Ribozyme Relationships: The Hammerhead, Hepatitis Delta, and Hairpin Ribozymes Have a Common Origin

The hammerhead and the hairpin ribozymes of plant viroids/virusoids and the hepatitis delta ribozyme were generally considered to be unrelated to one another. Here we report notable sequence interrelationships and some structural ones connecting all three. Received: 8 September 1999 / Accepted: 31 March 2000     

RNA Folding Argues Against a Hot-Start Origin of Life

Opinion is strongly divided on whether life arose on earth under hot or cold conditions, the hot-start and cold-start scenarios, respectively. The origin of life close to deep thermal vents appears as the majority opinion among biologists, but there is considerable biochemical evidence that high temperatures are incompatible with an RNA world. To be functional, RNA has to fold into a three-dimensional structure. We report both theoretical and experimental results on RNA folding and show that (as expected) hot conditions strongly reduce RNA folding. The theoretical results come from energy-minimization calculations of the average extent of folding of RNA, mainly from 0–90°C, for both random sequences and tRNA sequences. The experimental results are from circular-dichroism measurements of tRNA over a similar range of temperatures. The quantitative agreement between calculations and experiment is remarkable, even to the shape of the curves indicating the cooperative nature of RNA folding and unfolding. These results provide additional evidence for a lower temperature stage being necessary in the origin of life. Received: 1 March 2000 / Accepted: 14 June 2000     

The Evolution of the Ribonucleotide Reductases: Much Ado About Oxygen

Ribonucleotide reduction is the only known biological means for de novo production of deoxyribonucleotides, the building blocks of DNA. These are produced from ribonucleotides, the building blocks of RNA, and the direction of this reaction has been taken to support the idea that, in evolution, RNA preceded DNA as genetic material. However, an understanding of the evolutionary relationships among the three modern-day classes of ribonucleotide reductase and how the first reductase arose early in evolution is still far off. We propose that the diversification of this class of enzymes is inherently tied to microbial colonization of aerobic and anaerobic niches. The work is of broader interest, as it also sheds light on the process of adaptation to oxygenic environments consequent to the evolution of atmospheric oxygen.     

Ancient Mitochondrial DNA Reveals the Origin of Sus scrofa from Rebun Island, Japan

The Kabukai A site (5 to 8C A.D.) of the Okhotsk cultural area is on Rebun Island, a small island near the coast, north–northwest of Hokkaido, Japan. Specimens of Sus scrofa, called the Sakhalin pig, were discovered in five cultural layers at the Kabukai A site. Ancient DNA was extracted from the remains of 42 Sakhalin pig bones. Thirty-nine nucleotide sequences of the 574-bp mitochondrial DNA control region, estimated to have originated from at least 21 individuals, were amplified and analyzed phylogenetically. Nine distinct haplotypes (A1, A2, A3, B1, B2, C1, C2, D1, and D2) from this site were classified into four haplotype groups (A, B, C, and D) by parsimonious network analysis. Phylogenetic analysis of 9 ancient and 55 modern haplotypes indicated that the population of Sakhalin pigs at the Kabukai A site belonged to two distinct clusters; haplotype groups A and B formed a cluster comprised only of themselves, and haplotype groups C and D belonged to the cluster of one of the two genetic groups of Japanese wild boars uniquely distributed in the western part of Japan, including one northeast Mongolian wild boar. Analysis of the haplotype distribution among three archaeological sites and their historical transitions among the five layers reflecting the cultural periods at the Kabukai A site suggests that the Sakhalin pig populations were introduced from Sakhalin island and the Amur River basin in the northeastern Eurasian continent together with some cultural influences. Received: 18 April 2000 / Accepted: 24 November 2000     

Poxviruses and the Origin of the Eukaryotic Nucleus

A number of molecular forms of DNA polymerases have been reported to be involved in eukaryotic nuclear DNA replication, with contributions from α-, δ-, and ε-polymerases. It has been reported that δ-polymerase possessed a central role in DNA replication in archaea, whose ancestry are thought to be closely related to the ancestor of eukaryotes. Indeed, in vitro experiment shown here suggests that δ-polymerase has the potential ability to start DNA synthesis immediately after RNA primer synthesis. Therefore, the question arises, where did the α-polymerase come from? Phylogenetic analysis based on the nucleotide sequence of several conserved regions reveals that two poxviruses, vaccinia and variola viruses, have polymerases similar to eukaryotic α-polymerase rather than δ-polymerase, while adenovirus, herpes family viruses, and archaeotes have eukaryotic δ-like polymerases, suggesting that the eukaryotic α-polymerase gene is derived from a poxvirus-like organism, which had some eukaryote-like characteristics. Furthermore, the poxvirus's proliferation independent from the host-cell nucleus suggests the possibility that this virus could infect non-nucleated cells, such as ancestral eukaryotes. I wish to propose here a new hypothesis for the origin of the eukaryotic nucleus, posing symbiotic contact of an orthopoxvirus ancestor with an archaebacterium, whose genome already had a δ-like polymerase gene. Received: 26 October 2000 / Accepted: 16 January 2001     

The Prebiotic Synthesis of Modified Purines and Their Potential Role in the RNA World

Modified purines are found in all organisms in the tRNA, rRNA, and even DNA, raising the possibility of an early role for these compounds in the evolution of life. These include N ⁶-methyladenine, 1-methyladenine, N ⁶,N ⁶-dimethyladenine, 1-methylhypoxanthine, 1-methylguanine, and N ²-methylguanine. We find that these bases as well as a number of nonbiological modified purines can be synthesized from adenine and guanine by the simple reaction of an amine or an amino group with adenine and guanine under the concentrated conditions of the drying-lagoon or drying-beach model of prebiotic synthesis with yields as high as 50%. These compounds are therefore as prebiotic as adenine and guanine and could have played an important role in the RNA world by providing additional functional groups in ribozymes, especially for the construction of hydrophobic binding pockets. Received: 7 August 1998 / Accepted: 31 December 1998     

Ribonucleotide reductases and their occurrence in microorganisms: A link to the RNA/DNA transition

Abstract: The evolution of a deoxyribonucleotide synthesizing ribonucleotide reductase might have initiated the transition from the ancient RNA world into the prevailing DNA world. At least five classes of ribonucleotide reductases have evolved. The ancient enzyme has not been identified. A reconstruction of the first ribonucleotide reductase requires knowledge of contemporary enzymes and of microbial evolution. Experimental work on the former focuses on few organisms, whereas the latter is now well understood on the basis of ribosomal RNA sequences. Deoxyribonucleotide formation has not been investigated in many evolutionary important microorganisms. This review covers our knowledge on deoxyribonucleotide synthesis in microorganisms and the distribution of ribonucleotide reductases in nature. Ecological constraints on enzyme evolution and knowledge deficiencies emerge from complete coverage of the phylogenetic groups.     

On the Origin of Metabolic Pathways

The heterotrophic theory of the origin of life is the only proposal available with experimental support. This comes from the ease of prebiotic synthesis under strongly reducing conditions. The prebiotic synthesis of organic compounds by reduction of CO₂ to monomers used by the first organisms would also be considered an heterotrophic origin. Autotrophy means that the first organisms biosynthesized their cell constituents as well as assembling them. Prebiotic synthetic pathways are all different from the biosynthetic pathways of the last common ancestor (LCA). The steps leading to the origin of the metabolic pathways are closer to prebiotic chemistry than to those in the LCA. There may have been different biosynthetic routes between the prebiotic and the LCAs that played an early role in metabolism but have disappeared from extant organisms. The semienzymatic theory of the origin of metabolism proposed here is similar to the Horowitz hypothesis but includes the use of compounds leaking from preexisting pathways as well as prebiotic compounds from the environment.     

On Error Minimization in a Sequential Origin of the Standard Genetic Code

Distances between amino acids were derived from the polar requirement measure of amino acid polarity and Benner and co-workers' (1994) 74-100 PAM matrix. These distances were used to examine the average effects of amino acid substitutions due to single-base errors in the standard genetic code and equally degenerate randomized variants of the standard code. Second-position transitions conserved all distances on average, an order of magnitude more than did second-position transversions. In contrast, first-position transitions and transversions were about equally conservative. In comparison with randomized codes, second-position transitions in the standard code significantly conserved mean square differences in polar requirement and mean Benner matrix-based distances, but mean absolute value differences in polar requirement were not significantly conserved. The discrepancy suggests that these commonly used distance measures may be insufficient for strict hypothesis testing without more information. The translational consequences of single-base errors were then examined in different codon contexts, and similarities between these contexts explored with a hierarchical cluster analysis. In one cluster of codon contexts corresponding to the RNY and GNR codons, second-position transversions between C and G and transitions between C and U were most conservative of both polar requirement and the matrix-based distance. In another cluster of codon contexts, second-position transitions between A and G were most conservative. Despite the claims of previous authors to the contrary, it is shown theoretically that the standard code may have been shaped by position-invariant forces such as mutation and base content. These forces may have left heterogeneous signatures in the code because of differences in translational fidelity by codon position. A scenario for the origin of the code is presented wherein selection for error minimization could have occurred multiple times in disjoint parts of the code through a phyletic process of competition between lineages. This process permits error minimization without the disruption of previously useful messages, and does not predict that the code is optimally error-minimizing with respect to modern error. Instead, the code may be a record of genetic process and patterns of mutation before the radiation of modern organisms and organelles. Received: 28 July 1997 / Accepted: 23 January 1998     

Complex Evolution of Tandem-Repetitive DNA in the Chironomus thummi Species Group

The subspecies Chironomus thummi thummi and C. t. piger display dramatic differences in the copy number and chromosomal localization of a tandemly repeated DNA family (Cla elements). In order to analyze the evolutionary dynamics of this repeat family, we studied the organization of Cla elements in the related outgroup species C. luridus. We find three different patterns of Cla element organization in C. luridus, showing that Cla elements may be either strictly tandem-repetitive or be an integral part of two higher-order tandem repeats (i.e., Hinf[lur] elements, Sal[lur] elements). All three types of Cla-related repeats are localized in the centromeres of C. luridus chromosomes. This suggests that the dispersed chromosomal localization of Cla elements in C. t. thummi may be the result of an amplification and transposition during evolution of this subspecies. Received: 22 May 1996 / Accepted: 8 October 1996     

Mutation and Recombination in Cattle Satellite DNA: A Feedback Model for the Evolution of Satellite DNA Repeats

The cattle genome contains several distinct centromeric satellites with interrelated evolutionary histories. We compared these satellites in Bovini species that diverged 0.2 to about 5 Myr ago. Quantification of hybridization signals by phosphor imaging revealed a large variation in the relative amounts of the major satellites. In the genome of water buffalo this has led to the complete deletion of satellite III. Comparative sequencing and PCR-RFLP analysis of satellites IV, 1.711a, and 1.711b from the related Bos and Bison species revealed heterogeneities in 0.5 to 2% of the positions, again with variations in the relative amounts of sequence variants. Restriction patterns generated by double digestions suggested a recombination of sequence variants. Our results are compatible with a model of the life history of satellites during which homogeneity of interacting repeat units is both cause and consequence of the rapid turnover of satellite DNA. Initially, a positive feedback loop leads to a rapid saltatory amplification of homogeneous repeat units. In the second phase, mutations inhibit the interaction of repeat units and coexisting sequence variants amplify independently. Homogenization by the spreading of one of the variants is prevented by recombination and the satellite is eventually outcompeted by another, more homogeneous tandem repeat sequence. Received: 21 July 2000 / Accepted: 30 October 2000     

Variable and Invariable DNA Repeat Characters Revealed by Taxonprint Approach Are Useful for Molecular Systematics

A specially optimized restriction analysis of highly repetitive DNA elements, called DNA taxonprint, was applied for phylogenetic study of primates and lizards. It was shown that electrophoretic bands of DNA repeats revealed by the taxonprint technique have valuable properties for molecular systematics. Approximately half of taxonprint bands (TB) are invariable and do not disappear from the genomes during evolution or change spontaneously. Presumably these invariable bands are restriction fragments of dispersed DNA repeats. Another group represents variable taxonprint bands that differ even between closely related species. These variable bands are probably represented by tandem DNA repeats and could be used as species-specific markers. It was shown that taxonprint bands are independent characters since the appearance of a new taxonprint band does not change the previous band pattern. Phylogenetic reconstruction carried out on taxonprint data demonstrated that this approach could be of general utility for molecular systematics and species identification. Received: 12 January 1998 / Accepted: 16 May 1998     

A Phylogenetic Study of the Origin of the Domestic Pig Estimated from the Near-Complete mtDNA Genome

The near-complete pig mtDNA genome sequence (15,997 bp) was determined from two domestic pigs (one Chinese Meishan and one Swedish Landrace) and two European wild boars. The sequences were analyzed together with a previously published sequence representing a Swedish domestic pig. The sequences formed three distinct clades, denoted A, E1, and E2, with considerable sequence divergence between them (0.8–1.2%). The results confirm our previous study (based on the sequence of the cytochrome B gene and the control region only) and provide compelling evidence that domestication of pigs must have occurred from both an Asian and a European subspecies of the wild boar. We estimated the time since the divergence of clade A (found in Chinese Meishan pigs) and E1 (found in European domestic pigs) at about 900,000 years before present, long before domestication about 9000 years ago. The pattern of nucleotide substitutions among the sequences was in good agreement with previous interspecific comparisons of mammalian mtDNA; the lowest substitution rates were observed at nonsynonymous sites in protein-coding genes, in the tRNA and rRNA genes, while the highest rates were observed at synonymous sites and in the control region. The presence of Asian clade A in some major European breeds (Large White and Landrace) most likely reflects the documented introgression of Asian germplasm into European stocks during the 18th and 19th centuries. The coexistence of such divergent mtDNA haplotypes for 100+ generations is expected to lead to the presence of recombinant haplotypes if paternal transmission and recombination occur at a low frequency. We found no evidence of such recombination events in the limited sample studied so far. Received: 19 April 2000; Accepted: 15 November 2000     

Conserved Gene Clusters in Bacterial Genomes Provide Further Support for the Primacy of RNA

Five complete bacterial genome sequences have been released to the scientific community. These include four (eu)Bacteria, Haemophilus influenzae, Mycoplasma genitalium, M. pneumoniae, and Synechocystis PCC 6803, as well as one Archaeon, Methanococcus jannaschii. Features of organization shared by these genomes are likely to have arisen very early in the history of the bacteria and thus can be expected to provide further insight into the nature of early ancestors. Results of a genome comparison of these five organisms confirm earlier observations that gene order is remarkably unpreserved. There are, nevertheless, at least 16 clusters of two or more genes whose order remains the same among the four (eu)Bacteria and these are presumed to reflect conserved elements of coordinated gene expression that require gene proximity. Eight of these gene orders are essentially conserved in the Archaea as well. Many of these clusters are known to be regulated by RNA-level mechanisms in Escherichia coli, which supports the earlier suggestion that this type of regulation of gene expression may have arisen very early. We conclude that although the last common ancestor may have had a DNA genome, it likely was preceded by progenotes with an RNA genome. Received: 10 March 1996 / Accepted: 20 May 1997     

Structure and Origin of a Novel Dimeric Retroposon B1-dID

Here we describe a new short retroposon family of rodents. Like the primate Alu element consisting of two similar monomers, it is dimeric, but the left and right monomers are different and descend from B1 and ID short retroposons, respectively. Such elements (B1-dID) were found in the genomes of Gliridae, Sciuridae, Castoridae, Caviidae, and Hystricidae. Nucleotide sequences of this retroposon can be assigned to several structural variants. Phylogenetic analysis of B1-dID and related sequences suggests a possible scenario of B1-dID evolution in the context of rodent evolution. Received: 30 August 1999 / Accepted: 20 March 2000     

Mitochondrial DNA Phylogeny of the Family Cichlidae: Monophyly and Fast Molecular Evolution of the Neotropical Assemblage

A mitochondrial DNA (mtDNA) phylogeny of cichlid fish is presented for the most taxonomically inclusive data set compiled to date (64 taxa). 16S rDNA data establish with confidence relationships among major lineages of cichlids, with a general pattern congruent with previous morphological studies and less inclusive molecular phylogenies based on nuclear genes. Cichlids from Madagascar and India are the most basal groups of the family Cichlidae and sister to African–Neotropical cichlids. The cichlid phylogeny suggests drift-vicariance events, consistent with the fragmentation of Gondwana, to explain current biogeographic distributions. Important phylogenetic findings include the placement of the controversial genus Heterochromis basal among African cichlids, the South American genus Retroculus as the most basal taxon of the Neotropical cichlid assemblage, and the close relationship of the Neotropical genera Cichla with Astronotus rather than with the crenicichlines. Based on a large number of South American genera, the Neotropical cichlids are defined as a monophyletic assemblage and shown to harbor significantly higher levels of genetic variation than their African counterparts. Relative rate tests suggest that Neotropical cichlids have experienced accelerated rates of molecular evolution. But these high evolutionary rates were significantly higher among geophagine cichlids. Received: 18 September 1998 / Accepted: 16 December 1998     

Amino Acids as RNA Ligands: A Direct-RNA-Template Theory for the Code's Origin

Numerous RNA binding sites for specific amino acids are now known, coming predominantly from selection-amplification experiments. These sites are chemically discriminating despite being predominantly small, simple RNA structures: internal and bulge loops. Recent studies of sites for hydrophobic side chains suggest that there are other generalizable structural features which recur in hydrophobic RNA sites. Further, sites for hydrophobic side chains can contain codons for the bound amino acid, as has also long been known for the polar amino acid arginine. Such findings are comprehensively reviewed, and the implications for the origin of coded peptide synthesis are considered. An origins hypothesis which accommodates all the data, DRT (direct RNA templating), is formulated. Received: 22 December 1997 / Accepted: 13 February 1998     

Origin of the NEFA and Nuc Signal Sequences

The human protein NEFA binds calcium, contains a leucine zipper repeat that does not form a homodimer, and is proposed (along with the homologous Nuc protein) to have a common evolutionary history with an EF-hand ancestor. We have isolated and characterized the N-terminal domain of NEFA that contains a signal sequence inferred from both endoproteinase Asp-N (Asp-N) and tryptic digests. Analysis of this N-terminal sequence shows significant similarity to the conserved multiple domains of the mitochondrial carrier family (MCF) proteins. The leader sequence of Nuc is, however, most similar to the signal sequences of membrane and/or secreted proteins (e.g., mouse insulin-like growth factor receptor). We suggest that the divergent NEFA and Nuc N-terminal sequences may have independent origins and that the common high hydrophobicity governs their targeting to the ER. These results provide insights into signal sequence evolution and the multiple origins of protein targeting. Received: 20 February 1997 / Accepted: 28 July 1997     

Origin and Evolution of Viral Interleukin-10 and Other DNA Virus Genes with Vertebrate Homologues

Phylogenies of gene families including members in both vertebrates and DNA viruses of the poxvirus and/or herpesvirus families showed that the viral genes originated at widely different times over the history of life. Certain of these viral genes (for example, the genes encoding the large and small subunits of ribonucleoside–diphosphate reductase) originated before animals diverged from fungi, while others originated much more recently. The most striking examples of recent origin involved viral genes encoding the cytokine interleukin-10 (IL-10), which originated independently in viruses at least three times since the divergence of the orders of eutherian mammals, presumably by viral capture of host genes. In certain domains, viral IL-10 genes showed significantly higher rates of nonsynonymous substitution than their nearest mammalian homologues. Though the mutation rate in these viral genes is up to 20 times that of the corresponding mammalian genes, a high mutation rate alone did not account for these differences because they were not seen in all domains. Rather, in certain domains it appears that functional constraints present in the case of mammalian IL-10 are relaxed in the case of the viral homologues. Furthermore, a nonrandom pattern of change with respect to amino acid residue charge in the N-terminal portion of the mature protein has occurred repeatedly in independently derived viral IL-10 genes, strongly suggesting that positive selection has led to divergence of this functionally important domain in viral IL-10. Received: 11 January 2001 / Accepted: 23 May 2001