The sequence of the LWS opsin is shown in supplemental Figure 3. in lamprey in order to understand the molecular origins of rod and cone photoreceptor G proteins. Two Gt subunits, GtL and GtS, AZD-7648 were identified in the retina. GtL is usually equally distant from cone and rod G proteins and is expressed in the lampreys long photoreceptors. The short photoreceptor GtS is usually a rod-like transducin- that retains several unique features of cone transducins. Thus, the duplication of the ancestral transducin gene giving rise to rod transducins has already occurred in the last common ancestor of the jawed and jawless vertebrates. cones and rods, respectively, produced responses identical to native responses of photoreceptors, suggesting that the rod and cone pigment signaling is not different (Kefalov et al., 2003). Cones have been shown to express much higher levels of the RGS9 GAP complex than rods, leading to a hypothesis that RGS9-1 abundance controls rapid response kinetics in cones (Zhang, Wensel & Kraft, 2003). Overexpression of the GAP complex in mouse rods accelerated the recovery kinetics, but the activation phase and the sensitivity of flash responses were unchanged (Krispel et al., 2006). Thus, high GAP complex concentrations most likely contribute to the faster recovery in cones compared to rods, whereas additional mechanisms are required to explain the outstanding key differences. Certain lower vertebrate species AZD-7648 with uniquely evolved photoreceptor cells provide an opportunity to pinpoint potential significance of specific sequence variations between rod and cone components. One such example is usually Tokay gecko photoreceptors. These photoreceptors are rods in terms of their morphology and physiology, but utilize cone-like components, including pigments, Gt1, PDE6, arrestin, and cGMP-gated AZD-7648 channel subunits (Zhang, Wensel & Yuan, 2006). Therefore, critical sequences might be confined to a limited number of rod-only specific residues conserved in cone-like phototransduction molecules of the Tokay gecko (Zhang et al., 2006). As representatives of the earliest known vertebrate class of jawless fish, lampreys constitute a unique model to study the evolution of the vertebrate visual systems (Walls, 1942; Lamb, Collin & Pugh, 2007). Two morphologically distinct types of photoreceptor cells, short (SPs) and long photoreceptors (LPs), are described in the retina of sea lamprey (Dickson & Graves, 1979). Classification of SPs and LPs as cones or rods had long been debated (Ohman, 1976; Dickson & Graves, 1979; Govardovskii & Lychakov, 1984; Ishikawa, Takao, Washioka, Tokunaga, Watanabe & Tonosaki, 1987). The controversy has not been clarified with the identification of the rhodopsin gene apparently expressed in SPs (Zhang & Yokoyama, 1997). This pigment was initially categorized as an Rh1 opsin, indicative of rod function (Zhang & Yokoyama, 1997). A competing viewpoint emerged later suggesting that this lampreys Rh-like opsin gene diverged from an ancestral Rh-gene prior to its duplication into the Rh1 and Rh2 lineages (Collin, Knight, Davies, Potter, Hunt & Trezise, 2003; Collin & Trezise, 2004). Even so, by most morphological and electrophysiological criteria LPs are cones, whereas SPs are mixed cone/rod photoreceptors or unusual rods that operate under scotopic and photopic conditions (Govardovskii & Lychakov, 1984). We recently demonstrated expression of a single type of PDE6 catalytic subunit in with nearly equivalent relations to cone and rod PDE6s (Muradov, Boyd, Kerov & Artemyev, 2007). The PDE6 holoenzyme incorporates a cone-type P-subunit in LPs and a distinct mixed cone/rod-type P-subunit in SPs (Muradov et al., 2007). These findings indicated that lampreys represent an interesting model of evolution of cone and rod phototransduction components. Here, we investigated transducins in and examined the identity of the visual pigment expressed in LPs. Materials and methods Materials All restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA). AmpliTaq DNA polymerase was a product of Applied Biosystems (Foster City, CA), and cloned DNA polymerase was a product of Stratagene (La Jolla, CA). TRI Reagent and oligo(dT) Rabbit Polyclonal to CELSR3 column were purchased from Molecular Research Center (Cincinnati, OH). All other reagents were purchased.
