Linguistic measure of taxonomic and functional relatedness of nucleotide sequences

The frequencies of "words", oligonucleotides within nucleotide sequences, reflect the genetic information contained in the sequence "texts". Nucleotide sequences are characteristically represented by their contrast word vocabularies. Comparison of the sequences by correlating their contrast vocabularies is shown to reflect well the relatedness (unrelatedness) between the sequences. A single value, the linguistic similarity between the sequences, is suggested as a measure of sequence relatedness. Sequences as short as 1000 bases can be characterized and quantitatively related to other sequences by this technique. The linguistic sequence similarity value is used for analysis of taxonomically and functionally diverse nucleotide sequences. The similarity value is shown to be very sensitive to the relatedness of the source species, thus providing a convenient tool for taxonomic classification of species by their sequence vocabularies. Functionally diverse sequences appear distinct by their linguistic similarity values. This can be a basis for a quick screening technique for functional characterization of the sequences and for mapping functionally distinct regions in long sequences.     

Phylogeny and molecular fingerprinting of green sulfur bacteria

The 16S rDNA sequences of nine strains of green sulfur bacteria (Chlorobiaceae) were determined and compared to the four known sequences of Chlorobiaceae and to sequences representative for all eubacterial phyla. The sequences of the Chlorobiaceae strains were consistent with the secondary structure model proposed earlier for Chlorobium vibrioforme strain 6030. Similarity values > 90.1% and K_nuc values < 0.11 indicate a close phylogenetic relatedness among the green sulfur bacteria. As a group, these bacteria represent an isolated branch within the eubacterial radiation. In Chlorobiaceae, a similar morphology does not always reflect a close phylogenetic relatedness. While ternary fission is a morphological trait of phylogenetic significance, gas vesicle formation occurs also in distantly related species. Pigment composition is not an indicator of phylogenetic relatedness since very closely related species contain different bacteriochlorophylls and carotenoids. Two different molecular fingerprinting techniques for the rapid differentiation of Chlorobiaceae species were investigated. The 16S rDNA fragments of several species could not be separated by denaturing gradient gel electrophoresis. In contrast, all strains investigated during the present work gave distinct banding patterns when dispersed repetitive DNA sequences were used as targets in PCR. The latter technique is, therefore, well suited for the rapid screening of isolated pure cultures of green sulfur bacteria. Received: 26 August 1996 / Accepted: 8 January 1997     

Molecular cloning and characterization of ribosomal RNA genes from the brine shrimp: Nucleotide sequence analysis and evolution of the 5.8 S rRNA gene region and its flanking nucleotides

A detailed restriction endonuclease map was prepared for the cloned 5.8 S ribosomal RNA (rRNA) gene region of the brine shrimp Artemia. The nucleotide sequence of the 5.8 S rRNA gene and its flanking nucleotides was determined. This sequence differs in two positions from that of the previously reported 5.8 S rRNA. The primary structure of the Artemia 5.8 S rRNA gene, which, unlike in dipteran insects, is shown to contain no insertion sequence, is conserved according to the relatedness of the species compared. The 5.8 S rRNA gene flanking nucleotides, which were sequenced 176 nucleotide pairs upstream and 70 nucleotide pairs downstream from the gene, show no evidence of sequence conservation between evolutionarily diverse species by computer analysis. Direct nucleotide repeats are present within the flanking sequences at both ends of the gene at about the same distance upstream and downstream, which could serve as processing signals.     

Diversity of preferred nucleotide sequences around the translation initiation codon in eukaryote genomes

Understanding regulatory mechanisms of protein synthesis in eukaryotes is essential for the accurate annotation of genome sequences. Kozak reported that the nucleotide sequence GCCGCC(A/G)CCAUGG (AUG is the initiation codon) was frequently observed in vertebrate genes and that this 'consensus' sequence enhanced translation initiation. However, later studies using invertebrate, fungal and plant genes reported different 'consensus' sequences. In this study, we conducted extensive comparative analyses of nucleotide sequences around the initiation codon by using genomic data from 47 eukaryote species including animals, fungi, plants and protists. The analyses revealed that preferred nucleotide sequences are quite diverse among different species, but differences between patterns of nucleotide bias roughly reflect the evolutionary relationships of the species. We also found strong biases of A/G at position -3, A/C at position -2 and C at position +5 that were commonly observed in all species examined. Genes with higher expression levels showed stronger signals, suggesting that these nucleotides are responsible for the regulation of translation initiation. The diversity of preferred nucleotide sequences around the initiation codon might be explained by differences in relative contributions from two distinct patterns, GCCGCCAUG and AAAAAAAUG, which implies the presence of multiple molecular mechanisms for controlling translation initiation.     

From genomics to functional markers in the era of next-generation sequencing

The availability of complete genome sequences, along with other genomic resources for Arabidopsis, rice, pigeon pea, soybean and other crops, has revolutionized our understanding of the genetic make-up of plants. Next-generation DNA sequencing (NGS) has facilitated single nucleotide polymorphism discovery in plants. Functionally-characterized sequences can be identified and functional markers (FMs) for important traits can be developed at an ever-increasing ease. FMs are derived from sequence polymorphisms found in allelic variants of a functional gene. Linkage disequilibrium-based association mapping and homologous recombinants have been developed for identification of “perfect” markers for their use in crop improvement practices. Compared with many other molecular markers, FMs derived from the functionally characterized sequence genes using NGS techniques and their use provide opportunities to develop high-yielding plant genotypes resistant to various stresses at a fast pace.     

MOLECULAR DELINEATION OF SPECIES AND SYNGENS IN VOLVOCACEAN GREEN ALGAE (CHLOROPHYTA)1

Two species of the colonial green flagellate family Volvocaceae are worldwide in distribution yet exhibit contrasting species structure. Geographically disparate isolates of Gonium pectorale Mueller can interbreed while isolates of Pandorina morum Bory behave quite differently. More than 20 sexually isolated subpopulations occur within this species; these have been termed “syngens” (sensu Sonneborn). Because prezygotic barriers to mating cause intersyngen pairings to fail, breeding analyses cannot be used to estimate genetic relatedness among the syngens of P. morum. DNA comparisons provide an alternative method of assessing genetic relatedness. We compared the nucleotide sequence of the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat among clones of P. morum and of G. pectorale. Members of syngens of P. morum with distribution restricted to one small geographical area show great similarity. Likewise, members of any syngen of worldwide distribution show near uniformity, even those from different continents. However, the ITS sequence of each syngen differs from that of other syngens. In contrast, G. pectorale, which has an ITS region that is remarkably uniform throughout the world, appears to consist of a single syngen within North America and Europe by mating tests. The molecular data are in complete conformity with previous syngen assignment. Because the latter is based on mating affinity, with two complementary mating types per syngen, the evolution of new mating type pairs appears to be the basis of microevolution in these algae. We infer that either P. morum is a more ancient species than G. pectorale or that P. morum has a less stable genome. In either case, the biogeographic distribution of certain syngens may reflect climatological changes of the past.     

Globalizing Genomics: The Origins of the International Nucleotide Sequence Database Collaboration

Genomics is increasingly considered a global enterprise – the fact that biological information can flow rapidly around the planet is taken to be important to what genomics is and what it can achieve. However, the large-scale international circulation of nucleotide sequence information did not begin with the Human Genome Project. Efforts to formalize and institutionalize the circulation of sequence information emerged concurrently with the development of centralized facilities for collecting that information. That is, the very first databases build for collecting and sharing DNA sequence information were, from their outset, international collaborative enterprises. This paper describes the origins of the International Nucleotide Sequence Database Collaboration between GenBank in the United States, the European Molecular Biology Laboratory Databank, and the DNA Database of Japan. The technical and social groundwork for the international exchange of nucleotide sequences created the conditions of possibility for imagining nucleotide sequences (and subsequently genomes) as a “global” objects. The “transnationalism” of nucleotide sequence was critical to their ontology – what DNA sequences came to be during the Human Genome Project was deeply influenced by international exchange.     

Identification of a gene family of cadherin cell adhesion molecules

Cadherins are a group of functionally related glycoproteins responsible for the Ca²⁺-dependent cell-cell adhesion mechanism. They are divided into subclasses, such as E-, P- and N-cadherin, which are distinct in immunological specificities and tissue distribution. Cell aggregation experiments suggest that these molecules have subclass specificities in cell-cell binding and are involved in selective cell adhesions. Analysis of amino acid sequences deduced from the nucleotide sequences of cDNAs encoding cadherins demonstrated that they are integral membrane proteins and share common sequences throughout their entire length; average similarity in the sequences among them is in a range of 50–60%. This result provided evidence that cadherins constitute a gene family which encodes adhesion molecules with different specificities. We also showed that, when cells with little cadherin activity were transfected with cadherin cDNAs, they acquired the cadherin-mediated adhesion properties.     

Evidence of gene orthology and trans‐species polymorphism,but not of parallel evolution,despite high levels of concerted evolution in the major histocompatibility complex of flamingo species

The major histocompatibility complex (MHC) is a cornerstone in the study of adaptive genetic diversity. Intriguingly, highly polymorphic MHC sequences are often not more similar within species than between closely related species. Divergent selection of gene duplicates, balancing selection maintaining trans‐species polymorphism (TSP) that predate speciation and parallel evolution of species sharing similar selection pressures can all lead to higher sequence similarity between species. In contrast, high rates of concerted evolution increase sequence similarity of duplicated loci within species. Assessing these evolutionary models remains difficult as relatedness and ecological similarities are often confounded. As sympatric species of flamingos are more distantly related than allopatric species, flamingos represent an ideal model to disentangle these evolutionary models. We characterized MHC Class I exon 3, Class IIB exon 2 and exon 3 of the six extant flamingo species. We found up to six MHC Class I loci and two MHC Class IIB loci. As all six species shared the same number of MHC Class IIB loci, duplication appears to predate flamingo speciation. However, the high rate of concerted evolution has prevented the divergence of duplicated loci. We found high sequence similarity between all species regardless of codon position. The latter is consistent with balancing selection maintaining TSP, as under this mechanism amino acid sites under pathogen‐mediated selection should be characterized by fewer synonymous codons (due to their common ancestry) than under parallel evolution. Overall, balancing selection maintaining TSP appears to result in high MHC similarity between species regardless of species relatedness and geographical distribution.     

Ecogenomic characterization of widespread,closely-related SAR11 clades of the freshwater genus “Candidatus Fonsibacter” and proposal of Ca. Fonsibacter lacus sp. nov

The ubiquitous alpha-proteobacteria of the order “Candidatus Pelagibacterales” (SAR11) are highly abundant in aquatic environments, and among them, members of the monophyletic lineage LD12 (also known as SAR11 clade IIIb) are specifically found in lacustrine ecosystems. Clade IIIb bacteria are some of the most prominent members of freshwater environments, but little is known about their biology due to the lack of genome representatives. Only recently, the first non-marine isolate was cultured and described as “Candidatus Fonsibacter ubiquis”. Here, we expand the collection of freshwater IIIb representatives and describe a new IIIb species of the genus “Ca. Fonsibacter”. Specifically, we assembled a collection of 67 freshwater metagenomic datasets from the interconnected lakes of the Chattahoochee River basin (GA, USA) and obtained nearly complete metagenome-assembled genomes (MAGs) representing 5 distinct IIIb subclades, roughly equivalent to species based on genomic standards, including the previously described “Ca. F. ubiquis”. Genomic comparisons between members of the IIIb species revealed high similarity in gene content. However, when comparing their abundance profiles in the Chattahoochee basin and various aquatic environments, differences in temporal and spatial distributions among the distinct species were observed implying niche differentiation might be underlying the coexistence of the highly functionally similar representatives. The name Ca. Fonsibacter lacus sp. nov. is proposed for the most abundant and widespread species in the Chattahoochee River basin and various freshwater ecosystems.     

Comparative genomics by capture PCR

There is increasing demand for efficient methods to relate genomic information from model organisms to other species of interest. Comparative genetic analyses are particularly valuable to identify functionally important sequence features on the basis of their evolutionary conservation. We demonstrate here how a single segment of just 32 or less conserved coding nucleotide positions can be used to isolate homologous gene sequences from large numbers of species using a single-sided PCR technique. The method was used to isolate and determine the 3'-untranslated sequence of the somatostatin gene from vertebrate species ranging from human to hagfish. Two sequence motifs centered an average 40-145 nucleotides downstream of the translational stop codon have remained conserved for up to 350 million years. One of the conserved tetrapod segments was used to select a primer for amplification of so-called comparative anchor tagged sequences (CATS) in regular PCR, and shown to amplify homologous sequences from DNA samples from 30 out of 33 tetrapods. In conclusion, we present a useful procedure to reveal functionally relevant sequence elements, and to select primers for amplification of homologous sequences from a wide range of species.     

DNA polymerase gene sequences indicate western and forest tent caterpillar viruses form a new taxonomic group within baculoviruses

Baculoviruses infect larval lepidopterans, and thus have potential value as microbial controls of agricultural and forest pests. Understanding their genetic relatedness and host specificity is relevant to the risk assessment of viral insecticides if non-target impacts are to be avoided. DNA polymerase gene sequences have been demonstrated to be useful for inferring genetic relatedness among dsDNA viruses. We have adopted this approach to examine the relatedness among natural isolates of two uncharacterized caterpillar-infecting baculoviruses, Malacosoma californicum pluviale nucleopolyhedrovirus (McplMNPV) and Malacosoma disstria nucleopolyhedrovirus (MadiMNPV), which infect two closely related host species with little to no cross-infectivity. We designed two degenerate primers (BVP1 and BVP2) based on protein motifs conserved among baculoviruses. McplMNPV and MadiMNPV viral DNA was obtained from naturally infected caterpillars collected from geographically distinct sites in the Southern Gulf Islands and Prince George regions of British Columbia, Canada. Sequencing of 0.9 kb PCR amplicons from six McplMNPV and six MadiMNPV isolates obtained from a total of eight sites, revealed very low nucleotide variation among McplMNPV isolates (99.2-100% nucleotide identity) and among MadiMNPV isolates (98.9-100% nucleotide identity). Greater nucleotide variation was observed between viral isolates from the two different caterpillar species (only 84.7-86.1% nucleotide identity). Both maximum parsimony and maximum likelihood phylogenetic analyses support placement of McplMNPV and MadiMNPV in a clade that is distinct from other groups of baculoviruses.     

What type of diversity yields synergy during mixed litter decomposition in a natural forest ecosystem?

Investigating the relationship of biodiversity and ecosystem function in natural forests allows incorporation of established feedbacks between long-lived plants and soil processes. We studied forested stands in northern Arizona that vary in dominant species richness across small areas. We examined the effects of natural variation in dominant tree biodiversity on ecosystem parameters, particularly litter decomposition. We determined not only whether plant species decompose in mixture as predicted by their individual decomposition rates but also: (1) how particular species affect the decomposition rate of each other in mixture; and (2) whether litter decomposes more rapidly at its site of origin; i.e. is there a “home field advantage” to decomposition? Over a 2-year period, litter mixtures of functionally similar tree species decomposed more rapidly than expected from rates of the individual species alone. Mixtures of conifer species litter decomposed up to 50% faster than expected, with individual conifer members of those mixtures decomposing up to 85% faster than expected. In contrast, more functionally diverse mixtures of litter, which included a deciduous species, did not show synergistic effects during decomposition. We found no significant “home-field advantage” to decomposition. Our study is the first to demonstrate that litter mixtures from more closely related plant species give rise to the most synergistic effects of biodiversity on litter dynamics, indicating that more taxonomically and functionally diverse plant assemblages do not always drive greater emergent effects on ecosystem function.     

Towards theoretical formalisms

This article deals with the relationship between vocabulary (total number of distinct oligomers or “words”) and text-length (total number of oligomers or “words”) for a coding DNA sequence (CDS). For natural human languages, Heaps established a mathematical formula known as Heaps’ law, which relates vocabulary to text-length. Our analysis shows that Heaps’ law fails to model this relationship for CDSs. Here we develop a mathematical model to establish the relationship between the number of type of words (vocabulary) and the number of words sampled (text-length) for CDSs, when non-overlapping nucleotide strings with the same length are treated as words. We use tangent-hyperbolic function, which captures the saturation property of vocabulary. Based on the parameters of the model, we formulate a mathematical equation, known as “equation of word organization”, whose parameters essentially indicate that nucleotide organization of coding sequences are different from one another. We also compare the word organization of CDSs with the random word distribution and conclude that a CDS is neither similar to a natural human language nor to a random one. Moreover, these sequences have their unique nucleotide organization and it is completely structured for specific biological functioning.     

The dilemma of phage taxonomy illustrated by comparative genomics of Sfi21-like Siphoviridae in lactic acid bacteria

The complete genome sequences of two dairy phages, Streptococcus thermophilus phage 7201 and Lactobacillus casei phage A2, are reported. Comparative genomics reveals that both phages are members of the recently proposed Sfi21-like genus of Siphoviridae, a widely distributed phage type in low-GC-content gram-positive bacteria. Graded relatedness, the hallmark of evolving biological systems, was observed when different Sfi21-like phages were compared. Across the structural module, the graded relatedness was represented by a high level of DNA sequence similarity or protein sequence similarity, or a shared gene map in the absence of sequence relatedness. This varying range of relatedness was found within Sfi21-like phages from a single species as demonstrated by the different prophages harbored by Lactococcus lactis strain IL1403. A systematic dot plot analysis with 11 complete L. lactis phage genome sequences revealed a clear separation of all temperate phages from two classes of virulent phages. The temperate lactococcal phages share DNA sequence homology in a patchwise fashion over the nonstructural gene cluster. With respect to structural genes, four DNA homology groups could be defined within temperate L. lactis phages. Closely related structural modules for all four DNA homology groups were detected in phages from Streptococcus or Listeria, suggesting that they represent distinct evolutionary lineages that have not uniquely evolved in L. lactis. It seems reasonable to base phage taxonomy on data from comparative genomics. However, the peculiar modular nature of phage evolution creates ambiguities in the definition of phage taxa by comparative genomics. For example, depending on the module on which the classification is based, temperate lactococcal phages can be classified as a single phage species, as four distinct phage species, or as two if not three different phage genera. We propose to base phage taxonomy on comparative genomics of a single structural gene module (head or tail genes). This partially phylogeny-based taxonomical system still mirrors some aspects of the current International Committee on Taxonomy in Virology classification system. In this system the currently sequenced lactococcal phages would be grouped into five genera: c2-, sk1, Sfi11-, r1t-, and Sfi21-like phages.     

The genus Apsiphortica from China with DNA barcoding information (Diptera: Drosophilidae)

Two new species of the genus Apsiphortica are described from China: A. orthophallos n. sp. and A. sinuatipenis n. sp. Species delimitations are improved by integrating morphological and DNA barcoding information. The intra- and interspecific pairwise p-distances (proportional distance) are summarized for five Apsiphortica species from China. Furthermore, nucleotide sites with fixed status in the alignment of the COI sequences (639 nucleotide sites in length) are used as “pure” molecular diagnostic characters to delineate the five species. A key to all the Chinese species of the genus Apsiphortica is provided.     

The taxonomic position of Saccharomyces boulardii as evaluated by sequence analysis of the D1/D2 domain of 26S rDNA, the ITS1-5.8S rDNA-ITS2 region and the mitochondrial cytochrome-c oxidase II gene

A taxonomic study was carried out on eight strains of Saccharomyces boulardii. Morphological and physiological characteristics were consistent with those of Saccharomyces cerevisiae. Sequences of the D1/D2 domain of the 26S rDNA were identical for all strains examined and had a similarity value of 100% compared to sequences of the type strain of S. cerevisiae (CBS 1171T) and strain S288c. For all S. boulardii isolates was found the exact same ITS1-5.8S rDNA-ITS2 sequence, which displayed a close resemblance with the sequences published for S288c (99.9%), CBS 1171(T) (99.3%) and other S. cerevisiae strains. Sequence analysis of the mitochondrial cytochrome-c oxidase II gene (COX2) also resulted in identical sequences for the S. boulardii isolates and comparisons with available nucleotide sequences revealed close relatedness to strains of S. cerevisiae including S288c (99.5%) and CBS 1171(T) (96.6%). The electrophoretic karyotypes of the S. boulardii strains appeared quite uniform and although very typical of S. cerevisiae, they formed a cluster separate from strains of this species. The results of the present study strongly indicate a close relatedness of S. boulardii to S. cerevisiae and thereby support the recognition of S. boulardii as a member of S. cerevisiae and not as a separate species.     

Evolution of transfer RNA

Evolution by gene duplication and subsequent divergence is indicated by similarities common to 43 different transfer RNAs. Pairwise comparisons of these tRNAs reveal additional similarity, greatest for certain pairs of tRNAs for the same amino acid in the same organism, and also occurring in certain pairs of tRNAs for different amino acids in the same organism. Although tRNAs functionally interact with several other molecules, there have been surprisingly few restrictions on the divergence of their primary structures. This divergence has proceeded so far that clear phylogenetic separations are absent in most cases: it it impossible to construct a coherent phylogeny for most of the 43. Selection and stochastic processes have both been active in the evolution of tRNA. Selection has favored moderate change more than expected and has reduced radical change below that expected from stochastic processes alone. Two obvious effects of selection are nine invariant loci, another five that are always purines and five others that are always pyrimidines, in the tRNAs involved in protein synthesis. In addition to these constraints in the primary nucleotide sequence, the method of “identical site equivalents”, introduced here, demonstrates that further constraints exist equivalent to about 12 additional invariant loci. These “invisible” restraints reflect disperse chemical forces maintaining the tertiary structure and reducing evolutionary divergence to an extent quantitatively comparable to that of the nine observable invariant loci. The average divergence (49·4%) for pairs of tRNAs for different amino acids involved in protein synthesis represents an equilibrium between natural selection and stochastic processes. These tRNAs have had time to diverge nearly to the 75% maximum expected from stochastic process alone; this is shown by comparing the two glycine tRNAs involved in peptidoglycan synthesis with tRNAs for different amino acids participating in polypeptide synthesis. The rates of nucleotide replacements in genes coding for the tRNAs and the cytochromes c are about the same: 2 × 10 ^?10 replacements per nucleotide site per year.     

Evolution of the rat kallikrein gene family: Gene conversion leads to functional diversity

Summary Kallikrein-like simple serine proteases are encoded by closely related members of a gene family in several mammalian species. Molecular cloning and genomic Southern blot analysis after conventional and pulsed-field gel electrophoresis indicate that the rat kallikrein gene family comprises 15–20 members, probably closely linked at a single locus. Determination of the nucleotide sequences of the rGK-3,-4, and-6 genes here completes sequence data for a total of nine rat kallikrein family members. Comparison of the rat gene sequences to each other and to those of human and mouse kallikrein family genes reveals patterns of relatedness indicative of concerted evolution. Analysis of nucleotide sequence variants in kallikrein family members shows that most sequence variants are shared by multiple family members; the patterns of shared variants are complex and indicate multiple short gene conversions between family members. Sequence exchanges between family members generate novel assortments of variants in amino acid coding regions that may affect substrate specificity and thereby contribute to the diversity of enzyme activity. Furthermore, small sequence exchanges also may play a role in generating the diverse patterns of tissue-specific expression of rat family members. These analyses indicate an important role for gene conversion in the evolution of the functional diversity of these duplicated genes.