Supplementary Tables from Evolution of the shut-off steps of vertebrate phototransduction

Summary of transcripts examined, and multiple sequence alignment

Supplementary Figures from Evolution of the shut-off steps of vertebrate phototransduction

Additional phylogenetic analysis and molecular model

SDS-PAGE of <i>Gadus morhua</i> and <i>Coryphaenoides armatus</i> ldh-b.

<p>Panel A shows the Factor XA cleavage of <i>Coryphaenoides armatus</i> ldh-b. Lane 1 is Factor XA overnight digest of the Maltose binding protein-ldh-b fusion protein, lanes 2 is pure <i>C. armatus</i> ldh-b, lane 3 is maltose binding protein and lane 4 is Dalton VII marker. Panel B shows the thrombin cleavage of the <i>Gadus morhua</i> ldh; lane 1 is His-tagged ldh-b and lane 2 is ldh-b after removal of the His-tag and lane 3 is Dalton VII marker.</p

Effect of pressure on protein stability as monitored by CD at 222 nm.

<p>The effect of pressure on GM is given by the continuous line, on MA is given by the dashed-dotted line, on CA is given by the dashed line and on AM is given by the dotted line.</p

Primary structure of the LDH-b sequences from <i>C. armatus</i> and <i>G. morhua</i> and their modelling onto the human LDH-b structure.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002042#pone-0002042-g001" target="_blank">Figure 1a</a> shows the sequence alignment of the <i>C. armatus</i> (the abyssal grenadier, or macrourid/rat tail) and <i>G. morhua</i> (Atlantic cod) LDH-b. The corresponding relative position of the <i>Acc</i>I site is highlighted, from which the hybrid enzymes were generated. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002042#pone-0002042-g001" target="_blank">Figure 1b and c</a> are cartoon representations of the LDH structure showing the residue substitutions as red splashes on the green subunit. (b) Overview of the tetramer viewed down a 2-fold axis and (c) a close up of the green subunit in similar orientation to that of panel (b) Residues 7, 76, 267, 220 are hidden in this view and the subunits are in different colours: green, cyan, yellow, purple; sites of mutation shown on green subunit in red, and NAD is shown in stick representation. This Figure was made using PyMOL and based on the human LDH structure.</p

Effect of pressure on the specific activities of all four LDH-bs.

<p>Panel A is 1 µg/ml, panel B is 1 µg/ml 250 mM TMAO and panel C is 100 µg/ml. ▪ is GM, ▴ is MA, - is CA and • is AM. Error bars are standard error.</p

Kinetic parameters of the LDH-b enzymes measured in this study.

<p>Kinetic parameters of the LDH-b enzymes measured in this study.</p

Early symptoms of brain tumours

<div><p>Lampreys are one of the two surviving groups of the agnathan (jawless) stages in vertebrate evolution and are thus ideal candidates for elucidating the evolution of visual systems. This study investigated the retinal amino acid neurochemistry of the southern hemisphere lamprey <i>Geotria australis</i> during the downstream migration of the young, recently-metamorphosed juveniles to the sea and during the upstream migration of the fully-grown and sexually-maturing adults to their spawning areas. Glutamate and taurine were distributed throughout the retina, whilst GABA and glycine were confined to neurons of the inner retina matching patterns seen in most other vertebrates. Glutamine and aspartate immunoreactivity was closely matched to Müller cell morphology. Between the migratory phases, few differences were observed in the distribution of major neurotransmitters i.e. glutamate, GABA and glycine, but changes in amino acids associated with retinal metabolism i.e. glutamine and aspartate, were evident. Taurine immunoreactivity was mostly conserved between migrant stages, consistent with its role in primary cell functions such as osmoregulation. Further investigation of glutamate signalling using the probe agmatine (AGB) to map cation channel permeability revealed entry of AGB into photoreceptors and horizontal cells followed by accumulation in inner retinal neurons. Similarities in AGB profiles between upstream and downstream migrant of <i>G. australis</i> confirmed the conservation of glutamate neurotransmission. Finally, calcium binding proteins, calbindin and calretinin were localized to the inner retina whilst recoverin was localized to photoreceptors. Overall, conservation of major amino acid neurotransmitters and calcium-associated proteins in the lamprey retina confirms these elements as essential features of the vertebrate visual system. On the other hand, metabolic elements of the retina such as neurotransmitter precursor amino acids and Müller cells are more sensitive to environmental changes associated with migration.</p> </div

Localization of aspartate in the G. australis retina.

<p>Aspartate immunoreactivity in <b>A, C:</b> the downstream migrant and <b>B, D:</b> the upstream migrant of <i>G. australis. </i><b>A:</b> Aspartate immunoreactivity for the downstream animals was confined to the mid INL (white arrowheads) and punctate staining along the outer limiting membrane (white arrow). <b>B:</b> Aspartate immunoreactivity in the upstream animal was observed in different photoreceptors (white arrowheads) and some small cells in the INL (white arrows). <b>C:</b> Magnified image of the downstream migrant retina showing aspartate immunoreactivity across the outer limiting membrane (OLM). <b>D:</b> Magnified image of the INL in the upstream migrating <i>G. australis</i> showing immunoreactive horizontal cells (HC) and nerve fibers (NF) are indicated. Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058406#pone-0058406-g002" target="_blank">Figure 2</a>. Scale bar is 20 µm in all figures.</p

Amino acid primary antibodies used in this study.

<p>The antibodies used for this study were kindly donated by Dr R. E. Marc but are now also commercially available through Chemicon and Signature Immunologics. All antibodies are rabbit polyclonal.</p