Sequences used in our analysis. Sequences were aligned manually with the aid of the editing and dot-matrix capabilities of SnAP (Sequence Analysis Programs) package of computer programs. The aligned sequences were then used as input by the program MAKDAT, which creates output files suitable for phylogenetic analysis programs. We used version 3.0 of PAUP (Swofford 1990) running on a Macintosh computer to carry out parsimony analyses. The two phylogenetic trees shown in this presentation were drawn using the graphic capabilities of PAUP, followed by manual editing. All branch lengths as well as bootstrap values reflect PAUP\'s parsimony values.|~|/files/powerpoints_images/node12869/Slide2.JPG|~|563|~|422|~|0
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Sequences used in our analysis. Sequences were aligned manually with ...
A phylogenetic tree showing the voltage-gated ion channel gene superfamily based on comparison of nucleotide sequences of S4 segments only. Included are two cyclic nucleotide-gated ion channel genes, four Drosophila Kv channel genes (Shaker, Shab, Shaw and Shal), three Nav channel genes (Nav1.1, Nav1.2, Nav1.3) and three Cav channel genes (Cav1.1,Cav1.2, Cav2.1). The indicated branch lengths (bootstrap values in parentheses) were generated by PAUP using domain II of the Nav and Cav channels. Analyses by other methods supported this topology. The total length is 301 substitutions. We chose the S4 segment because it was the only region where we felt we could make a satisfactory alignment for all the genes. We recognize that using such a small region may not yield very robust trees because it represents such a small proportion of the entire length of these genes.The tree shows three groups. The first consists of the two cyclic-nucleotide gated channels, the second comprises the Kv channels and the third consists of the Nav and Cav channels. Since the Nav and Cav channels each have four separate S4 segments, we analyzed separate trees by using each S4 segment in turn, as well as combinations of them. In each case PAUP (heuristic search) detected a single most parsimonious tree (of length 293-308) and a total of 4-7 near-parsimonious tree (within 2 substitutions of the best tree). For domain II of Nav and Cav, the most parsimonious tree had the topology shown in the figure. The same topology was included among the near-parsimonious trees by using domains I and III of Nav and Cav. Domain IV did not maintain this topology. The near-parsimonious trees all keep the Nav and Cav channel genes together, and also the two cyclic nucleotide-gated channels, but break up or vary the rooting of the Kv channels. The best tree identified by WTDPARS using Nav and Cav domains I, II and IV showed the topology presented in the figure. Here again, several near-best trees differed only in the rooting of the Kv group. The neighbor joining method also yieled the topology shown in the figure when domains I or II of Nav and Cav were used. Thus several methods converge on the phylogenetic topology shown in the figure. |~|/files/powerpoints_images/node12869/Slide3.JPG|~|563|~|422|~|0
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A phylogenetic tree showing the voltage-gated ion channel gene superf...
This figure shows the relationship between the four domains of the Cav and the Nav channel genes. The most parsimonious tree shown is based on amino acid sequence alignment. The total tree length is 791 substitutions. The Kv and the cyclic nucleotide-gated channel genes each encode a single domain that contains N- and C-termini and six transmembrane segments, S1 to S6. Four of these subunits assemble together to form a functional channel. The Cav and Nav channel genes have four such domains repeated in tandem which assemble around a central axis to form the functional channel. To examine the relationship between the four domains of Cav and Nav channel genes, we analyzed amino acid alignments of Nav1.1 and Cav1.1 by using PAUP protein parsimony (heuristic search). The topology shown in the figure was favored by several other different combinations of Cav and Nav channel genes (Nav1.1/Nav1.2./Cav1.1; Nav1.2/Cav1.1; Nav1.1/Cav1.2; Nav1.2/Cav1.2). This topology places domains I and III into one monophylectic group and domains II and IV into another monophylectic group. A parsimony bootstrap analysis (PAUP) using Nav1.1 and Cav1.1 supports this topology at the 75% level. Hille (1989) proposed that a common ancestral single-domain gene gave rise to the primordial Cav channel gene by two intragenic duplications and then gave rise to the Nav gene by further divergence. The phylogenetic tree presented in this figure supports this scheme. |~|/files/powerpoints_images/node12869/Slide4.JPG|~|563|~|422|~|0
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This figure shows the relationship between the four domains of the Ca...
A primordial single-domain gene (A) underwent an internal duplication to create a two-domain gene (A and A). This gene underwent sequence divergence to yield A and B. A second duplication gave rise to a four-domain gene (A, B, A, B). Further divergence led to the four-domain gene known today (I, II, III, IV). Thus, domains I and III of Cav and Nav channel genes are more similar to each other, while domains II and IV of the Nav and Cav channel genes are more to each other. An implication of this evolutionary scheme is that an ancestral two-domain channel gene was capable of encoding a functional protein, because it would have existed for long enough for sequence divergence followed by gene duplication. The discover of a rabbit Cav channel protein with a two-domain structure, produced by alternate splicing of a four-domain transcript, supports this idea. K and Ca channels have been detected in bacteria, protozoa (Paramecium) and plants, while Nav channels appear to be absent in those organisms that diverged early in the eukaryotic radiation. It is most likely that the first four-domain channel was a Cav channel. The Nav channel-gene evolved from the four-domain Cav channel-gene by sequence divergence. |~|/files/powerpoints_images/node12869/Slide5.JPG|~|563|~|422|~|0
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A primordial single-domain gene (A) underwent an internal duplication...
Analysis of amino acid sequence alignments of the four-domain cation-selective ion channels (Cav and Nav) indicates that they evolved from a single domain gene by two sequential gene duplications. The existence of a rabbit Cav channel protein with a two-domain structure, produced by alternate splicing of a four-domain transcript, supports this evolutionary scheme.
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References
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