Convergence of marine megafauna movement patterns in coastal and open oceans.

The extent of increasing anthropogenic impacts on large marine vertebrates partly depends on the animals' movement patterns. Effective conservation requires identification of the key drivers of movement including intrinsic properties and extrinsic constraints associated with the dynamic nature of the environments the animals inhabit. However, the relative importance of intrinsic versus extrinsic factors remains elusive. We analyze a global dataset of ∼2.8 million locations from >2,600 tracked individuals across 50 marine vertebrates evolutionarily separated by millions of years and using different locomotion modes (fly, swim, walk/paddle). Strikingly, movement patterns show a remarkable convergence, being strongly conserved across species and independent of body length and mass, despite these traits ranging over 10 orders of magnitude among the species studied. This represents a fundamental difference between marine and terrestrial vertebrates not previously identified, likely linked to the reduced costs of locomotion in water. Movement patterns were primarily explained by the interaction between species-specific traits and the habitat(s) they move through, resulting in complex movement patterns when moving close to coasts compared with more predictable patterns when moving in open oceans. This distinct difference may be associated with greater complexity within coastal microhabitats, highlighting a critical role of preferred habitat in shaping marine vertebrate global movements. Efforts to develop understanding of the characteristics of vertebrate movement should consider the habitat(s) through which they move to identify how movement patterns will alter with forecasted severe ocean changes, such as reduced Arctic sea ice cover, sea level rise, and declining oxygen content.

Development and characterization of 11 novel microsatellite loci for the roundscale spearfish Tetrapturus georgii and their cross-species amplification among other istiophorid species.

Eleven novel polymorphic microsatellite loci were developed and characterized for the recently validated roundscale spearfish Tetrapturus georgii. Characterization of these markers, based on 35 roundscale spearfish from the western North Atlantic, revealed two to 21 alleles per locus with an average expected heterozygosity (H(E) ) of 0·09-0·94, and all loci conformed to Hardy-Weinberg expectations. Cross-amplification of these 11 loci against all other eight known istiophorid species indicates promising prospects for the utility of these markers for istiophorids in general.

Morphometric convergence and molecular divergence: the taxonomic status and evolutionary history of Gymnura crebripunctata and Gymnura marmorata in the eastern Pacific Ocean.

To clarify the taxonomic status of Gymnura crebripunctata and Gymnura marmorata, the extent of morphological and nucleotide variation between these nominal species was examined using multivariate morphological and mitochondrial DNA comparisons of the same characters with congeneric species. Discriminant analysis of 21 morphometric variables from four species (G. crebripunctata, G. marmorata, Gymnura micrura and Gymnura poecilura) successfully distinguished species groupings. Classification success of eastern Pacific species improved further when specimens were grouped by species and sex. Discriminant analysis of size-corrected data generated species assignments that were consistently accurate in separating the two species (100% jackknifed assignment success). Nasal curtain length was identified as the character which contributed the most to discrimination of the two species. Sexual dimorphism was evident in several characters that have previously been relied upon to distinguish G. crebripunctata from G. marmorata. A previously unreported feature, the absence of a tail spine in G. crebripunctata, provides an improved method of field identification between these species. Phylogenetic and genetic distance analyses based on 698 base pairs of the mitochondrial cytochrome b gene indicate that G. crebripunctata and G. marmorata form highly divergent lineages, supporting their validity as distinct species. The closely related batoid Aetoplatea zonura clustered within the Gymnura clade, indicating that it may not represent a valid genus. Strong population structuring (overall Phi(ST) = 0.81, P < 0.01) was evident between G. marmorata from the Pacific coast of the Baja California peninsula and the Gulf of California, supporting the designation of distinct management units in these regions.

An increase in DNA double-strand breaks, induced by Ku70 depletion, is associated with human papillomavirus 16 episome loss and de novo viral integration events.

Integration of human papillomavirus type 16 (HPV16) is a common event in cervical carcinogenesis, although mechanisms of integration are poorly understood. We have tested the hypothesis that an increased number of DNA double-strand breaks (DSBs) affect HPV16 episome maintenance and integration in cervical keratinocytes. Increased DSBs were generated over prolonged periods of up to 50 population doublings in the unique polyclonal cervical keratinocyte cell line W12, which stably maintains HPV16 episomes. This was achieved using repeated treatments with short interfering RNA to obtain sustained depletion of Ku70, a key mediator of DNA non-homologous end joining. An increase in DSBs was seen shortly after commencement of Ku70 depletion. Continuous depletion was reproducibly associated with loss of HPV16 episomes and also with a new viral integration event, which was rapidly selected in outgrowing W12 cells. Despite the prolonged presence of DSBs, high-level chromosomal instability (detected by marked changes in genomic copy number) was not observed until cells containing the new integrant were almost fully selected, with no evidence of such chromosomal instability prior to integration. Our data show that increased DNA DSBs are associated with HPV16 episomal loss and integration in cervical keratinocytes. We found no evidence to support the notion that major chromosomal instability precedes HPV16 integration, although such instability is an important consequence of the integration event.

A thermostable endonuclease III homolog from the archaeon Pyrobaculum aerophilum.

Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration. The biological outcome includes increased mutation rate and potential lethality. A major DNA N:-glycosylase responsible for the excision of modified pyrimidine bases is the base excision repair (BER) glycosylase endonuclease III, for which functional homologs have been identified and characterized in Escherichia coli, yeast and humans. So far, little is known about how hyperthermophilic Archaea cope with such pyrimidine damage. Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100 degrees C. The predicted product of 223 amino acids shares significant sequence homology with several [4Fe-4S]-containing DNA N:-glycosylases including E.coli endonuclease III (EcNth). The histidine-tagged recombinant protein was expressed in E.coli and purified. Under optimal conditions of 80-160 mM NaCl and 70 degrees C, PaNth displays DNA glycosylase/ss-lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT). This activity is enhanced when DHT is paired with G. Our data, showing the structural and functional similarity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea. Conserved amino acid residues are identified for five subfamilies of endonuclease III/UV endonuclease homologs clustered by phylogenetic analysis.

Rapid and simultaneous identification of body parts from the morphologically similar sharks Carcharhinus obscurus and Carcharhinus plumbeus (Carcharhinidae) using multiplex PCR.

Many commercially exploited carcharhinid sharks are difficult to identify to species owing to extensive morphological similarities. This problem is severely exacerbated when it comes to identifying detached shark fins, and the finless and headless shark carasses typically sold in markets. To assist in the acquisition of urgently needed conservation and management data on shark catch and trade, we have developed a highly streamlined approach based on multiplex polymerase chain reaction (PCR) that uses species-specific primers derived from nuclear ribosomal ITS2 sequences to achieve rapid species identification of shark body parts. Here we demonstrate the utility of this approach for identifying fins and flesh from two globally distributed, morphologically very similar carcharhinid sharks (Carcharhinus obscurus and Carcharhinus plumbeus) intensively targeted in fisheries worldwide, and often confused for each other even as whole animals. The assay is conducted in a 4-primer multiplex format that is structured to simultaneously achieve the following efficiency and cost-reduction objectives: it requires only a single-tube amplification reaction for species diagnosis, it incorporates an internal positive control to allow detection of false-negative results, and it is novel in that it allows species identification even when DNAs from two species are combined in the same tube during the PCR reaction. The latter innovation reduces the required effort for screening a set of unknown samples by 50%. The streamlined approach illustrated here should be amenable for use in a shark conservation and management context where large numbers of samples typically need to be screened; the approach shown may also provide a model for a rapid diagnostic method applicable to species identification in general.

Disruption of the developmentally regulated Rev3l gene causes embryonic lethality.

The REV3 gene encodes the catalytic subunit of DNA polymerase (pol) zeta, which can replicate past certain types of DNA lesions [1]. Saccharomyces cerevisiae rev3 mutants are viable and have lower rates of spontaneous and DNA-damage-induced mutagenesis [2]. Reduction in the level of Rev31, the presumed catalytic subunit of mammalian pol zeta, decreased damage-induced mutagenesis in human cell lines [3]. To study the function of mammalian Rev31, we inactivated the gene in mice. Two exons containing conserved DNA polymerase motifs were replaced by a cassette encoding G418 resistance and beta-galactosidase, under the control of the Rev3l promoter. Surprisingly, disruption of Rev3l caused mid-gestation embryonic lethality, with the frequency of Rev3l(-/-) embryos declining markedly between 9.5 and 12.5 days post coitum (dpc). Rev3l(-/-) embryos were smaller than their heterozygous littermates and showed retarded development. Tissues in many areas were disorganised, with significantly reduced cell density. Rev3l expression, traced by beta-galactosidase staining, was first detected during early somitogenesis and gradually expanded to other tissues of mesodermal origin, including extraembryonic membranes. Embryonic death coincided with the period of more widely distributed Rev3l expression. The data demonstrate an essential function for murine Rev31 and suggest that bypass of specific types of DNAlesions by pol zeta is essential for cell viability during embryonic development in mammals.

The evolutionarily conserved zinc finger motif in the largest subunit of human replication protein A is required for DNA replication and mismatch repair but not for nucleotide excision repair.

The largest subunit of the replication protein A (RPA) contains an evolutionarily conserved zinc finger motif that lies outside of the domains required for binding to single-stranded DNA or forming the RPA holocomplex. In previous studies, we showed that a point mutation in this motif (RPAm) cannot support SV40 DNA replication. We have now investigated the role of this motif in several steps of DNA replication and in two DNA repair pathways. RPAm associates with T antigen, assists the unwinding of double-stranded DNA at an origin of replication, stimulates DNA polymerases alpha and delta, and supports the formation of the initial short Okazaki fragments. However, the synthesis of a leading strand and later Okazaki fragments is impaired. In contrast, RPAm can function well during the incision step of nucleotide excision repair and in a full repair synthesis reaction, with either UV-damaged or cisplatin-adducted DNA. Two deletion mutants of the Rpa1 subunit (eliminating amino acids 1-278 or 222-411) were not functional in nucleotide excision repair. We report for the first time that wild type RPA is required for a mismatch repair reaction in vitro. Neither the deletion mutants nor RPAm can support this reaction. Therefore, the zinc finger of the largest subunit of RPA is required for a function that is essential for DNA replication and mismatch repair but not for nucleotide excision repair.

Resistance of human nucleotide excision repair synthesis in vitro to p21Cdn1.

The p21Cdn1 protein (cip1/waf1/sdi1) plays an important role as an inhibitor of mammalian cell proliferation in response to DNA damage. By interacting with and inhibiting the function of cyclin-Cdk complexes, p21 can block entry into S phase. p21 can also directly inhibit replicative DNA synthesis by binding to the DNA polymerase sliding clamp factor PCNA. When cells are damaged and p21 is induced, DNA nucleotide excision repair (NER) continues, even though this pathway is PCNA-dependent. We investigated features of p21-resistant NER using human cell extracts. A direct end-labelling approach was used to measure the excision of damaged oligonucleotides by NER and no inhibition by p21 was found. By contrast, filling of the approximately 30 nt gaps created by NER could be inhibited by pre-binding p21 to PCNA, but only when gap filling was uncoupled from incision. Binding p21 to PCNA could also inhibit filling of model 30 nt gaps by both purified DNA polymerases delta and epsilon. When p21 was incubated in a cell extract before addition of PCNA, inhibition of repair synthesis was gradually relieved with time. This incubation gives p21 the opportunity to associate with other targets. As p21 blocks association of DNA polymerases with PCNA but does not prevent loading of PCNA onto DNA, repair gap filling can occur rapidly as soon as p21 dissociates from PCNA. A synthetic PCNA-binding p21 peptide was an efficient inhibitor of NER synthesis in cell extracts.

Which DNA polymerases are used for DNA-repair in eukaryotes?

There are five well-characterized nuclear DNA polymerases in eukaryotes (DNA polymerases alpha, beta, delta, epsilon and zeta) and this short review summarizes our current knowledge concerning the participation of each in DNA-repair. The three major DNA excision-repair pathways involve a DNA synthesis step that replaces altered bases or nucleotides removed during repair. Base excision-repair removes many modified bases and abasic sites, and in mammalian cells this mainly involves DNA polymerase beta. An alternative means for completion of base excision-repair, involving DNA polymerases delta or epsilon, may also operate and be even more important in yeast. Nucleotide excision-repair uses DNA polymerases delta or epsilon to resynthesize the bases removed during repair of pyrimidine dimers and other bulky adducts in DNA. Similarly, mismatch-repair of replication errors appears to involve DNA polymerases delta or epsilon. DNA polymerase alpha is required for semi-conservative replication of DNA but not for repair of DNA. A more recently discovered enzyme, DNA polymerase zeta, appears to be involved in the bypass of damage, without excision, and occurs during DNA replication of a damaged template.

Mammalian evolution and the interphotoreceptor retinoid binding protein (IRBP) gene: convincing evidence for several superordinal clades.

Phylogenetic relationships of 25 mammalian species representing 17 of the 18 eutherian orders were examined using DNA sequences from a 1.2-kb region of the 5' end of exon 1 of the single-copy nuclear gene known as interphotoreceptor retinoid binding protein (IRBP). A wide variety of methods of analysis of the DNA sequence, and of the translated products, all supported a five-order clade consisting of elephant shrew (Macroscelidea)/aardvark (Tubulidentata)/and the paenungulates (hyracoids, sirenians, and elephants), with bootstrap support in all cases of 100%. The Paenungulata was also strongly supported by these IRBP data. In the majority of analyses this monophyletic five-order grouping was the first branch off the tree after the Edentata. These results are highly congruent with two other recent sources of molecular data. Another superordinal grouping, with similar 100% bootstrap support in all of the same wide-ranging types of analyses, was Artiodactyla/Cetacea. Other superordinal affinities, suggested by the analyses, but with less convincing support, included a Perissodactyla/Artiodactyla/Cetacea clade, an Insectivora/Chiroptera clade, and Glires (an association of rodents and lagomorphs).

Presence of the seven transmembrane thrombin receptor on human tumour cells: effect of activation on tumour adhesion to platelets and tumor tyrosine phosphorylation.

Thrombin-treated tumour cells enhance their adhesion to platelets, fibronectin and von Willebrand factor in vitro, and enhanced their pulmonary metastasis in mice in vivo. A unique seven transmembrane spanning thrombin receptor has recently been cloned which is activated following thrombin proteolysis of the N-terminal end of the receptor with exposure of a tethered ligand. An N-terminal 14-mer (SFLLRNPNKYEPF) or 6-mer (SFLLRN) of the tethered ligand can serve as a thrombin receptor activation peptide (TRAP) by mimicking the action of thrombin on platelets, endothelial cells and smooth muscle cells. We have examined six human tumour cell lines for their response to TRAP, for the presence of this thrombin receptor mRNA by RT-PCR, protein by immunoblot and for their in vitro and in vivo response to TRAP. All six cell lines contain the receptor mRNA, and when treated with 100 microM 6-mer TRAP or 1 u/ml thrombin increase their adhesion to platelets 2-3-fold. Four of the six cell lines undergo tyrosine phosphorylation within 30 s to 1 min after exposure to 6-mer TRAP or thrombin. Thus tumour cells respond to thrombin via activation of their seven transmembrane spanning thrombin receptor.

Nucleotide excision repair DNA synthesis by DNA polymerase epsilon in the presence of PCNA, RFC, and RPA.

In eukaryotes, nucleotide excision repair of DNA is a complex process that requires many polypeptides to perform dual incision and remove a segment of about 30 nucleotides containing the damage, followed by repair DNA synthesis to replace the excised segment. Nucleotide excision repair DNA synthesis is dependent on proliferating cell nuclear antigen (PCNA). To study gap-filling DNA synthesis during DNA nucleotide excision repair, UV-damaged DNA was first incubated with PCNA-depleted human cell extracts to create repair incisions. Purified DNA polymerase delta or epsilon, with DNA ligase, was then used to form the repair patch. DNA polymerase delta could perform repair synthesis and was strictly dependent on the presence of both PCNA and replication factor C, but gave rise to a very low proportion of complete, ligated circles. The presence of replication protein A (which is also required for nucleotide excision repair) did not alter this result, while addition of DNase IV increased the fraction of ligated products. DNA polymerase epsilon, on the other hand, could fill the repair patch in the absence of PCNA and replication factor C, and most of the products were ligated circles. Addition of replication protein A changed the situation dramatically, and synthesis by polymerase epsilon became dependent on both PCNA and replication factor C. A combination of DNA polymerase epsilon, PCNA, replication factor C, replication protein A, and DNA ligase I appears to be well-suited to the task of creating nucleotide excision repair patches.

Mammalian DNA nucleotide excision repair reconstituted with purified protein components.

Nucleotide excision repair is the principal way by which human cells remove UV damage from DNA. Human cell extracts were fractionated to locate active components, including xeroderma pigmentosum (XP) and ERCC factors. The incision reaction was then reconstituted with the purified proteins RPA, XPA, TFIIH (containing XPB and XPD), XPC, UV-DDB, XPG, partially purified ERCC1/XPF complex, and a factor designated IF7. UV-DDB (related to XPE protein) stimulated repair but was not essential. ERCC1- and XPF-correcting activity copurified with an ERCC1-binding polypeptide of 110 kDa that was absent in XP-F cell extract. Complete repair synthesis was achieved by combining these factors with DNA polymerase epsilon, RFC, PCNA, and DNA ligase I. The reconstituted core reaction requires about 30 polypeptides.

Planar-motion correction with use of K-space data acquired in Fourier MR imaging.

The authors have developed a method for reducing magnetic resonance (MR) image artifacts caused by planar motion. Segments of k-space acquired with the subject stationary are detected automatically. Each k-space segment is Fourier transformed into an image in which rotational and translational displacements are measured manually. Before correction, k-space is made as Hermitian as allowed by the largest symmetric range of low spatial frequencies acquired with the subject stationary. Segments of k-space acquired with the subject in different positions are corrected separately. Although translation corrections can be applied effectively to both k-space and the spatial domain, rotation corrections are applied in the spatial domain to avoid image artifacts. To complement the correction, data corrupted by rotation are replaced by the complex conjugate of data of the opposite kappa x and kappa y, provided that these data have not been corrupted by rotation. The method reduced ghosts and blurring substantially on sagittal head images acquired with a standard spin-echo pulse sequence while a volunteer subject nodded his head.

Cip1 inhibits DNA replication but not PCNA-dependent nucleotide excision-repair.

BACKGROUND: DNA that is damaged by ultraviolet (UV) light is repaired predominantly by nucleotide excision-repair, a process requiring the DNA polymerase auxiliary factor PCNA. UV-irradiation also induces the production of Cip1 protein via activation of p53. Cip1 is an inhibitor of the cyclin-dependent kinases, which are required for the cell cycle to proceed through the G1/S-phase transition and initiate DNA replication. Inhibition by Cip1 probably causes the block to initiation of DNA replication that is seen in irradiated cells. Cip1 also directly inhibits the function of PCNA during DNA synthesis. As nucleotide excision-repair requires PCNA, the physiological relevance of PCNA inhibition by Cip1 is currently unclear. RESULTS: We show that nucleotide excision-repair of UV-damaged DNA occurs in extracts of Xenopus eggs, and that this reaction is PCNA-dependent. The repair reaction is not inhibited by Cip1, even when the level of PCNA is reduced 100-fold so that it becomes limiting for DNA repair. By contrast, Cip1 strongly suppresses the function of PCNA in replicative DNA synthesis under these conditions. CONCLUSIONS: Cip1 can potentially inhibit DNA replication in Xenopus egg extracts by inhibiting the cyclin-dependent kinase function required for the initiation of replication forks, and also by inhibiting PCNA function. The inhibition of PCNA is selective for its function in DNA replication, however, as Cip1 does not affect PCNA function in nucleotide excision-repair. The induction of Cip1 in response to DNA damage, therefore, allows repair to continue in the genome under conditions in which replication is severely inhibited.