How the past impacts the future: modelling the performance of evolutionarily distinct mammals through time.

How does past evolutionary performance impact future evolutionary performance? This is an important question not just for macroevolutionary biologists who wish to chart the phenomena that describe deep-time changes in biodiversity but also for conservation biologists, as evolutionarily distinct species-which may be deemed 'low-performing' in our current era-are increasingly the focus of conservation efforts. Contrasting hypotheses exist to account for the history and future of evolutionarily distinct species: on the one hand, they may be relicts of large radiations, potentially 'doomed' to extinction; or they may be slow-evolving, 'living fossils', likely neither to speciate nor go extinct; or they may be seeds of future radiations. Here, we attempt to test these hypotheses in Mammalia by combining a molecular phylogenetic supertree with fossil record occurrences and measuring change in evolutionary distinctness (ED) at different time slices. With these time slices, we modelled future ED as a function of past ED. We find that past evolutionary performance does indeed have an impact on future evolutionary performance: the most evolutionarily isolated clades tend to become more evolutionarily distinct with time, indicating that low-performing clades tend to remain low-performing throughout their evolutionary history. This article is part of a discussion meeting issue 'The past is a foreign country: how much can the fossil record actually inform conservation?'

A soft-bodied lophophorate from the Silurian of England.

Soft-bodied taxa comprise an important component of the extant lophophorate fauna, but convincing fossils of soft-bodied lophophorates are extremely rare. A small fossil lophophorate, attached to a brachiopod dorsal valve, is described from the Silurian (Wenlock Series) Herefordshire Lagerstätte of England. This unmineralized organism was bilaterally symmetrical and comprised a subconical body attached basally to the host and partially enclosed by a broad 'hood'; the body bore a small, coiled lophophore. Where the hood attached laterally, there is a series of transverse ridges and furrows. The affinities of this organism probably lie with Brachiopoda; the hood is interpreted as the homologue of a dorsal valve/mantle lobe, and the attachment as the homologue of the ventral valve and/or pedicle. The ridges are arranged in a manner that suggests constructional serial repetition, indicating that they are unlikely to represent mantle canals. Extant brachiopods are not serially structured, but morphological and molecular evidence suggests that their ancestors were. The new organism may belong to the brachiopod stem group, and might also represent a significant element of the Palaeozoic lophophorate fauna.

Fossilized soft tissues in a Silurian platyceratid gastropod.

Gastropod shells are common in the fossil record, but their fossil soft tissues are almost unknown, and have not been reported previously from the Palaeozoic. Here, we describe a Silurian (approx. 425 Myr) platyceratid gastropod from the Herefordshire Lagerstätte that preserves the oldest soft tissues yet reported from an undoubted crown-group mollusc. The digestive system is preserved in detail, and morphological data on the gonads, digestive gland, pedal muscle, radula, mouth and foot are also available. The specimen is preserved three-dimensionally, and has been reconstructed digitally following serial grinding. Platyceratids are often found attached to echinoderms, and have been interpreted as either commensal coprophages or kleptoparasites. The new data provide support for an attached mode of life, and are suggestive of a coprophagous feeding strategy. The affinities of the platyceratids are uncertain; they have been compared to both the patellogastropods and the neritopsines. Analysis of the new material suggests that a patellogastropod affinity is the more plausible of these hypotheses.

A starfish with three-dimensionally preserved soft parts from the Silurian of England.

Palaeozoic asteroids represent a stem-group to the monophyletic post-Palaeozoic Neoasteroidea, but many aspects of their anatomy are poorly known. Using serial grinding and computer reconstruction, we describe fully articulated Silurian (ca 425 Myr) specimens from the Herefordshire Lagerstätte, preserved in three dimensions complete with soft tissues. The material belongs to a species of Bdellacoma, a genus previously assigned to the ophiuroids, but has characters that suggest an asteroid affinity. These include a pyloric system in the gut, and the presence of large bivalved pedicellariae, the latter originally described under the name Bursulella from isolated valves. Ampullae are external and occur within podial basins; the radial canal is also external. Podia are elongate and lack terminal suckers. The peristome is large relative to the mouth. Aspects of the morphology are comparable to that of the extant Paxillosida, supporting phylogenetic schemes that place this order at the base of the asteroid crown group.

A three-dimensionally preserved fossil polychaete worm from the Silurian of Herefordshire, England.

Polychaete body fossils are rare, and are almost invariably compressed and too poorly preserved for meaningful comparison with extant forms. We here describe Kenostrychus clementsi gen. et sp. nov. from the Silurian Herefordshire Konservat-Lagerstätte of England, in which three-dimensional external morphology is preserved with a fidelity unprecedented among fossil polychaetes. The fossils, which are preserved in calcite, were serially ground and photographed at 30 microm intervals to produce computer-generated reconstructions of the original external surface. The new genus has a generalized polychaete morphology with large biramous parapodia, unspecialized anterior segments and a small prostomium with median and lateral antennae and ventral prostomial palps. Cirriform branchiae arise from the ventral surface of each notopodium, and may be homologous with the inter-ramal branchiae of the extant nephtyids. Through cladistic analysis, Kenostrychus is interpreted as a member of a stem group of either the Phyllodocida or the Aciculata (Phyllodocida + Eunicida). Direct comparison with other fossil forms is difficult, but hints that inter-ramal respiratory structures may be primitive within the Phyllodocida and/or the Aciculata.

Managing DNA polymerases: coordinating DNA replication, DNA repair, and DNA recombination.

Two important and timely questions with respect to DNA replication, DNA recombination, and DNA repair are: (i) what controls which DNA polymerase gains access to a particular primer-terminus, and (ii) what determines whether a DNA polymerase hands off its DNA substrate to either a different DNA polymerase or to a different protein(s) for the completion of the specific biological process? These questions have taken on added importance in light of the fact that the number of known template-dependent DNA polymerases in both eukaryotes and in prokaryotes has grown tremendously in the past two years. Most notably, the current list now includes a completely new family of enzymes that are capable of replicating imperfect DNA templates. This UmuC-DinB-Rad30-Rev1 superfamily of DNA polymerases has members in all three kingdoms of life. Members of this family have recently received a great deal of attention due to the roles they play in translesion DNA synthesis (TLS), the potentially mutagenic replication over DNA lesions that act as potent blocks to continued replication catalyzed by replicative DNA polymerases. Here, we have attempted to summarize our current understanding of the regulation of action of DNA polymerases with respect to their roles in DNA replication, TLS, DNA repair, DNA recombination, and cell cycle progression. In particular, we discuss these issues in the context of the Gram-negative bacterium, Escherichia coli, that contains a DNA polymerase (Pol V) known to participate in most, if not all, of these processes.

Genetic interactions between the Escherichia coli umuDC gene products and the beta processivity clamp of the replicative DNA polymerase.

The Escherichia coli umuDC gene products encode DNA polymerase V, which participates in both translesion DNA synthesis (TLS) and a DNA damage checkpoint control. These two temporally distinct roles of the umuDC gene products are regulated by RecA-single-stranded DNA-facilitated self-cleavage of UmuD (which participates in the checkpoint control) to yield UmuD' (which enables TLS). In addition, even modest overexpression of the umuDC gene products leads to a cold-sensitive growth phenotype, apparently due to the inappropriate expression of the DNA damage checkpoint control activity of UmuD(2)C. We have previously reported that overexpression of the epsilon proofreading subunit of DNA polymerase III suppresses umuDC-mediated cold sensitivity, suggesting that interaction of epsilon with UmuD(2)C is important for the DNA damage checkpoint control function of the umuDC gene products. Here, we report that overexpression of the beta processivity clamp of the E. coli replicative DNA polymerase (encoded by the dnaN gene) not only exacerbates the cold sensitivity conferred by elevated levels of the umuDC gene products but, in addition, confers a severe cold-sensitive phenotype upon a strain expressing moderately elevated levels of the umuD'C gene products. Such a strain is not otherwise normally cold sensitive. To identify mutant beta proteins possibly deficient for physical interactions with the umuDC gene products, we selected for novel dnaN alleles unable to confer a cold-sensitive growth phenotype upon a umuD'C-overexpressing strain. In all, we identified 75 dnaN alleles, 62 of which either reduced the expression of beta or prematurely truncated its synthesis, while the remaining alleles defined eight unique missense mutations of dnaN. Each of the dnaN missense mutations retained at least a partial ability to function in chromosomal DNA replication in vivo. In addition, these eight dnaN alleles were also unable to exacerbate the cold sensitivity conferred by modestly elevated levels of the umuDC gene products, suggesting that the interactions between UmuD' and beta are a subset of those between UmuD and beta. Taken together, these findings suggest that interaction of beta with UmuD(2)C is important for the DNA damage checkpoint function of the umuDC gene products. Four possible models for how interactions of UmuD(2)C with the epsilon and the beta subunits of DNA polymerase III might help to regulate DNA replication in response to DNA damage are discussed.

An exceptionally preserved vermiform mollusc from the Silurian of England.

Studies of the origin and radiation of the molluscs have yet to resolve many issues regarding their nearest relatives, phylogeny and ancestral characters. The Polyplacophora (chitons) and the Aplacophora are widely interpreted as the most primitive extant molluscs, but Lower Palaeozoic fossils of the former lack soft parts, and the latter were hitherto unrecognized as fossils. The Herefordshire Lagerstätte is a Silurian (about 425 Myr bp) deposit that preserves a marine biota in remarkable three-dimensional detail. The external surface of even non-biomineralized cuticle was preserved by entombment in volcanic ash, subsequent incorporation into concretions, and infilling of the fossils with sparry calcite. Here we describe, from this deposit, a complete vermiform mollusc, which we interpret as a plated aplacophoran. Serial grinding at intervals of tens of micrometres, combined with computer-based reconstruction methods, renders the fossils in the round.

umuDC-dnaQ Interaction and its implications for cell cycle regulation and SOS mutagenesis in Escherichia coli.

The Escherichia coli SOS-regulated umuDC gene products participate in a DNA damage checkpoint control and in translesion DNA synthesis. Specific interactions involving the UmuD and UmuD' proteins, both encoded by the umuD gene, and components of the replicative DNA polymerase, Pol III, appear to be important for regulating these two biological activities of the umuDC gene products. Here we show that overproduction of the epsilon proofreading subunit of Pol III suppresses the cold sensitivity normally associated with overexpression of the umuDC gene products. Our results suggest that this suppression is attributable to specific interactions between UmuD or UmuD' and the C-terminal domain of epsilon.

umuDC-mediated cold sensitivity is a manifestation of functions of the UmuD(2)C complex involved in a DNA damage checkpoint control.

The umuDC genes are part of the Escherichia coli SOS response, and their expression is induced as a consequence of DNA damage. After induction, they help to promote cell survival via two temporally separate pathways. First, UmuD and UmuC together participate in a cell cycle checkpoint control; second, UmuD'(2)C enables translesion DNA replication over any remaining unrepaired or irreparable lesions in the DNA. Furthermore, elevated expression of the umuDC gene products leads to a cold-sensitive growth phenotype that correlates with a rapid inhibition of DNA synthesis. Here, using two mutant umuC alleles, one that encodes a UmuC derivative that lacks a detectable DNA polymerase activity (umuC104; D101N) and another that encodes a derivative that is unable to confer cold sensitivity but is proficient for SOS mutagenesis (umuC125; A39V), we show that umuDC-mediated cold sensitivity can be genetically separated from the role of UmuD'(2)C in SOS mutagenesis. Our genetic and biochemical characterizations of UmuC derivatives bearing nested deletions of C-terminal sequences indicate that umuDC-mediated cold sensitivity is not due solely to the single-stranded DNA binding activity of UmuC. Taken together, our analyses suggest that umuDC-mediated cold sensitivity is conferred by an activity of the UmuD(2)C complex and not by the separate actions of the UmuD and UmuC proteins. Finally, we present evidence for structural differences between UmuD and UmuD' in solution, consistent with the notion that these differences are important for the temporal regulation of the two separate physiological roles of the umuDC gene products.

Genetic and biochemical characterization of a novel umuD mutation: insights into a mechanism for UmuD self-cleavage.

Most translesion DNA synthesis (TLS) in Escherichia coli is dependent upon the products of the umuDC genes, which encode a DNA polymerase, DNA polymerase V, with the unique ability to replicate over a variety of DNA lesions, including cyclobutane dimers and abasic sites. The UmuD protein is activated for its role in TLS by a RecA-single-stranded DNA (ssDNA)-facilitated self-cleavage event that serves to remove its amino-terminal 24 residues to yield UmuD'. We have used site-directed mutagenesis to construct derivatives of UmuD and UmuD' with glycines in place of leucine-101 and arginine-102. These residues are extremely well conserved among the UmuD-like proteins involved in mutagenesis but are poorly conserved among the structurally related LexA-like transcriptional repressor proteins. Based on both the crystal and solution structures of the UmuD' homodimer, these residues are part of a solvent-exposed loop. Our genetic and biochemical characterizations of these mutant UmuD and UmuD' proteins indicate that while leucine-101 and arginine-102 are critical for the RecA-ssDNA-facilitated self-cleavage of UmuD, they serve only a minimal role in enabling TLS. These results, and others, suggest that the interaction of RecA-ssDNA with leucine-101 and arginine-102, together with numerous other contacts between UmuD(2) and the RecA-ssDNA nucleoprotein filaments, serves to realign lysine-97 relative to serine-60, thereby activating UmuD(2) for self-cleavage.

The SOS response: recent insights into umuDC-dependent mutagenesis and DNA damage tolerance.

Be they prokaryotic or eukaryotic, organisms are exposed to a multitude of deoxyribonucleic acid (DNA) damaging agents ranging from ultraviolet (UV) light to fungal metabolites, like Aflatoxin B1. Furthermore, DNA damaging agents, such as reactive oxygen species, can be produced by cells themselves as metabolic byproducts and intermediates. Together, these agents pose a constant threat to an organism's genome. As a result, organisms have evolved a number of vitally important mechanisms to repair DNA damage in a high fidelity manner. They have also evolved systems (cell cycle checkpoints) that delay the resumption of the cell cycle after DNA damage to allow more time for these accurate processes to occur. If a cell cannot repair DNA damage accurately, a mutagenic event may occur. Most bacteria, including Escherichia coli, have evolved a coordinated response to these challenges to the integrity of their genomes. In E. coli, this inducible system is termed the SOS response, and it controls both accurate and potentially mutagenic DNA repair functions [reviewed comprehensively in () and also in ()]. Recent advances have focused attention on the umuD(+)C(+)-dependent, translesion DNA synthesis (TLS) process that is responsible for SOS mutagenesis (). Here we discuss the SOS response of E. coli and concentrate in particular on the roles of the umuD(+)C(+) gene products in promoting cell survival after DNA damage via TLS and a primitive DNA damage checkpoint.

A new arthropod from the Silurian Konservat-Lagerstätte of Herefordshire, UK.

A small, non-biomineralized, macrophagous arthropod with chelicerate affinities, Offacolus kingi gen. et sp. nov., from the Silurian (Wenlock Series) of Herefordshire, UK, is described. The dorsal exoskeleton comprises an arch-like cephalic shield, a thorax of three free tergites and a triangular posterior tagma of five fused tergites, the last with a stout postero-dorsally directed medial spine. Seven pairs of appendages beneath the cephalic shield surround a postero-medially sited oral cavity on the ventral surface of the head. Appendages I and, probably II are uniramous and project antero-ventrally; I was sensory and II sensory and/or ambulatory. Appendages III-VI are biramous, each with an antero-ventrally projecting ramus and a robust, highly geniculate, horizontally oriented ramus that projects through an anterior gape. The former rami were ambulatory and the latter have spinose terminal podomeres and functioned as a unit for trapping food and transferring it towards the oral cavity. Appendage VII, which is probably uniramous, is posteroventrally directed and flap like. Each tergite of the thorax and posterior tagma covers at least a pair (probably two pairs) of probably biramous appendages with each ramus flap like and setose.

The Escherichia coli SOS mutagenesis proteins UmuD and UmuD' interact physically with the replicative DNA polymerase.

The Escherichia coli umuDC operon is induced in response to replication-blocking DNA lesions as part of the SOS response. UmuD protein then undergoes an RecA-facilitated self-cleavage reaction that removes its N-terminal 24 residues to yield UmuD'. UmuD', UmuC, RecA, and some form of the E. coli replicative DNA polymerase, DNA polymerase III holoenzyme, function in translesion synthesis, the potentially mutagenic process of replication over otherwise blocking lesions. Furthermore, it has been proposed that, before cleavage, UmuD together with UmuC acts as a DNA damage checkpoint system that regulates the rate of DNA synthesis in response to DNA damage, thereby allowing time for accurate repair to take place. Here we provide direct evidence that both uncleaved UmuD and UmuD' interact physically with the catalytic, proofreading, and processivity subunits of the E. coli replicative polymerase. Consistent with our model proposing that uncleaved UmuD and UmuD' promote different events, UmuD and UmuD' interact differently with DNA polymerase III: whereas uncleaved UmuD interacts more strongly with beta than it does with alpha, UmuD' interacts more strongly with alpha than it does with beta. We propose that the protein-protein interactions we have characterized are part of a higher-order regulatory system of replication fork management that controls when the umuDC gene products can gain access to the replication fork.

Mutations affecting the ability of the Escherichia coli UmuD' protein to participate in SOS mutagenesis.

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli, a process that results from a translesion synthesis mechanism. The UmuD protein is activated for its role in mutagenesis by a RecA-facilitated autodigestion that removes the N-terminal 24 amino acids. A previous genetic screen for nonmutable umuD mutants had resulted in the isolation of a set of missense mutants that produced UmuD proteins that were deficient in RecA-mediated cleavage (J. R. Battista, T. Ohta, T. Nohmi, W. Sun, and G. C. Walker, Proc. Natl. Acad. Sci. USA 87:7190-7194, 1990). To identify elements of the UmuD' protein necessary for its role in translesion synthesis, we began with umuD', a modified form of the umuD gene that directly encodes the UmuD' protein, and obtained missense umuD' mutants deficient in UV and methyl methanesulfonate mutagenesis. The D39G, L40R, and T51I mutations affect residues located at the UmuD'2 homodimer interface and interfere with homodimer formation in vivo. The D75A mutation affects a highly conserved residue located at one end of the central strand in a three-stranded beta-sheet and appears to interfere with UmuD'2 homodimer formation indirectly by affecting the structure of the UmuD' monomer. When expressed from a multicopy plasmid, the L40R umuD' mutant gene exhibited a dominant negative effect on a chromosomal umuD+ gene with respect to UV mutagenesis, suggesting that the mutation has an effect on UmuD' function that goes beyond its impairment of homodimer formation. The G129D mutation affects a highly conserved residue that lies at the end of the long C-terminal beta-strand and results in a mutant UmuD' protein that exhibits a strongly dominant negative effect on UV mutagenesis in a umuD+ strain. The A30V and E35K mutations alter residues in the N-terminal arms of the UmuD'2 homodimer, which are mobile in solution.

Escherichia coli DnaA protein. The N-terminal domain and loading of DnaB helicase at the E. coli chromosomal origin.

Initiation of DNA replication at the Escherichia coli chromosomal origin occurs through an ordered series of events that depends first on the binding of DnaA protein, the replication initiator, to DnaA box sequences followed by unwinding of an AT-rich region. A step that follows is the binding of DnaB helicase at oriC so that it is properly positioned at each replication fork. We show that DnaA protein actively mediates the entry of DnaB at oriC. One region (amino acids 111-148) transiently binds to DnaB as determined by surface plasmon resonance. A second functional domain, possibly involving formation of a unique nucleoprotein structure, promotes the stable binding of DnaB during the initiation process and is inactivated in forming an intermediate termed the prepriming complex by removal of the N-terminal 62 residues. Based on similarities in the replication process between prokaryotes and eukaryotes, these results suggest that a similar mechanism may load the eukaryotic replicative helicase.

A comparative genetic analysis of the Irish greyhound population using multilocus DNA fingerprinting, canine single locus minisatellites and canine microsatellites.

Pairwise analysis of HinfI/33.6 DNA fingerprints from a total of one hundred and fifty-three Irish greyhounds of known pedigree were used to determine band-share estimates of unrelated, first-degree and second-degree relationships. Forty-eight unrelated Irish greyhounds were used to determine allele frequencies for three single-locus minisatellites, and following a preliminary screen, eight of the most polymorphic tetra-nucleotide microsatellites from a panel of 15. The results indicated that both band-share estimates by DNA fingerprinting and microsatellite allele frequencies are highly effective in resolving parentage in this greyhound population, while single-locus minisatellites showed limited polymorphism and could not be used alone for routine parentage testing in this breed. The present study also demonstrated that, to obtain optimal resolution of parentage, sample sets of known pedigree status are required to determine the band-share distribution and/or microsatellite allele frequencies.

Threonine 435 of Escherichia coli DnaA protein confers sequence-specific DNA binding activity.

The Escherichia coli DnaA protein, as a sequence-specific DNA binding protein, promotes the initiation of chromosomal replication by binding to four asymmetric 9-mer sequences termed DnaA boxes in oriC. Characterization of N-terminal, C-terminal, and internal in-frame deletion mutants identified residues near the C terminus of DnaA protein required for DNA binding. Furthermore, genetic and biochemical characterization of 11 missense mutations mapping within the C-terminal 89 residues indicated that they were defective in DNA binding. Detailed biochemical characterization of one mutant protein bearing a threonine to methionine substitution at position 435 (T435M) revealed that it retained only nonspecific DNA binding activity, suggesting that threonine 435 imparts specificity in binding. Finally, T435M was inactive on its own for in vitro replication of an oriC plasmid but was able to augment limiting levels of wild type DnaA protein, consistent with the proposal that not all of the DnaA monomers in the initial complex are bound specifically to oriC and that direct interaction occurs among monomers.

Novel alleles of the Escherichia coli dnaA gene.

The Escherichia coli dnaA gene is required for replication of the bacterial chromosome. To identify residues critical for its replication activity, a method to select novel mutations was developed that relied on lytic growth of lambda from an inserted pSC101 replication origin. Replication from the lambda origin was inhibited by lysogen-encoded cI repressor. Replication from the pSC101 origin that resulted in lytic growth was dependent on active DnaA protein encoded by a plasmid in a host strain lacking the chromosomal dnaA gene. With this approach, a large collection of missense, nonsense, and a few internal deletion mutations were obtained. Nucleotide sequence analysis of the missense mutations indicated that 28 of 50 were unique. Of these, one was identical to the dnaA205 allele whereas the remainder are novel. These missense mutations were clustered into three regions, suggesting three functional domains of DnaA protein required for its replication activity. Many of the missense mutations mapping to the C-terminal 61 residues were inactive for replication from the pSC101 origin. These are defective in DNA binding. Mutations that mapped elsewhere were temperature-sensitive.

The Escherichia coli dnaA gene: four functional domains.

The Escherichia coli DnaA protein is a sequence-specific DNA binding protein that promotes the initiation of replication of the bacterial chromosome, and of several plasmids including pSC101. Twenty-eight novel missense mutations of the E. coli dnaA gene were isolated by selecting for their inability to replicate a derivative of pSC101 when contained in a lambda vector. Characterization of these as well as seven novel nonsense mutations and one in-frame deletion mutation are described here. Results suggest that E. coli DnaA protein contains four functional domains. Mutations that affect residues in the P-loop or Walker A motif thought to be involved in ATP binding identify one domain. The second domain maps to a region near the C terminus and is involved in DNA binding. The function of the third domain that maps near the N terminus is unknown but may be involved in the ability of DnaA protein to oligomerize. Two alleles encoding different truncated gene products retained the ability to promote replication from the pSC101 origin but not oriC, identifying a fourth domain dispensable for replication of pSC101 but essential for replication from the bacterial chromosomal origin, oriC.