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     

Evolution in a chronic RNA virus infection: selection on HTLV-I tax protein differs between healthy carriers and patients with tropical spastic paraparesis

HTLV-I causes T-cell leukemia and tropical spastic paraparesis (TSP) in a minority of infected people, whereas the majority remain healthy. The virus differs little in sequence between isolates but has been shown to have a quasispecies structure. Using the Nei and Gojobori algorithm, we have shown that the proportion of nonsynonymous to synonymous changes in HTLV-I proviral tax gene sequences from healthy seropositive subjects (Dn/Ds= 0.9 to 1.3) is significantly higher than those from TSP patients (Dn/Ds= 0.3 to 0.6). Here we show that the distinction between healthy seropositives and TSP patients can only be seen with proviral tax sequences, but not with cDNA, the amino-terminal or carboxy-terminal half of tax, or the rex gene. The Dn/Ds ratio of proviral tax sequences was used to analyze two TSP patients with atypical features and to investigate the influence of cytotoxic T cells (CTL) on the viral quasispecies. Received: 18 January 1995 / Accepted: 7 November 1995     

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     

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