Systematics of Mukdenia and Oresitrophe (Saxifragaceae): Insights from genome skimming data

Oresitrophe and Mukdenia (Saxifragaceae) are epilithic sister genera used in traditional Chinese medicine. The taxonomy of Mukdenia, especially of M. acanthifolia, has been controversial. To address this, we produced plastid and mitochondrial data using genome skimming for Mukdenia acanthifolia and Mukdenia rossii, including three individuals of each species. We assembled complete plastomes, mitochondrial CDS and nuclear ribosomal ETS/ITS sequences using these data. Comparative analysis shows that the plastomes of Mukdenia and Oresitrophe are relatively conservative in terms of genome size, structure, gene content, RNA editing sites and codon usage. Five plastid regions that represent hotspots of change (trnH-psbA, psbC-trnS, trnM-atpE, petA-psbJ and ccsA-ndhD) are identified within Mukdenia, and six regions (trnH-psbA, petN-psbM, trnM-atpE, rps16-trnQ, ycf1 and ndhF) contain a higher number of species-specific parsimony-informative sites that may serve as potential DNA barcodes for species identification. To infer phylogenetic relationships between Mukdenia and Oresitrophe, we combined our data with published data based on three different datasets. The monophyly of each species (Oresitrophe rupifraga, M. acanthifolia and M. rossii) and the inferred topology ((M. rossii, M. acanthifolia), O. rupifraga) are well supported in trees reconstructed using the complete plastome sequences, but M. acanthifolia and M. rossii did not form a separate clade in the trees based on ETS + ITS data, while the mitochondrial CDS trees are not well-resolved. We found low recovery of genes in the Angiosperms353 target enrichment panel from our unenriched genome skimming data. Hybridization or incomplete lineage sorting may be the cause of discordance between trees reconstructed from organellar and nuclear data. Considering its morphological distinctiveness and our molecular phylogenetic results, we strongly recommend that M. acanthifolia be treated as a distinct species.     

First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.     

The freshwater planarian Dugesia (G.) tigrina contains a great diversity of homeobox genes

To identify potential pattern control and cell determination and/or differentiation genes in the freshwater planarian Dugesial (G.) tigrina, we searched for homeobox genes of different types in the genome of this primitive metazoan. We applied two basic approaches: 1) Screening the cDNA library with degenerate oligonucleotides corresponding to the most conserved amino acid sequence from helix-3 of the homeodomain of each family; and 2) PCR amplification of genomic DNA or cDNA, using two sets of degenerated oligonucleotides corresponding to helices 1 and 3 of the homeodomain or two specific domains of the POU family. Using the first strategy we have identified and characterized two tissue-specific cell determination and/or differentiation NK-type homeobox genes. Using the second strategy we have identified several homeobox genes that belong to the HOM/Hox, paired (prd) or POU families.     

Glycoproteins in bacterial membranes. In vivo labeling of the sugar portion of energy-transducing ATPase and a low-molecular-weight fraction fromMicrococcus lysodeikticus membranes

The energy-transducing ATPase and a low-molecular-weight fraction ofMicrococcus lysodeikticus membranes incorporated¹⁴C label fromd-[U-¹⁴C]glucose fed to the bacteria in synthetic medium. The specific radioactivity of the sugar portion of the ATPase and low-molecular-weight fraction was, respectively, 2.65 and 2.88 times that of their amino acids. Glucose and mannose in approximately equimolar amounts were identified as the main sugars of the glycoprotein ATPase, thus confirming previous structural studies. Glucose, galactose, and mannose (1:1:2) were identified as the main sugars of the low-molecular-weight glycopeptides. These results confirm and extend the notion that glycoprotein are constituents of prokaryotic membranes.     

Membrane adenosine triphosphatase of Micrococcus lysodeikticus. Isolation of two forms of the enzyme complex and correlation between enzymatic stability,latency and activity.

Summary Two new forms of the plasma membrane ATP-ase ofMicrococcus lysodeikticus NCTC 2665 were isolated from a sub-strain of the microorganism by polyacrylamide gel electrophoresis. One of them had a mol.wt of 368,000 and a very low specific activity (0.80 µ mol.min^–1.mg protein^–1) that could not be stimulated by trypsin. This form has been called B^I (strain B, inactive). If the electrophoresis was carried out in the presence of reducing agents (i.e., dithiothreitol) and the pH of the effluent maintained at a value of 8.5 another form of the enzyme was obtained. This had a mol.wt of 385,000 and a specific activity of 2.5–5.0 µ mol.min^–1.mg protein^–1 that could be stimulated by trypsin to 5–10 µ mol.min^–1.mg protein^–1. This preparation of the ATPase has been called form B_A (strain B, enzyme active). The subunit composition of both forms has been studied by sodium dodecyl sulphate and urea gel electrophoresis and compared to that of the enzyme previously purified from the original strain (form A). The three forms of the enzyme had similar and subunits, with mol.wt of about 50,000 and 30,000 dalton, respectively. They also had in common the component(s) of relative mobility 1.0, whose status as true subunit(s) of the enzyme remains yet to be established. However, subunit, that had a mol.wt of about a 52,500 in form A (Andreu et al. Eur. J. Biochem. (1973) 37, 505–515), had a mol.wt similar to in form B_I and about 60,000 in form B_A. Furthermore B_A usually showed two types of this subunit ( and) and an additional peptide chain () with a mol.wt of about 25,000 dalton. This latter subunit seemed to account for the stimulation by trypsin of form B_A.Forms B_A could be converted to B_I by storage and freezing and thawing. Conventional protease activity could not be detected in any of the purified ATPase forms and addition of protease inhibitors to form B_A failed to prevent its conversion to form B_I. The low activity form (B_I) was more stable than the active forms of the enzyme and also differed in its circular dichroism. These results show thatM. lysodeikticus ATPase can be isolated in several forms. Although these variations may be artifacts caused by the purification procedures, they provide model systems for understanding the structural and functional relationships of the enzyme and for drawing some speculations about its functionin vivo.     

Micrococcus lysodeikticus membrane ATPase. effect of trypsin on stimulation of a purified form of the enzyme and identification of its natural inhibitor

A soluble purified form of Micrococcus lysodeikticus ATPase (form B_AT, from strain B, active, trypsin-stimulated) was stimulated 100% by trypsin and this stimulation was inhibited by preincubation of the protease with phenyl methyl sulphonylfluoride. This form of the enzyme was also stimulated 125–150% by filtration on Sephadex G-200. Analysis by sodium dodecyl sulphate-gel electrophoresis showed that stimulation of this form of M. lysodeikticus ATPase was always accompanied by the disappearance of a subunit of mol. wt. 25 000 (ε subunit). It suggests that this subunit is the natural inhibitor of M. lysodeikticus ATPase. In the case of ATPase stimulation by trypsin, a partial and limited degradation of the α subunit was also observed. The interaction between the ε subunit and the rest of the ATPase complex was reversibly affected by pH, suggesting its non-covalent nature.     

Confomational and molecular responses to pH variation of the purified membrane adenosine triphospate of Micrococcus lysodeikticus

A preparation of ATPase from the membranes of Micrococcus lysodeikticus, solubilized and more than 95 %. pure, showed two main bands in analytical polyacrylamide gel electrophoresis. They did not correspond to isoenzymes because one band could be converted into the other by exposure to a mildly alkaline pH value. The conversion was paralleled by changes in molecular weight, circular dichroism and catalytic properties. Denaturation by pH at 25 °C was followed by means of circular dichroism, ultracentrifugation and polyacrylamide gel electrophoresis. A large conformational transition took place in the acid range with midpoints at about pH = 3.6 (I = 10^?4 M), 4.3 (I = 0.03 M) and 5.3 (I = 0.1 M). The transition was irreversible. Strong aggregation of the protein occurred in this range of pH. The final product was largely random coil, but even at pH 1.5 dissociation into individual subunits was not complete. However, partial dissociation took place at pH 5 (I = 0.028 M). At this pH value the enzyme was inactive, but 20–30 % of the activity could be recovered when the pH was returned to 7.5.In the alkaline region the midpoint of the transition occurred near pH = 11 (I = 0.028 M). The pK of most of the tyrosine residues of the protein was about 10.9. The unfolding was irreversible and the protein was soon converted into peptide species with molecular weights lower than those determined for the subunits by gel clectrophoresis in the presence of sodium dodecyl sulphate. Conventional proteolysis did not account for the transformation.     

Micrococcus lysodeikticus ATPase. Purification by preparative gel electrophoresis and subunit structure studied by urea and sodium dodecylsulfate gel electrophoresis.

Micrococcus lysodeikticus ATPase was purified by preparative gel electrophoresis after its "shodk wash" release from the membrane. The method afforded the highest yield of pure protein in the minimum time as compared with former purification procedures. The pure protein had a specific activity of 7 mumol Pi-min- minus 1-mg- minus 1 with incubation times not longer than 3 min, 345 000 mol. wt and was not stimulated by trypsin. By gel electrophoresis at alkaline pH (8.5) in 8 M urea or in sokium dodecylsulfate, the ATPase revealed a complex pattern with two major subunits (alpha and beta) and two minor ones (gamma and delta). The non-identity between the major subunits was demonstrated.