The complete mitochondrial DNA sequence of the shark Mustelus manazo: evaluating rooting contradictions to living bony vertebrates

A remarkable example of a misleading mitochondrial protein tree is presented, involving ray-finned fishes, coelacanths, lungfishes, and tetrapods, with sea lampreys as an outgroup. In previous molecular phylogenetic studies on the origin of tetrapods, ray-finned fishes have been assumed as an outgroup to the tetrapod/lungfish/coelacanth clade, an assumption supported by morphological evidence. Standard methods of molecular phylogenetics applied to the protein-encoding genes of mitochondria, however, give a bizarre tree in which lamprey groups with lungfish and, therefore, ray-finned fishes are not the outgroup to a tetrapod/lungfish/coelacanth clade. All of the dozens of published phylogenetic methods, including every possible modification to maximum likelihood known to us (such as inclusion of site heterogeneity and exclusion of potentially misleading hydrophobic amino acids), fail to place the ray-finned fishes in a biologically acceptable position. A likely cause of this failure may be the use of an inappropriate outgroup. Accordingly, we have determined the complete mitochondrial DNA sequence from the shark, Mustelus manazo, which we have used as an alternative and more proximal outgroup than the lamprey. Using sharks as the outgroup, lungfish appear to be the closest living relative of tetrapods, although the possibility of a lungfish/coelacanth clade being the sister group of tetrapods cannot be excluded.      

Synthesis of 21-carboxy-D-arabinitol-1-phosphate in French bean (Phaseolus vulgaris L.): a search for precursors

Discs of French bean leaves were vacuum infiltrated with solutionscontaining ¹⁴C-labelled substances. The infiltrated discs wereeither transferred immediately to darkness or first illuminatedfor 2 h and then transferred to darkness. After 6 h in darknessthe discs were extracted with buffer containing CO₂, Mg²⁺ andadditional ribulose-1, 5-bisphosphate carboxylase/oxygenase(Rubisco; EC 4.1.1.39 [EC] ). Protein in the extracts was separatedfrom substances of low molecular weight by gel filtration andcoagulated by heating to 100C. Coagulated protein was removedby centrifugation and cations in the supernatant solution wereremoved by ion exchange resin. The non-volatile anions in theresulting solutions, among which was 2¹-carboxy-D-arabinitol-1-phosphate(CA1P), were separated by HPLC. The amount of CA1P was determinedfrom the signal of a pulsed amperometric detector and its radioactivityby scintillation counting. Vacuum infiltration of [2¹–¹⁴C]2¹-carboxy-D-arabinitol (CA) resulted in 12.6% of the radioactivityin the leaf discs being in CA1P after 6 h in darkness and 21.6%when 2 h light was given before the dark treatment. Where radioactiveglucose, fructose, sucrose, hamamelose, glycerate, glycine oracetate were infiltrated, ¹⁴C in CA1P was less than 1% of thetotal present after the dark period with or without a precedingperiod of light. Incorporation of ¹⁴C from [¹⁴C] CA into CA1Pin darkness was strongly inhibited by 2,4-dinitrophenol andalso to a lesser extent by tentoxin. With both inhibitors themain effect was a decreased uptake of the substrate. Illuminationprior to darkness stimulated the incorporation of radioactivityfrom CA, glycine, glucose, sucrose, and hamamelose into CA1Pin subsequent darkness. Unlike the other substrates, which wereextensively metabolized, CA and hamamelose were converted tofew products; CA was converted almost exclusively to CA1P andCA1P was a major product of hamamelose metabolism. Key words: CA1P, Phaseolus vulgaris, precursors, synthesis