Marsupials exhibit great diversity in ecology and morphology. However, compared with their sister group, the placental mammals, our understanding of many aspects of marsupial evolution remains limited. We use 101 mitochondrial genomes and data from 26 nuclear loci to reconstruct a dated phylogeny including 97% of extant genera and 58% of modern marsupial species. This tree allows us to analyze the evolution of habitat preference and geographic distributions of marsupial species through time. We found a pattern of mesic-adapted lineages evolving to use more arid and open habitats, which is broadly consistent with regional climate and environmental change. However, contrary to the general trend, several lineages subsequently appear to have reverted from drier to more mesic habitats. Biogeographic reconstructions suggest that current views on the connectivity between Australia and New Guinea/Wallacea during the Miocene and Pliocene need to be revised. The antiquity of several endemic New Guinean clades strongly suggests a substantially older period of connection stretching back to the Middle Miocene and implies that New Guinea was colonized by multiple clades almost immediately after its principal formation.

Osteoarthritis (OA) is characterized by alterations to subchondral bone as well as articular cartilage. Changes to bone in OA have also been identified at sites distal to the affected joint, which include increased bone volume fraction and reduced bone mineralization. Altered bone remodelling has been proposed to underlie these bone changes in OA. To investigate the molecular basis for these changes, we performed microarray gene expression profiling of bone obtained at autopsy from individuals with no evidence of joint disease (control) and from individuals undergoing joint replacement surgery for either degenerative hip OA, or fractured neck of femur (osteoporosis [OP]). The OP sample set was included because an inverse association, with respect to bone density, has been observed between OA and the low bone density disease OP. Compugen human 19K-oligo microarray slides were used to compare the gene expression profiles of OA, control and OP bone samples. Four sets of samples were analyzed, comprising 10 OA-control female, 10 OA-control male, 10 OA-OP female and 9 OP-control female sample pairs. Print tip Lowess normalization and Bayesian statistical analyses were carried out using linear models for microarray analysis, which identified 150 differentially expressed genes in OA bone with t scores above 4. Twenty-five of these genes were then confirmed to be differentially expressed ( P < 0.01) by real-time PCR analysis. A substantial number of the top-ranking differentially expressed genes identified in OA bone are known to play roles in osteoblasts, osteocytes and osteoclasts. Many of these genes are targets of either the WNT (wingless MMTV integration) signalling pathway ( TWIST1 , IBSP , S100A4 , MMP25 , RUNX2 and CD14 ) or the transforming growth factor (TGF)-β/bone morphogenic protein (BMP) signalling pathway ( ADAMTS4 , ADM , MEPE , GADD45B , COL4A1 and FST ). Other differentially expressed genes included WNT ( WNT5B , NHERF1 , CTNNB1 and PTEN ) and TGF-β/BMP ( TGFB1 , SMAD3 , BMP5 and INHBA ) signalling pathway component or modulating genes. In addition a subset of genes involved in osteoclast function ( GSN , PTK9 , VCAM1 , ITGB2 , ANXA2 , GRN , PDE4A and FOXP1 ) was identified as being differentially expressed in OA bone between females and males. Altered expression of these sets of genes suggests altered bone remodelling and may in part explain ...

The rich fossil record of the family Equidae (Mammalia: Perissodactyla) over the past 55 MY has made it an icon for the patterns and processes of macroevolution. Despite this, many aspects of equid phylogenetic relationships and taxonomy remain unresolved. Recent genetic analyses of extinct equids have revealed unexpected evolutionary patterns and a need for major revisions at the generic, subgeneric, and species levels. To investigate this issue we examine 35 ancient equid specimens from four geographic regions (South America, Europe, Southwest Asia, and South Africa), of which 22 delivered 87-688 bp of reproducible aDNA mitochondrial sequence. Phylogenetic analyses support a major revision of the recent evolutionary history of equids and reveal two new species, a South American hippidion and a descendant of a basal lineage potentially related to Middle Pleistocene equids. Sequences from specimens assigned to the giant extinct Cape zebra, Equus capensis, formed a separate clade within the modern plain zebra species, a phenotypicically plastic group that also included the extinct quagga. In addition, we revise the currently recognized extinction times for two hemione-related equid groups. However, it is apparent that the current dataset cannot solve all of the taxonomic and phylogenetic questions relevant to the evolution of Equus. In light of these findings, we propose a rapid DNA barcoding approach to evaluate the taxonomic status of the many Late Pleistocene fossil Equidae species that have been described from purely morphological analyses.DNA taxonomy ͉ equid evolution ͉ macroevolution ͉ phylogeny ͉ ancient DNA T he original sequence of horse fossils found in the 1870s by paleontologist Othaniel Charles Marsh, and popularized by Thomas Huxley (1), has been enriched by a large fossil record over the years and has now become one of the most widely known examples of macroevolutionary change (2). The original linear model of gradual modification of fox-sized animals (Hyracothere horses) to the modern forms has been replaced by a more complex tree, showing periods of explosive diversification and branch extinctions over 55 MY (3). The end of the Early Miocene (15-20 MYA) marks a particularly important transition, separating an initial phase of small leafy browsers from a second phase of more diverse animals, exhibiting tremendous body-size plasticity and modifications in tooth morphology (4). This explosive diversification has been accompanied by several stages of geographic extension from North America to the rest of the New and Old Worlds, so that by the end of the Miocene (5 MYA) more than a dozen distinct genera are represented in the fossil record (4) (Astrohippus,

We report the isolation and characterization of CDC45, which encodes a polypeptide of650 amino acids that is essential for the initiation of chromosomal DNA replication in the budding yeast, Saccharomyces cerevisiae. CDC45 genetically interacts with at least two members of the MCM (minichromosome maintenance) family of replication genes, CDC46 and CDC47, which are proposed to perform a role in restricting initiation of DNA replication to once per cell cycle. family of polypeptides are essential for the initiation of chromosomal DNA replication, minichromosome maintenance, and, in S. cerevisiae, are only present in the nucleus from the end of mitosis until the G1-S transition (4-6). However, MCM homologues in Schizosaccharomyces pombe (7,8), Drosophila melanogaster (9), X laevis (10) and mammals (11)(12)(13)(14) are located in the nucleus throughout the cell cycle where they associate with chromatin during GI and dissociate during S-phase. A common feature of all MCM proteins, however, is that they bind to, and are displaced from, chromatin in a manner consistent with them licensing DNA (15-18).It is clear from characterization of RLF activity in Xenopus that components besides MCM polypeptides are required for licensing activity (19). Cdc45p is a likely component of MCM complexes in yeast based on observations showing that CDC45 is essential for chromosomal replication and that two MCMs genetically interact with CDC45 (20). In this report we describe the isolation and characterization of the CDC45 gene and show that Cdc45p is assembled into a complex with one member of the MCM family, Cdc46p/Mcm5p. This suggests Cdc45p as an additional component of RLF complexes in budding yeast. MATERIALS AND METHODSYeast Strains, Media, Isolation of CDC45 and DNA Manipulations. Yeast media used in this study are as described in Guthrie and Fink (27). All yeast strains used in this study were isogenic with W303-la (MATa, ura3-52, trpl-1, ade2-1, lys2-801, leu2-3, his3-11, 15, canl-100 [psi']) and were either generated by direct gene replacement or by backcrossing the original mutant to W303-la at least three times. The CDC45 gene was isolated by rescue of the cdc45-1 cold-sensitive mutation at 12°C in the strain DBY2027 (from D. Botstein, Stanford University) essentially as described (6). Approximately 25,000 transformants were screened. Plasmids were recovered from primary yeast transformants showing plasmid-dependent cold-sensitive rescue by transformation into bacteria, and then tested for complementing activity by retransformation of plasmid DNA into the original cold-sensitive strain. Rescuing plasmids were characterized by restriction mapping. All sequencing was performed on both strands with customized primers using Applied Biosystems Taq Dye Deoxy Terminator cycle sequencing kits according to the manufacturers instructions.A strain expressing Cdc45p(1-650)-GFP was constructed by inserting the gene fusion at the CDC45 locus by a one-step gene replacement (6). A single HA epitope tag was inserted directly after c...