Relaxed Neighbor Joining: A Fast Distance-Based Phylogenetic Tree Construction Method

Our ability to construct very large phylogenetic trees is becoming more important as vast amounts of sequence data are becoming readily available. Neighbor joining (NJ) is a widely used distance-based phylogenetic tree construction method that has historically been considered fast, but it is prohibitively slow for building trees from increasingly large datasets. We developed a fast variant of NJ called relaxed neighbor joining (RNJ) and performed experiments to measure the speed improvement over NJ. Since repeated runs of the RNJ algorithm generate a superset of the trees that repeated NJ runs generate, we also assessed tree quality. RNJ is dramatically faster than NJ, and the quality of resulting trees is very similar for the two algorithms. The results indicate that RNJ is a reasonable alternative to NJ and that it is especially well suited for uses that involve large numbers of taxa or highly repetitive procedures such as bootstrapping. [Reviewing Editor: Dr. James Bull]     

Gene Order Breakpoint Evidence in Animal Mitochondrial Phylogeny

Multiple genome rearrangement methodology facilitates the inference of animal phylogeny from gene orders on the mitochondrial genome. The breakpoint distance is preferable to other, highly correlated but computationally more difficult, genomic distances when applied to these data. A number of theories of metazoan evolution are compared to phylogenies reconstructed by ancestral genome optimization, using a minimal total breakpoints criterion. The notion of unambiguously reconstructed segments is introduced as a way of extracting the invariant aspects of multiple solutions for a given ancestral genome; this enables a detailed reconstruction of the evolution of non-tRNA mitochondrial gene order. Received: 15 July 1998 / Accepted: 5 March 1999     

后基因组时代生物信息学的发展趋势

介绍生物信息学产生背景、发展过程以及研究现状,讨论了后基因组时代分子生物学的主要研究领域功能基因组学、蛋白质组学、比较基因组学、药物基因组学之间的关系。在分析基因组时代和后基因组时代生物信息学所研究内容的差异基础上．说明了基于分层递阶结构的系统结构、特征分析方法以及相应的软件系统开发将成为生物信息学发展的基本趋势之一。     

Extensive Rearrangements in the Chloroplast Genome of <Emphasis Type="Italic">Trachelium caeruleum</Emphasis> Are Associated with Repeats and tRNA Genes

Chloroplast genome organization, gene order, and content are highly conserved among land plants. We sequenced the chloroplast genome of Trachelium caeruleum L. (Campanulaceae), a member of an angiosperm family known for highly rearranged genomes. The total genome size is 162,321 bp, with an inverted repeat (IR) of 27,273 bp, large single-copy (LSC) region of 100,114 bp, and small single-copy (SSC) region of 7,661 bp. The genome encodes 112 different genes, with 17 duplicated in the IR, a tRNA gene (trnI-cau) duplicated once in the LSC region, and a protein-coding gene (psbJ) with two duplicate copies, for a total of 132 putatively intact genes. ndhK may be a pseudogene with internal stop codons, and clpP, ycf1, and ycf2 are so highly diverged that they also may be pseudogenes. ycf15, rpl23, infA, and accD are truncated and likely nonfunctional. The most conspicuous feature of the Trachelium genome is the presence of 18 internally unrearranged blocks of genes inverted or relocated within the genome relative to the ancestral gene order of angiosperm chloroplast genomes. Recombination between repeats or tRNA genes has been suggested as a mechanism of chloroplast genome rearrangements. The Trachelium chloroplast genome shares with Pelargonium and Jasminum both a higher number of repeats and larger repeated sequences in comparison to eight other angiosperm chloroplast genomes, and these are concentrated near rearrangement endpoints. Genes for tRNAs occur at many but not all inversion endpoints, so some combination of repeats and tRNA genes may have mediated these rearrangements.     

Phylogeny, Genome Evolution, and Host Specificity of Single-Stranded RNA Bacteriophage (Family Leviviridae)

Bacteriophage of the family Leviviridae have played an important role in molecular biology where representative species, such as Qβ and MS2, have been studied as model systems for replication, translation, and the role of secondary structure in gene regulation. Using nucleotide sequences from the coat and replicase genes we present the first statistical estimate of phylogeny for the family Leviviridae using maximum-likelihood and Bayesian estimation. Our analyses reveal that the coliphage species are a monophyletic group consisting of two clades representing the genera Levivirus and Allolevivirus. The Pseudomonas species PP7 diverged from its common ancestor with the coliphage prior to the ancient split between these genera and their subsequent diversification. Differences in genome size, gene composition, and gene expression are shown with a high probability to have changed along the lineage leading to the Allolevivirus through gene expansion. The change in genome size of the Allolevivirus ancestor may have catalyzed subsequent changes that led to their current genome organization and gene expression. Received: 3 March 2000 / Accepted: 17 October 2000     

Genome editing with CompoZr custom zinc finger nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes. Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA. Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency. While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site. The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator's cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.     

Phylogeny of Plastids Based on Cladistic Analysis of Gene Loss Inferred from Complete Plastid Genome Sequences

Based on the recent hypothesis on the origin of eukaryotic phototrophs, red algae, green plants, and glaucophytes constitute the primary photosynthetic eukaryotes (whose plastids may have originated directly from a cyanobacterium-like prokaryote via primary endosymbiosis), whereas the plastids of other lineages of eukaryotic phototrophs appear to be the result of secondary or tertiary endosymbiotic events (involving a phototrophic eukaryote and a host cell). Although phylogenetic analyses using multiple plastid genes from a wide range of eukaryotic lineages have been carried out, some of the major phylogenetic relationships of plastids remain ambiguous or conflict between different phylogenetic methods used for nucleotide or amino acid substitutions. Therefore, an alternative methodology to infer the plastid phylogeny is needed. Here, we carried out a cladistic analysis of the loss of plastid genes after primary endosymbiosis using complete plastid genome sequences from a wide range of eukaryotic phototrophs. Since it is extremely unlikely that plastid genes are regained during plastid evolution, we used the irreversible Camin-Sokal model for our cladistic analysis of the loss of plastid genes. The cladistic analysis of the 274 plastid protein-coding genes resolved the 20 operational taxonomic units representing a wide range of eukaryotic lineages (including three secondary plastid-containing groups) into two large monophyletic groups with high bootstrap values: one corresponded to the red lineage and the other consisted of a large clade composed of the green lineage (green plants and Euglena) and the basal glaucophyte plastid. Although the sister relationship between the green lineage and the Glaucophyta was not resolved in recent phylogenetic studies using amino acid substitutions from multiple plastid genes, it is consistent with the rbcL gene phylogeny and with a recent phylogenetic study using multiple nuclear genes. In addition, our analysis robustly resolved the conflicting/ambiguous phylogenetic positions of secondary plastids in previous phylogenetic studies: the Euglena plastid was sister to the chlorophycean (Chlamydomonas) lineage, and the secondary plastids from the diatom (Odontiella) and cryptophyte (Guillardia) were monophyletic within the red lineage.     

Plastid Genome Phylogeny and a Model of Amino Acid Substitution for Proteins Encoded by Chloroplast DNA

Maximum likelihood (ML) phylogenies based on 9,957 amino acid (AA) sites of 45 proteins encoded in the plastid genomes of Cyanophora, a diatom, a rhodophyte (red algae), a euglenophyte, and five land plants are compared with respect to several properties of the data, including between-site rate variation and aberrant amino acid composition in individual species. Neighbor-joining trees from AA LogDet distances and ML analyses are seen to be congruent when site rate variability was taken into account. Four feasible trees are identified in these analyses, one of which is preferred, and one of which is almost excluded by statistical criteria. A transition probability matrix for the general reversible Markov model of amino acid substitutions is estimated from the data, assuming each of these four trees. In all cases, the tree with diatom and rhodophyte as sister taxa was clearly favored. The new transition matrix based on the best tree, called cpREV, takes into account distinct substitution patterns in plastid-encoded proteins and should be useful in future ML inferences using such data. A second rate matrix, called cpREV*, based on a weighted sum of rate matrices from different trees, is also considered. Received: 3 June 1999 / Accepted: 26 November 1999     

新方法新技术与新型抗生素发现

日趋严峻的细菌耐药性问题给“抗生素传奇时代”带来了严重威胁, 传统的抗菌药物开发手段已很难应对, 因此急需新思路来寻找新型抗生素。近年来, 一些新方法新技术如新型筛选模型的构建、基因组挖掘技术以及组合生物合成等给天然产物的筛选引发了革命性的变革, 也为放线菌来源的新型抗生素的发现带来了新的希望。本文将对这些新技术及其在新型抗生素研发中的应用进展进行阐述。     

Genetic and electron-microscopic characterization of Rickettsiella bacteria from the manuka beetle, Pyronota setosa (Coleoptera: Scarabaeidae)

Larvae of manuka beetles, Pyronota spp. (Coleoptera: Scarabaeidae) cause pasture damage in New Zealand by feeding on the roots of grasses. Surveys for potential biocontrol agents revealed a putative disease, expressed as whitened larvae of one of the outbreak species, Pyronota setosa. Microbial diagnosis indicated an intracoelomic, intracellular infection, and intracellular bacteria have been identified with subcellular structures characteristic of infection by Rickettsiella-like microorganisms. These bacteria were rod-shaped, often slightly bent with a mean of 628 nm in length and 220 nm in width. Numerous associated protein crystals of variable size and shape occurred within round to oval shaped “giant bodies” either singly or as clusters of smaller crystals. Molecular phylogenetic analysis based on 16S ribosomal RNA and signal recognition particle receptor (FtsY) encoding sequences demonstrates that the manuka beetle pathogen belongs to the taxonomic genus Rickettsiella. Therefore, the pathotype designation ‘Rickettsiella pyronotae’ is proposed to refer to this organism. Moreover, genetic analysis makes it likely that - on the basis of the currently accepted organization of the genus Rickettsiella - this new pathotype should be considered a synonym of the nomenclatural type species, Rickettsiella popilliae.     

Long-Term Adverse Effects of Cigarette Smoking on the Incidence Risk of Metabolic Syndrome With a Dose-Response Relationship: Longitudinal Findings of the Korean Genome and Epidemiology Study Over 12 Years

ObjectiveTo investigate the association between the intensity and cumulative dose of cigarette smoking and incidence risk of metabolic syndrome (MetS) in a longitudinal prospective study over 12 years of follow-up.MethodsThis study included 3151 men aged 40 to 69 years from the Korean Genome and Epidemiology Study. MetS was defined as proposed by the Joint Interim Statement of the Circulation 2009 report. The hazard ratios (HRs) and 95% confidence intervals (95% CIs) for incidence risk of MetS were calculated from 2 separate perspectives: (1) number of cigarettes smoked per day (intensity) and (2) total number of cigarettes smoked over a person’s lifetime (cumulative dose) using multiple logistic regression analyses.ResultsIn comparison with never smokers, the HRs (95% CIs) were 0.97 (0.78-1.21) for former smokers and 1.50 (1.07-2.01) with 0 to 9 cigarettes per day, 1.66 (1.34-2.06) with 10 to 19 cigarettes per day, and 1.75 (1.34-2.29) with ≥20 cigarettes per day for current smokers after adjusting for confounding variables. Similar positive dose-response relationships were also observed when the cumulative dose of cigarette smoking was categorized into former and current smokers, with subcategories of <20 and >20 pack-years (PYs). The HRs (95% CIs) were 0.99 (0.77-1.23) for <20 PYs and 0.99 (0.77-1.28) for ≥20 PYs for former smokers and 1.63 (1.32-2.02) for <20 PYs and 1.67 (1.30-2.14) for ≥20 PYs for current smokers after adjusting for the same covariables.ConclusionCigarette smoking intensity and cumulative dose were both found to be positively associated with the incidence risk of MetS in men.     

Ultrastructural characterization and multilocus sequence analysis (MLSA) of ‘Candidatus Rickettsiella isopodorum’, a new lineage of intracellular bacteria infecting woodlice (Crustacea: Isopoda)

The taxonomic genus Rickettsiella (Gammaproteobacteria; Legionellales) comprises intracellular bacteria associated with a wide range of arthropods including insects, arachnids and crustaceans. The present study provides ultrastructural together with genetic evidence for a Rickettsiella bacterium in the common rough woodlouse, Porcellio scaber (Isopoda, Porcellionidae), occurring in Germany, and shows that this bacterium is very closely related to one of the same genus occurring in California that infects the pill bug, Armadillidium vulgare (Isopoda, Armadillidiidae). Both bacterial isolates displayed the ultrastructural features described previously for crustacean-associated bacteria of the genus Rickettsiella, including the absence of well-defined associated protein crystals; occurrence of the latter is a typical characteristic of infection by this type of bacteria in insects, but has not been reported in crustaceans. A molecular systematic approach combining multilocus sequence analysis (MLSA) with likelihood-based significance testing demonstrated that despite their distant geographic origins, both bacteria form a tight sub-clade within the genus Rickettsiella. In the 16S rRNA gene trees, this sub-clade includes other bacterial sequences from woodlice. Moreover, the bacterial specimens from P. scaber and A. vulgare are found genetically or morphologically different from each of the four currently recognized Rickettsiella species. Therefore, the designation ‘Candidatus Rickettsiella isopodorum’ is introduced for this new lineage of isopod-associated Rickettsiella bacteria.