed for the humans. Not shown is also the data for <i>Rattus norwegicus</i>, where LIMK1 was co localized with LMO7, but no other combination was found. No combinations whatsoever were found in Mus musculus and in Canis familaris. It must be noted that for some of the species investigated the genomes are not completely sequenced and/or fully assembled.</p

Figure 1

<p>Exon structure, domain composition and the six basic forms of PDZ/LIM genes. (A) The exon composition of the human PDZ-LIM domain encoding genes in alphabetical order. Indicated are <i>ALP</i> (ENSG00000154553), <i>ZASP</i> (ENSG00000122367, Elfin (ENSG00000107438), ENIGMA (ENSG00000196923), <i>Enigma-Homolog</i> (ENH) (ENSG00000163110) <i>LIMK1</i> (ENSG00000106683, OTTHUMG00000023448), <i>LIMK2</i> (ENSG00000182541), <i>LMO7</i> (ENSG00000136153), <i>Mystique</i> (ENSG00000120913) and RIL (ENSG00000131435). Domains are color coded on the exons: LIM yellow, PDZ blue, CH red and ZM motif green, while transcription start sites are indicated after non coding regions (colored white) with a small arrow on top. (B) Presence of domain architectures for PDZ and LIM genes and their species distribution. Six basic gene structures can be found amongst the different taxons. The <i>tungus gene</i>, found in the two <i>arthropod species investigated and the nematode homolog Eat-1 both</i> encode one N-terminal PDZ and four C-terminal LIM domains. Eat-1 has been described earlier as the <i>Caenorhabditis elegans</i> ALP/Enigma gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000189#pone.0000189-McKeown1" target="_blank">[50]</a>. Only a single <i>LIMK</i> gene was found per invertebrate species examined, and the <i>LMO7</i> homolog lacks the CH domain (<i>CG31534</i>). The LMO7 gene of <i>Drosophila</i> melanogaster lacks not only the CH domain but also the PDZ domain (not shown, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000189#pone.0000189.s002" target="_blank">Supplemental table S1</a>). As not all ALP and Enigma subfamily members share the ZM domain (ZASP and ALP contain 2 and Elfin one ZM motif) we have excluded the ZM motif from these groups and show only a ZM motif for eat-1/tungus in this figure.</p

Figure 2

<p>Phylogenetic trees of the Enigma and ALP subfamilies, the LIM kinases and LMO7 using full length sequences. (A,B,D and E) and PDZ domain sequences (C). In all phylogenetic trees shown, are the percentages for Bayesian posterior probability (first number) and for Maximum Likelihoods (second number) indicated at the branches. In figure C only the major branches are labeled for better overview. The two letter abbreviations used refer to genus and species, with the first capital letter and the second non-capital letter, respectively. All abbreviations used are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000189#s4" target="_blank">material and methods</a>. ENH is used for Enigma homolog (A) Shown is a phylogenic tree based on the full-length sequences of members of the Enigma subfamily. (B) Phylogenetic analysis for the ALP subfamily, based on full length sequences of selected homologs. (C) Evolutionary tree based on the PDZ domains of both ALP and Enigma subfamilies. (D) Phylogeny of the LIM domain kinases based on full length amino acid sequences. The root is placed on the <i>Drosophila melanogaster</i> homolog. The fruitfly homolog (which roots with the Uruchordate homolog found in <i>Ciona intestinalis</i>) gives rise to the common ancestor for the vertebrate LIM kinases. This ancestor then duplicates into LIMK1 and LIMK2, which are both present in all the vertebrate species investigated. (E) Evolutionary tree of LMO7 genes. The tree is rooted to the <i>Caenorhabditis elegans</i> sequence of tag-204, which has a similar structure to LMO7, albeit without the CH domain.</p

Figure 6

<p>Evolutionary model for the PDZ and LIM encoding genes. The most parsimonious model derived from our phylogenetic analysis shows that the PDZ domain of all 10 different PDZ/LIM encoding genes share a common ancestor, with closest homology to LMO7. The combination of this PDZ domain with a LIM domain formed the common ancestor for both the LMO7 and the ALP/Enigma lineages. The single LIM domain in the ALP/Enigma lineage (closest to LIM2 in <i>eat-1/tungus)</i> then duplicated and gave rise to a PDZ two LIM domain structure. Subsequently the duplicated LIM (closest to LIM3 in <i>eat-1/tungus)</i>, duplicated twice and generated a PDZ four LIM structure similar to <i>eat-1/tungus</i>. From this gene structure, through gene duplication and subsequent domain loss (loosing either three LIM domains (LIM2–4) or only 1 LIM domain (LIM1) for the ALP and Enigma subfamilies, respectively), the ALP/Enigma subfamilies evolved. The color code used for domains is PDZ (blue), LIM (yellow and green), CH (red), Kinase domain (black) and AM-motif (pink).</p

Figure 4

<p>Dendogram of a representative set of LIM domains. Sequence comparisons of LIM domains in a Dendogram, with different clusters color-coded. We included all non-redundant LIM domains found in <i>Dictyostelium discoideum</i> and all from <i>C. elegans</i> and added the closest homologs of PDZ/LIM family LIM domains from different species, which were identified via BLAST search. The Dictyostelium discoideum sequences are marked in red and cluster all together and not with any other of the LIM domain clusters.</p