Restriction Endonuclease Analysis of Highly Repetitive DNA as a Phylogenetic Tool

Multiple band patterns of DNA repeats in the 20–500-nucleotide range can be detected by digesting genomic DNA with short—cutting restriction endonucleases, followed by end labeling of the restriction fragments and fractionation in nondenaturing polyacrylamide gels. We call such band patterns obtained from genomic DNA ``taxonprints' (Fedorov et al. 1992). Here we show that taxonprints for the taxonomic groups studied (mammals, reptiles, fish, insects—altogether more than 50 species) have the following properties: (1) All individuals from the same species have identical taxonprints. (2) Taxonprint bands can be subdivided into those specific for a single species and those specific for groups of closely related species, genera, and even families. (3) Each restriction endonuclease produces unique band patterns; thus, five to ten restriction enzymes (about 100 bands) may be sufficient for a statistical treatment of phylogenetic relationships based on polymorphisms of restriction endinuclease sites. We demonstrate that taxonprint analysis allows one to distinguish closely related species and to establish the degree of similarity among species and among genera. These characteristics make taxonprint analysis a valuable tool for taxonomic and phylogenetic studies. Received: 10 February 1997 / Accepted: 10 March 1997     

Phylogeny of the Restriction Endonuclease-Like Superfamily Inferred from Comparison of Protein Structures

To date all attempts to derive a phyletic relationship among restriction endonucleases (ENases) from multiple sequence alignments have been limited by extreme divergence of these enzymes. Based on the approach of Johnson et al. (1990), I report for the first time the evolutionary tree of the ENase-like protein superfamily inferred from quantitative comparison of atomic coordinates of structurally characterized enzymes. The results presented are in harmony with previous comparisons obtained by crystallographic analyses. It is shown that λ-exonuclease initially diverged from the common ancestor and then two ``endonucleolytic' families branched out, separating ``blunt end cutters' from ``5′ four-base overhang cutters.' These data may contribute to a better understanding of ENases and encourage the use of structure-based methods for inference of phylogenetic relationship among extremely divergent proteins. In addition, the comparison of three-dimensional structures of ENase-like domains provides a platform for further clustering analyses of sequence similarities among different branches of this large protein family, rational choice of homology modeling templates, and targets for protein engineering. Received: 14 June 1999 / Accepted: 11 August 1999     

Insights into the Origin of Clostridiumbotulinum Strains: Evolution of Distinct Restriction Endonuclease Sites in rrs (16S rRNA gene)

Diversity analysis of Clostridium botulinum strains is complicated by high microheterogeneity caused by the presence of 9–22 copies of rrs (16S rRNA gene). The need is to mine genetic markers to identify very closely related strains. Multiple alignments of the nucleotide sequences of the 212 rrs of 13 C. botulinum strains revealed intra- and inter-genomic heterogeneity. Low intragenomic heterogeneity in rrs was evident in strains 230613, Alaska E43, Okra, Eklund 17B, Langeland, 657, Kyoto, BKT015925, and Loch Maree. The most heterogenous rrs sequences were those of C. botulinum strains ATCC 19397, Hall, H04402065, and ATCC 3502. In silico restriction mapping of these rrs sequences was observable with 137 type II Restriction endonucleases (REs). Nucleotide changes (NC) at these RE sites resulted in appearance of distinct and additional sites, and loss in certain others. De novo appearances of RE sites due to NC were recorded at different positions in rrs gene. A nucleotide transition A>G in rrs of C. botulinum Loch Maree and 657 resulted in the generation of 4 and 10 distinct RE sites, respectively. Transitions A>G, G>A, and T>C led to the loss of RE sites. A perusal of the entire NC and in silico RE mapping of rrs of C. botulinum strains provided insights into their evolution. Segregation of strains on the basis of RE digestion patterns of rrs was validated by the cladistic analysis involving six house keeping genes: dnaN, gyrB, metG, prfA, pyrG, and Rho.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0514-z) contains supplementary material, which is available to authorized users.     

Divergence of Noncoding Sequences and of Insertions Encoding Nonglobular Domains at a Genomic Region Well Conserved in Plasmodia

To identify conserved features in the rapidly diverging portions of a well-conserved locus, completely sequenced in Plasmodium falciparum and Plasmodium berghei, a computational method based on recurrence analysis was exploited. At the level of the genomic sequence, in both species, introns and intergenic sequences—though subject to rapid diversification—do not drift without constraints, but rather coevolve, in the sense that they maintain not only an AT-rich base composition, but also a consistent use of recurring (AT) _n tracts. One of the two genes present in the conserved locus encodes a protein that exhibits blocks of high similarity to the first enzyme in glutathione biosynthesis (γ-glutamylcysteine synthetase) but bears long low-complexity insertions, absent in other organisms. From an analysis of the aminoacid sequence, different constraints appear to act on the borders and on the central part of the insertions. Albeit maintaining a strong bias toward hydrophylic residues, central portions diverge more rapidly than borders, through point mutation and differential presence of entire tracts. Received: 20 September 1999 / Accepted: 9 February 2000     

Phylogenetic Analysis of a Retroposon Family in African Great Apes

The SINE-R retroposon family has been identified by its relationship with the long terminal repeats (LTRs) of human endogenous retrovirus class K (HERV-K) as a mobile element that has evolved recently in the human genome. Here we examined the recent evolutionary history of this class of elements by a PCR approach to genomic DNA from the African great apes and by phylogenetic analysis including comparison with the HERV K10 parent sequence. With primers derived from a cDNA sequence from human brain, we identified 27 sequences from the chimpanzee and 16 from the gorilla. Phylogenetic comparisons with previously recognized sequences from the human and from the orangutan and gibbon revealed wide overlap of elements across species, suggesting multiple origins in the course of hominoid evolution. Two human elements SINE-R.C2 and HS307 were the furthest removed from the HERV-K10 sequence but these two elements were closely related to three elements from the chimpanzee and four elements from the gorilla. This group of elements (our clusters 14 and 15) appears to have transposed late in hominoid evolution. One element (Ch-M16) showed 99.1% sequence identity with the SINE-R.C2 element, which is human-specific. Thus the SINE-R family appears to have continued to be active in transposition throughout the course of primate evolution. Received: 12 March 1999 / Accepted: 25 May 1999     

Anopheles stephensi Dox-A2 Shares Common Ancestry with Genes from Distant Groups of Eukaryotes Encoding a 26S Proteasome Subunit and Is in a Conserved Gene Cluster

The sequence of a cloned Anopheles stephensi gene showed 72% inferred amino acid identity with Drosophila melanogaster Dox-A2 and 93% with its putative ortholog in Anopheles gambiae. Dox-A2 is the reported but herein disputed structural locus for diphenol oxidase A2. Database searches identified Dox-A2 related gene sequences from 15 non-insect species from diverse groups. Phylogenetic trees based on alignments of inferred protein sequences, DNA, and protein motif searches and protein secondary structure predictions produced results consistent with expectations for genes that are orthologous. The only inconsistency was that the C-terminus appears to be more primitive in the yeasts than in plants. In mammals, plants, and yeast these genes have been shown to code for a non-ATPase subunit of the PA700 (19S) regulatory complex of 26S proteasome. The analyses indicated that the insect genes contain no divergent structural features, which taken within an appraisal of all available data, makes the reported alternative function highly improbable. A plausible additional role, in which the 26S proteasome is implicated in regulation of phenol oxidase, would also apply to at least the mammalian genes. No function has yet been reported for the other included sequences. These were from genome projects and included Caenorhabiditus elegans, Arabidopsis thaliana, Fugu rubripes, and Toxoplasma gondii. A consensus of the results predicts a protein containing exceptionally long stretches of helix with a hydrophilic C-terminus. Phosphorylation site motifs were identified at two conserved positions. Possible SRY and GATA-1 binding motifs were found at conserved positions upstream of the mosquito genes. The location of A. stephensi Dox-A2 was determined by in situ hybridization at 34D on chromosome arm 3R. It is in a conserved gene cluster with respect to the other insects. However, the A. stephensi cluster contains a gene showing significant sequence identity to human and pigeon carnitine acetyltransferase genes, therefore showing divergence with the distal end of the D. melanogaster cluster. Received: 3 July 1998 / Accepted: 22 December 1999     

Low Divergence in rDNA ITS Sequences Among Five Species of Fucus (Phaeophyceae) Suggests a Very Recent Radiation

Sequences from the two ribosomal DNA internal transcribed spacers (ITS1 and ITS2) were compared among five species of Fucus. Based on the present taxon sampling, parsimony analysis showed that Fucus serratus is the sister-group of the remaining Fucus species when Ascophyllum nodosum was used as an outgroup. The topology of the tree was (Fucus serratus (F. lutarius (F. vesiculosus (F. spiralis+F. ceranoides)))). The extremely low variation observed suggests a very recent radiation of the genus which supports the view widely accepted that the Fucales are among the most evolutionarily advanced of the brown algae. We further note that sequence differences between Fucus and Ascophyllum were 28%: this does not rule out the utility of ITS sequences within the Fucaceae. The very low number of informative positions allows to demonstrate empirically that distance matrix methods group on the basis of symplesiomorphies. Received: 17 February 1997 / Accepted: 17 April 1997     

Full-Length L1 Elements Have Arisen Recently in the Same 1-kb Region of the Gorilla and Human Genomes

New copies of the mammalian retrotransposon L1 arise in the germline at an undetermined rate. Each new L1 copy appears at a specific evolutionary time point that can be estimated by phylogenetic analysis. In humans, the active L1 sequence L1.2 resides at the genomic locus LRE1. Here we analyzed the region surrounding the LRE1 locus in humans and gorillas to determine the evolutionary history of the region and to estimate the age of L1.2. We found that the region was composed of an ancient L1, L1Hs-Lrg, which was significantly divergent from all other L1 sequences available in the databases. We also determined that L1.2 was absent from the gorilla genome and arose in humans after the divergence of gorilla and human lineages. In the gorilla LRE1 region, we discovered a different full-length L1 element, L1Gg-1, which was allelic and present at a high gene frequency in gorillas but absent from other primates. We determined the nucleotide sequence of L1Gg-1 and found that it was 98% identical to L1.2, suggesting a close relationship between active L1s in gorillas and humans. Received: 28 December 1997 / Accepted: 20 March 1998     

Sequence Variation in the Gene Encoding the Nonstructural 3 Protein of Hepatitis C Virus: Evidence for Immune Selection

To determine whether the persistent nature of hepatitis C infection is related to the emergence of antigenic variants driven by immune selection, we examined the sequence heterogeneity in a portion of the hepatitis C virus (HCV) nonstructural 3 (NS3) gene of a patient infected over the course of more than 2 years. By PCR amplification, cloning, and sequencing, we observed several variable and conserved regions in the NS3 segment of the HCV genome. All variable regions had higher ratios of nonsynonymous/synonymous mutations and encompassed immunodominant epitopes, and their locations were not essential to maintain the known function of HCV RNA helicase. In contrast, the regions that are critical for HCV RNA helicase activity were found to be conserved with lower heterogeneity or lower ratios of nonsynonymous/synonymous mutations, and none except one of these regions was encoded within immunodominant epitopes. Our results are consistent with immune selection of viral variants at the epitope and molecular levels that may enable HCV to evade host defenses over time. Plotting the relatedness of sequence variants revealed a star topology suggesting that a wild-type HCV sequence is maintained, unlike HIV. Received: 2 November 2000 / Accepted: 1 October 2001     

A New Method for Locating Changes in a Tree Reveals Distinct Nucleotide Polymorphism vs Divergence Patterns in Mouse Mitochondrial Control Region

A new, model-based method was devised to locate nucleotide changes in a given phylogenetic tree. For each site, the posterior probability of any possible change in each branch of the tree is computed. This probabilistic method is a valuable alternative to the maximum parsimony method when base composition is skewed (i.e., different from 25% A, 25% C, 25% G, 25% T): computer simulations showed that parsimony misses more rare → common than common → rare changes, resulting in biased inferred change matrices, whereas the new method appeared unbiased. The probabilistic method was applied to the analysis of the mutation and substitution processes in the mitochondrial control region of mouse. Distinct change patterns were found at the polymorphism (within species) and divergence (between species) levels, rejecting the hypothesis of a neutral evolution of base composition in mitochondrial DNA. Received: 15 March 1999 / Accepted: 7 October 1999     

Intraspecies Analysis: Comparison of ITS Sequence Data and Gene Intron Sequence Data with Breeding Data for a Worldwide Collection of Gonium pectorale

The morphologically uniform species Gonium pectorale is a colonial green flagellate of worldwide distribution. The affinities of 25 isolates from 18 sites on five continents were assessed by both DNA sequence comparisons and sexual compatibility. Complete sequences were obtained (i) for the internal transcribed spacer ITS-1 and ITS-2 regions of ribosomal DNA and (ii) for each of three single-copy spliceosomal introns, two in a small G protein and one in the actin gene. ITS sequences appeared to homogenize sufficiently rapidly to behave as a single copy gene. Intron sequence differences between isolates in this species reached nucleotide substitution saturation, while ITS sequences did not. Parsimony and evolutionary distance analysis of the two types of DNA data gave essentially the same tree conformation. By all these criteria, the group of G. pectorale isolates fell into two main clades, A and B. Clade A, with isolates from four continents, was comprised of four subclades of quite closely related isolates, plus one strain of ambiguous affinity. Clade B was comprised of two subclades represented by South African and South American isolates, respectively; thus, only subclades of clade B showed geographical localization. With respect to mating, all isolates except one homothallic strain and one apparently sterile strain fell into either one or the other of two mating types. Pairings in all possible combinations revealed that isolates from the same site formed abundant zygotes, which germinated to produce new, sexually active organisms. Zygotes were also formed in many pairings of other combinations, including crosses of clade A with clade B organisms, but none of the latter produced viable germlings. The ability to mate and produce viable progeny that were themselves capable of sexual reproduction was restricted to members of subclades established on the basis of DNA sequence similarities. Thus, the grades of difference in both nuclear intron sequences and rDNA ITS sequences paralleled those observed in the sexual analysis. Received: 9 March 1998 / Accepted: 1 June 1998     

Identification of Multiple Gypsy LTR-Retrotransposon Lineages in Vertebrate Genomes

Gypsy LTR-retrotransposons have been identified in the genomes of many organisms, but only a small number of vertebrate examples have been reported to date. Here we show that members of this family are likely to be widespread in many vertebrate classes with the possible exceptions of mammals and birds. Phylogenetic analyses demonstrate that although there are several distinct lineages of vertebrate gypsy LTR-retrotransposons, the majority clusters into one monophyletic clade. Groups of fungal, plant, and insect elements were also observed, suggesting horizontal transfer between phyla may be infrequent. However, in contrast to this, there was little evidence to support sister relationships between elements derived from vertebrate and insect hosts. In fact, the majority of the vertebrate elements appeared to be most closely related to a group of gypsy LTR-retrotransposons present within fungi. This implies either that at least one horizontal transmission between these two phyla has occurred previously or that a gypsy LTR-retrotransposon lineage has been lost from insect taxa. Received: 22 December 1998 / Accepted: 6 April 1999     

A Complex Organization of the Gene Encoding Cytochrome Oxidase Subunit 1 in the Mitochondrial Genome of the Dinoflagellate, Crypthecodinium cohnii: Homologous Recombination Generates Two Different cox1 Open Reading Frames

In the course of investigating mitochondrial genome organization in Crypthecodinium cohnii, a non-photosynthetic dinoflagellate, we identified four EcoRI fragments that hybridize to a probe specific for cox1, the gene that encodes subunit 1 of cytochrome oxidase. Cloning and sequence characterization of the four fragments (5.7, 5.1, 4.1, 3.5 kilobase pairs) revealed that cox1 exists in four distinct but related contexts in C. cohnii mtDNA, with a central repeat unit flanked by one of two possible upstream (flanking domain 1 or 2) and downstream (flanking domain 3 or 4) regions. The majority of the cox1 gene is located within the central repeat; however, the C-terminal portion of the open reading frame extends into flanking domains 3 and 4, thereby creating two distinct cox1 coding sequences. The 3′-terminal region of one of the cox1 reading frames can assume an elaborate secondary structure, which potentially could act to stabilize the mature mRNA against nucleolytic degradation. In addition, a high density of small inverted repeats (15–22 base pairs) has been identified at the 5′-end of cox1, further suggesting that hairpin structures could be important for gene regulation. The organization of cox1 in C. cohnii mtDNA appears to reflect homologous recombination events within the central repeat between different cox1 sequence contexts. Such recombining repeats are a characteristic feature of plant (angiosperm) mtDNA, but they have not previously been described in the mitochondrial genomes of protists. Received: 21 December 2000 / Accepted: 30 January 2001     

Voltage-dependent Conductance Changes in a Nonvoltage-activated Sodium Current from a Mast Cell Line

Nonexcitable cells do not express voltage-activated Na⁺ channels. Instead, selective Na⁺ influx is accomplished through GTP-activated Na⁺ channels, the best characterized of which are found in renal epithelia. We have described recently a GTP-dependent Na⁺ current in rat basophilic leukemia (RBL) cells that differs from previous reported Na⁺ channels in several ways including selectivity, pharmacology and mechanism of activation. In this report, we have investigated the biophysical properties of the RBL cell Na⁺ current using the whole cell patch-clamp technique. Following activation by 250–500 μm GTPγS, hyperpolarizing steps to a fixed potential (−100 mV) from a holding potential of 0 mV evoked transient inward Na⁺ currents that declined during the pulse. If the holding potential was made more positive (range 0 to +100 mV), then the amplitude of the transient inward current evoked by the hyperpolarization increased steeply, demonstrating that the conductance of the channels was voltage-dependent. Using a paired pulse protocol (500 msec pulses to −100 mV from a holding potential of 0 mV), it was found that the peak amplitude of the current during the second pulse became larger as the interpulse potential became more positive. In addition, increasing the time at which the cells were held at positive potentials also resulted in larger currents, indicating a time-dependent conductance change. With symmetrical Na⁺ solutions, outward currents were recorded at positive potentials and these demonstrated both a time- and voltage-dependent increase in conductance. The results show that a nonvoltage activated Na⁺ channel in an electrically nonexcitable cell undergoes prominent voltage-dependent transitions. Possible mechanisms underlying this voltage dependency are discussed. Received: 12 March 1998/Revised: 5 June 1998     

Extracellular ATP activates a P2 receptor in necturus erythrocytes during hypotonic swelling

We recently reported that ATP is released from Necturus erythrocytes via a conductive pathway during hypotonic swelling and that extracellular ATP potentiates regulatory volume decrease (RVD). This study was designed to determine whether extracellular ATP exerts its effect via a purinoceptor. This was accomplished using three different experimental approaches: 1) hemolysis studies to examine osmotic fragility, 2) a Coulter counter to assess RVD, and 3) the whole-cell patch-clamp technique to measure membrane currents. We found extracellular ATP and ATPγS, two P2 agonists, decreased osmotic fragility, enhanced cell volume recovery in response to hypotonic shock, and increased whole-cell currents. In addition, 2-methylthio-ATP potentiated RVD. In contrast, UTP, α,β-methylene-ATP, and 2′-& 3′-O-(4-benzoyl-benzoyl) adenosine 5′-triphosphate and the P1 agonist adenosine had no effect regardless of experimental approach. Furthermore, the P2 antagonist suramin increased osmotic fragility, inhibited RVD, and reduced whole-cell conductance in swollen cells. Consistent with a previous study that indicated cell swelling activates a K⁺ conductance, suramin had no effect in the presence of gramicidin (a cationophore used to maintain a high K⁺ permeability). We also found the P2 antagonist pyridoxal-5-phosphate-6-azophenyl-2′4-disulfonic acid (PPADS) increased osmotic fragility; however, reactive blue 2 and the P1 antagonists caffeine and theophylline had no effect. Our results show that extracellular ATP activated a P2 receptor in Necturus erythrocytes during hypotonic swelling, which in turn potentiated RVD by stimulating K⁺ efflux. Pharmacological evidence suggested the presence of a P2X receptor subtype. Received: 6 January 2001/Revised: 17 April 2001     

Enhanced Closed-state Inactivation in a Mutant Shaker K+ Channel

Many mutations that shift the voltage dependence of activation in Shaker channels cause a parallel shift of inactivation. The I2 mutation (L382I in the Shaker B sequence) is an exception, causing a 45 mV activation shift with only a 9 mV shift of inactivation midpoint relative to the wildtype (WT) channel. We compare the behavior of WT and I2 Shaker 29-4 channels in macropatch recordings from Xenopus oocytes. The behavior of WT channels can be described by both simple and detailed kinetic models which assume that inactivation proceeds only from the open state. The behavior of I2 channels requires that they inactivate from closed states as well, a property characteristic of voltage-gated sodium channels. A detailed ``multiple-state inactivation' model is presented that describes both activation and inactivation of I2 channels. The results are consistent with the view that residue L382 is associated with the receptor for the inactivation particles in Shaker channels. Received: 16 December 1996/Revised: 5 February 1997