最大似然法和贝叶斯推论与似然比检验探讨泽泻目系统发育关系

应用最大似然法(ML)、贝叶斯推论(BI)、邻接法(NJ)和似然比检验(hLRTs)进行泽泻目分子系统学研究。所用的rbcL基因序列代表了泽泻目14科46属以及作为外类群的6相关属。研究结果表明,*等级制似然比检验表明泽泻目rbcL序列最适合的DNA进化模型为GTR+I+G,最大似然法、贝叶斯法和邻接法构建的系统发育树拓扑结构相似,没有显著的差异,但贝叶斯树支持率较高;泽泻目为一单系类群,由两个主要谱系分支构成,深层分布格局由5个主要分支构成。基于分子系统发育树,文中对泽泻目科间、水鳖科+茨藻科、泽泻科+花蔺科+黄花蔺科、和"Cymodoeaceae complex"的系统发育关系进行了讨论。研究结果还表明,泽泻目系统发育关系可能还需要更多的证据进一步的澄清。     

Constructed deletions in lumen-exposed regions of the D1 protein in the cyanobacterium Synechocystis 6803: Effects on D1 insertion and accumulation in the thylakoid membrane, and on Photosystem II assembly

Modified forms of the D1 protein with deletions in lumen-exposed regions, were constructed in the cyanobacterium Synechocystis 6803 using site-directed mutagenesis. Integration and stability of the mutated D1 proteins in the thylakoid membrane were studied by immunoblot and pulse-chase analyses. It was found that in (N325-E333), the D1 protein with a deletion in the C-terminal tail, could insert in the thylakoids to normal amounts but its stability in the membrane was dramatically reduced. Insertion of D1 in (V58-D61) or (D103-G109);G110R, with deletions in the A-B loop, was severely obstructed, For (P350-T354), with a deletion in the processed region of the C-terminus of D1, no phenotypic effects were observed. The effects of failed D1 insertion or accumulation on Photosystem II assembly was monitored by immunoblot analysis. The conclusions from these experiments are that the extrinsic 33 kDa protein, CP43, and the subunit of cytochrome b559 accumulate in the thylakoid membrane independently of the D1 protein, and that accumulation of the D2 protein and CP47 requires insertion but not necessarily accumulation of the D1 protein.Abbreviations PSI II Photosystem II - PCR Polymerase Chain reaction Present address: Université Joseph Fourier, Sciences Technologie Médecine, BP 53, 38041 Grenoble Cedex 9, France     

Partial sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase and the phylogeny of Prochloron and Prochlorococcus (Prochlorales)

The prochlorophytes, oxygenic photosynthetic prokaryotes having no phycobiliprotein but possessing chlorophylls a and b, have been proposed to have a common ancestry with green chloroplasts, yet this is still controversal. We report here that partial sequence comparisons of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, including sequence data from two prochlorophytes, Prochlorococcus and Prochloron, indicate that Prochlorococcus is more closely related to a photosynthetic bacterium, Chromatium vinosum (-purple bacteria), than to cyanobacteria, while Prochloron is closely related to the prochlorophyte Prochlorothrix and to cyanobacteria. The molecular phylogenetic tree indicates that a common ancestor of Prochlorococcus and -purple bacteria branched off from the land plant lineage earlier than Prochloron, Prochlorothrix, and cyanobacteria.Correspondence to: A. Shimada     

Isozyme and seed protein phylogeny of the genusCitrullus (Cucurbitaceae)

Electrophoretic analysis of 26 enzyme coding genes was conducted on accessions of threeCitrullus species and the relatedPraecitrullus fistulosus andAcanthosicyos naudinianus. The isozyme phylogeny of the genusCitrullus and the related species was constructed based on pairwise measurements of the respective genetic distances between the species and races.P. fistulosus andA. naudinianus form two distinct outgroups toCitrullus which is characterized by two main clusters: The first includes twoC. colocynthis races and the second,C. lanatus andC. lanatus var.citroides, which are more closely related to each other than they are toC. ecirrhosus. The isozyme phylogeny is consistent with the variability in six seed protein bands and with the crossability relations among the examined species.     

Purification and characterization of Mr 43,000 protein similar to Mr 25,000 protein, a substrate for protein Ser/Thr kinases, identified as a part of Xenopus laevis vitellogenin B1

Mr 25,000 protein (pp25), a substrate for protein Ser/Thr kinases, was recently shown to consist of a portion of the Xenopus laevis vitellogenin B1 protein. By Western blot analyses using antibodies against pp25, a minor protein band with Mr 43,000 (pp43) was detected in purified preparations of pp25. In this study, pp43 was highly purified through several column chromatography steps and its protein structure was analyzed. The amino acid sequence of the amino-terminal region of pp43 was the same as that of pp25. pp43 contained about two times more phosphates than pp25. These phosphates in pp43 were more resistant to acid phosphatase attack than those of pp25. pp43 was able to bind to pNiXa, a binding protein of pp25. Alpha-chymotryptic digestion generated a common fragment with molecular mass of 23,000 from both pp43 and pp25. These results suggest that pp43 may be a precursor of pp25 generated during processing of vitellogenin B1.     

Characterization of the photosystem II 32 kDa protein in Synechococcus PCC7942

A rapidly labeled photosynthetic membrane protein was identified in the cyanobacterium Synechococcus PCC7942 R2 as the 32 kDA protein that is involved in electron transport and quinone binding in the photosystem II complex. Partial proteolysis of the membrane-bound protein indicates that the internal architecture and the topology of the Synechococcus 32 kDa protein resembles the analogous protein of higher plants. In addition to the R2 wild-type strain, we characterized three psbA-inactivated Synechococcus strains, in which two of the three endogenous psbA genes were inactivated. In all strains, a 32 kDa protein cross-reacts with an antiserum that was raised against a higher-plant 32 kDa protein and displays in vivo light-dependent turnover. In Synechococcus, the herbicide DCMU inhibits the 32 kDa protein turnover at similar concentration ranges as in higher plants; however, a fraction of the molecules always displays a DCMU-insensitive degradation.     

Neutral evolution of ten types of mariner transposons in the genomes of Caenorhabditis elegans and Caenorhabditis briggsae

Ten types of mariner transposable elements (232 individual sequences) are present in the completed genomic DNA sequence of Caenorhabditis elegans and the partial sequence of Caenorhabditis briggsae. We analyze these replicated instances of mariner evolution and find that elements of a type have evolved within their genomes under no selection on their transposase genes. Seven of the ten reconstructed ancestral mariners carry defective transposase genes. Selection has acted during the divergence of some ancestral elements. The neutrally-evolving mariners are used to analyze the pattern of molecular evolution in Caenorhabditis. There is a significant mutational bias against transversions and significant variation in rates of change across sites. Deletions accumulate at a rate of 0.034 events/bp per substitution/site, with an average size of 166 bp (173 gaps observed). Deletions appear to obliterate preexisting deletions over time, creating larger gaps. Insertions accumulate at a rate of 0.019 events/bp per substitution/site, with an average size of 151 bp (61 events). Although the rate of deletion is lower than most estimates in other species, the large size of deletions causes rapid elimination of neutral DNA: a mariners half-life (the time by which half an elements sequence should have been deleted) is ~0.1 subsitutions/site. This high rate of DNA deletion may explain the compact nature of the nematode genome. When this work was done, both authors were affiliated with the University of Illinois at Urbana-Champaign. Dr. Witherspoon is now working in the private sector, Dr. Robertson remains affiliated with the University of Illinois.     

cDNA and deduced amino acid sequences of cytochrome c fromChlamydomonas reinhardtii: Unexpected functional and phylogenetic implications

Summary We have isolated complementary DNA (cDNA) clones for apocytochrome c from the green algaChlamydomonas reinhardtii and shown that they are encoded by a single nuclear gene termedcyc.Cyc mRNA levels are found to depend primarily on the presence of acetate as a reduced carbon source in the culture medium. The deduced amino acid sequence shows that, apart from the probable removal of the initiating methionine,C. reinhardtii apocytochrome c is syntheszed in its mature form. Its structure is generally similar to that of cytochromes c from higher plants. Several punctual deviations from the general pattern of cytochrome c sequences that is found in other organisms have interesting structural and functional implications. These include, in particular, valines 19 and 39, asparagine 78, and alanine 83. A phylogenetic tree was constructed by the matrix method from cytochrome c data for a representative range of species. The results suggest thatC. reinhardtii diverged from higher plants approximately 700–750 million years ago; they also are not easy to reconcile with the current attribution ofChlamydomonas reinhardtii andEnteromorpha intestinalis to a unique phylum, because these two species probably diverged from one another at about the same time as they diverged from the line leading to higher plants.     

The pattern of insertion/deletion polymorphism in Arabidopsis thaliana

Little is known about variation of nucleotide insertion/deletions (indels) within species. In Arabidopsis thaliana, we investigated indel polymorphism patterns between two genome sequences and among 96 accessions at 1215 loci. Our study identified patterns in the variation of indel density, size, GC content and distribution, and a correlation between indels and substitutions. We found that the GC content in indel sequences was lower than that in non-indel sequences and that indels typically occur in regions with lower GC content. Patterns of indel frequency distribution among populations were more consistent with neutral expectation than substitution patterns. We also found that the local level of substitutions is positively correlated with indel density and negatively correlated with their distance to the closed indel, suggesting that indels play an important role in nucleotide variation.     

A molecular phylogeny of the genus Gagea (Liliaceae) in Germany inferred from non-coding chloroplast and nuclear DNA sequences

The systematics of the mainly yellow flowered Gagea species complex (Liliaceae) has long been considered difficult because only a few phenotypic features within this genus and as a result of hypothesized interspecific hybridisation. A molecular phylogenetic study of seven Gagea species (G. bohemica, G. lutea, G. minima, G. pomeranica, G. pratenis, G. spathacea and G. villosa) from Germany has been undertaken, based on plastid DNA sequences (trnL(UAA)-trnF(GAA), psbA-trnH) and on the nuclear ribosomal internal transcribed spacer (ITS). Sequence divergence within the Gagea species ranges up to 15.5% for psbA-trnH, 22.0% for trnL-trnF and 23.7% for ITS (ITS1 + 5.8S rRNA + ITS2). Two subspecies of Gagea bohemica: G. bohemica subsp. saxatilis and G. bohemica subsp. bohemica are characterized by trnL-trnF data and morphological features. Analysis of the ITS region shows that G. pomeranica represents a hybrid of G. pratensis and G. lutea. Lloydia serotina was initially used as an outgroup species, but it was placed within the investigated Gagea species in the psbA-trnH and the trnL-trnF phylogenetic tree.     

Phospholipin, a novel heterodimeric phospholipase A2 from Pandinus imperator scorpion venom

The primary structure of a phospholipase A2, with unique structural and functional characteristics, was determined. The large subunit has 108 amino acid residues, linked by a disulfide bridge to the small subunit, which contains 17 residues. Its gene was cloned from a cDNA library. The nucleotide sequence showed that the same RNA messenger encodes both subunits, separated only by a pentapeptide, that is processed during maturation.     

Purification,primary structure,and evolution of cytochrome c-550 from the magnetic bacterium,Magnetospirillum magnetotacticum

Cytochrome c-550 was purified from Magnetospirillum magnetotacticum to an electrophoretically homogeneous state, and some of its properties were determined. The cytochrome showed absorption peaks at 528 and 409 nm in the oxidized form, and at 550, 521, and 414 nm in the reduced form. Its midpoint redox potential at pH 7.0 was determined to be +289 mV. The primary structure of cytochrome c-550 was determined. Cytochrome c is composed of 97 amino acid residues, and its molecular weight was calculated to be 10,873, including heme c. Its primary structure is very similar to those of Rhodospirillum fulvum and Rhodospirillum molischianum cytochromes c ₂, suggesting that M. magnetotacticum is phylogenetically related to photosynthetic bacteria.     

Chloroplastrps16 intron phylogeny of the tribeSileneae (Caryophyllaceae)

Intron sequences of the chloroplast generps16 from 46 species were used to examine phylogenetic relationships indicated by nrDNA ITS sequence variation in the tribeSileneae (Caryophyllaceae, Caryophylloideae). This region has previously not been utilized for phylogenetic purposes but the results presented here suggest that it is a consistent and valuable complement to the ITS sequences. Therps16 intron trees are largely congruent with the ITS trees. All the major hypotheses suggested by the ITS data are supported, often at similar bootstrap levels. The joint usage ofrps16 intron and ITS sequences provides a powerful tool for resolving many of the difficult taxonomic issues in the tribeSileneae. Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

The amino acid sequence of the peptide moiety of the pseudomurein from Methanobacterium thermoautotrophicum

The amino acid sequence of the peptide subunits of the peptide moiety of the sacculus polymer (pseudomurein) of Methanobacterium thermoautotrophicum was elucidated by analysing overlapping peptides obtained from partial acid hydrolysates of isolated sacculi. It is suggested that the peptide subunits are attached to glycan strands via one of their glutamyl residues. Another glutamyl residue may crosslink two adjacent peptide subunits to form a dimer. The calculated molar ratios of the amino acids and the percentages of the N-or C-terminal amino acid residues of the supposed dimers are compatible with those actually found in the sacculus polymer.     

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.     

Biogeography and evolution of flower color in Veratrum (Melanthiaceae) through inference of a phylogeny based on multiple DNA markers

Veratrum (Melanthiaceae) comprises ca. 27 species with highly variable morphology. This study aims to construct the molecular phylogeny of this genus to infer its floral evolution and historical biogeography, which have not been examined in detail before. Maximum parsimony, maximum likelihood, and Bayesian analyses were performed on the separate and combined ITS, trnL-F, and atpB-rbcL sequences to reconstruct the phylogenetic tree of the genus. All Veratrum taxa formed a monophyletic group, within which two distinct clades were distinguished: species with white-to-green perianth formed one highly supported clade, and the species with black-purple perianth constituted another highly supported clade. Phylogenetic inference on flower color evolution suggested that white-to-green perianth was a plesiomorphic state and black-purple perianth was apomorphic for Veratrum. When species distribution areas were traced as a multi-state character, parsimonious optimization inferred that Veratrum possibly originated in East Asia. Our study confirmed previous phylogenetic and taxonomic suggestions on this genus and provided a typical example of plant radiation across the Northern Hemisphere.     

The plastid rpoA gene encoding a protein homologous to the bacterial RNA polymerase alpha subunit is expressed in pea chloroplasts

Summary The gene rpoA, encoding a protein homologous to the alpha subunit of RNA polymerase from Escherichia coli has been located in pea chloroplast DNA downstream of the petD gene for subunit IV of the cytochrome b-f complex. Nucleotide sequence analysis has revealed that rpoA encodes a polypeptide of 334 amino acid residues with a molecular weight of 38916. Northern blot analysis has shown that rpoA is co-transcribed with the gene for ribosomal protein S11. A lacZ-rpoA gene-fusion has been constructed and expressed in E. coli. Antibodies raised against the fusion protein have been employed to demonstrate the synthesis of the rpoA gene product in isolated pea chloroplasts. Western blot analysis using these antibodies and antibodies against the RNA polymerase core enzyme from the cyanobacterium, Anabaena 7120, has revealed the presence of the gene product in a crude RNA polymerase preparation from pea chloroplasts.     

Phylogenetic analysis of the thiolase family. Implications for the evolutionary origin of peroxisomes

Summary The thiolase family is a widespread group of proteins present in prokaryotes and three cellular compartments of eukaryotes. This fact makes this family interesting in order to study the evolutionary process of eukaryotes. Using the sequence of peroxisomal thiolase from Saccharomyces cerevisiae recently obtained by us and the other known thiolase sequences, a phylogenetic analysis has been carried out. It shows that all these proteins derived from a primitive enzyme, present in the common ancestor of eubacteria and eukaryotes, which evolved into different specialized thiolases confined to various cell compartments. The evolutionary tree obtained is compatible with the endosymbiotic theory for the origin of peroxisomes. Offprint requests to: J.E. Pérez-Ortín     

The nucleotide sequence of five ribosomal protein genes from the cyanelles ofCyanophora paradoxa: Implications concerning the phylogenetic relationship between cyanelles and chloroplasts

Summary The nucleotide sequences of the ribosomal protein genesrps18, rps19, rpl2, rpl33, and partial sequence ofrpl22 from cyanelles, the photosynthetic organelles of the protistCyanophora paradoxa, have been determined. These genes form two clusters oriented in opposite and divergent directions. One cluster contains therpl33 andrps18 genes; the other contains therpl2, rps19, andrpl22 genes, in that order. Phylogenetic trees were constructed from both the DNA sequences and the deduced protein sequences of cyanelles,Euglena gracilis and land plant chloroplasts, andEscherichia coli, using parsimony or maximum likelihood methods. In addition, a phylogenetic tree was built from a distance matrix comparing the number of nucleotide substitutions per site. The phylogeny inferred from all these methods suggests that cyanelles fall within the chloroplast line of evolution and that the evolutionary distances between cyanelles and land plant chloroplasts are shorter than betweenE. gracilis chloroplasts and land plant chloroplasts.     

Identification of psbA and psbD gene products,D1 and D2, as reaction centre proteins of photosystem 2

A recent report (Nanba O, Satoh K: Proc. Natl. Acad. Sci. USA 84: 109–112, 1987) described the isolation from spinach of a putative photosystem 2 reaction centre which contained cytochrome b-559 and three other electrophoretically resolvable polypeptide bands, two of which have molecular weights comparable to the D1 and D2 polypeptides. We have used in vivo labelling with radioactive methionine and probed with D1 and D2 monospecific antibodies (raised against synthetically expressed sequences of the psbA and psbD genes) for specific detection of these proteins in a similarly prepared photosystem 2 reaction centre preparation. These techniques identified a 32 000 dalton D1 band, a 30 000 dalton D2 band and a 55 000 dalton D1/D2 aggregate, the latter apparently arising from the detergent treatments employed. Digestions with a lysine-specific protease further confirmed the identification of the lysine-free D1 polypeptide and also confirmed that the D1 molecules in the 55 000 dalton band were in aggregation with other bands and not in self-aggregates. The D1 and D2 polypeptides (including the aggregate) are considerably enriched in the photosystem two reaction centre preparation compared to the other resolved fractions.