Stable carbon, nitrogen and sulphur isotope analysis of permafrost preserved human hair from rescue excavations (2009, 2010) at the precontact site of Nunalleq, Alaska

Acknowledgments This work was funded by an Arts and Humanities Research Council (AH/K006029/1) grant awarded to Rick Knecht, Kate Britton and Charlotta Hillerdal (Aberdeen); an AHRC-LabEx award (AH/N504543/1) to KB, RK, Keith Dobney (Liverpool) and Isabelle Sidéra (Nanterre); the Carnegie Trust to the Universities of Scotland (travel grant to KB); and the Max Planck Institute for Evolutionary Anthropology. The onsite collection of samples was carried out by staff and students from the University of Aberdeen, volunteer excavators and the residents of Quinhagak. We had logistical and planning support for fieldwork by the Qanirtuuq Incorporated, Quinhagak, Alaska, and the people of Quinhagak, who we also thank for sampling permissions. Special thanks to Warren Jones and Qanirtuuq Incorporated (especially Michael Smith and Lynn Church), and to all Nunalleq project team members, in Aberdeen and at other institutions, particularly Charlotta Hillerdal and Edouard Masson-Maclean (Aberdeen) for comments on earlier versions of this manuscript, and also to Véronique Forbes, Ana Jorge, Carly Ameen and Ciara Mannion (Aberdeen) for their inputs. Thanks also to Michelle Alexander (York). Finally, thank you to Ian Scharlotta (Alberta) for inviting us to contribute to this special issue, to the Editor, and to three anonymous reviewers, whose suggestions and recommended changes to an earlier version of this manuscript greatly improved the paper.

Cloning and Sequencing of a Protein Involved in Phagosomal Membrane Fusion in Paramecium

An mAb was raised to the C5 phagosomal antigen in Paramecium multimicronucleatum. To determine its function, the cDNA and genomic DNA encoding C5 were cloned. This antigen consisted of 315 amino acid residues with a predicted molecular weight of 36,594, a value similar to that determined by SDS-PAGE. Sequence comparisons uncovered a low but significant homology with a Schizosaccharomyces pombe protein and the C-terminal half of the β-fructofuranosidase protein of Zymomonas mobilis. Lacking an obvious transmembrane domain or a possible signal sequence at the N terminus, C5 was predicted to be a soluble protein, whereas immunofluorescence data showed that it was present on the membranes of vesicles and digestive vacuoles (DVs). In cells that were minimally permeabilized but with intact DVs, C5 was found to be located on the cytosolic surface of the DV membranes. Immunoblotting of proteins from the purified and KCl-washed DVs showed that C5 was tightly bound to the DV membranes. Cryoelectron microscopy also confirmed that C5 was on the cytosolic surface of the discoidal vesicles, acidosomes, and lysosomes, organelles known to fuse with the membranes of the cytopharynx, the DVs of stages I (DV-I) and II (DV-II), respectively. Although C5 was concentrated more on the mature than on the young DV membranes, the striking observation was that the cytopharyngeal membrane that is derived from the discoidal vesicles was almost devoid of C5. Approximately 80% of the C5 was lost from the discoidal vesicle-derived membrane after this membrane fused with the cytopharyngeal membrane. Microinjection of the mAb to C5 greatly inhibited the fusion of the discoidal vesicles with the cytopharyngeal membrane and thus the incorporation of the discoidal vesicle membranes into the DV membranes. Taken together, these results suggest that C5 is a membrane protein that is involved in binding and/or fusion of the discoidal vesicles with the cytopharyngeal membrane that leads to DV formation.

Evolutionary specialization of the nuclear targeting apparatus

The α- and β-karyopherins (Kaps), also called importins, mediate the nuclear transport of proteins. All α-Kaps contain a central domain composed of eight approximately 40 amino acid, tandemly arranged, armadillo-like (Arm) repeats. The number and order of these repeats have not changed since the common origin of fungi, plants, and mammals. Phylogenetic analysis suggests that the various α-Kaps fall into two groups, α1 and α2. Whereas animals encode both types, the yeast genome encodes only an α1-Kap. The β-Kaps are characterized by 14–15 tandemly arranged HEAT motifs. We show that the Arm repeats of α-Kaps and the HEAT motifs of β-Kaps are similar, suggesting that the α-Kaps and β-Kaps (and for that matter, all Arm and HEAT repeat-containing proteins) are members of the same protein superfamily. Phylogenetic analysis indicates that there are at least three major groups of β-Kaps, consistent with their proposed cargo specificities. We present a model in which an α-independent β-Kap progenitor gave rise to the α-dependent β-Kaps and the α-Kaps.

Potential responses of soil organic carbon to global environmental change

Recent improvements in our understanding of the dynamics of soil carbon have shown that 20–40% of the approximately 1,500 Pg of C stored as organic matter in the upper meter of soils has turnover times of centuries or less. This fast-cycling organic matter is largely comprised of undecomposed plant material and hydrolyzable components associated with mineral surfaces. Turnover times of fast-cycling carbon vary with climate and vegetation, and range from <20 years at low latitudes to >60 years at high latitudes. The amount and turnover time of C in passive soil carbon pools (organic matter strongly stabilized on mineral surfaces with turnover times of millennia and longer) depend on factors like soil maturity and mineralogy, which, in turn, reflect long-term climate conditions. Transient sources or sinks in terrestrial carbon pools result from the time lag between photosynthetic uptake of CO2 by plants and the subsequent return of C to the atmosphere through plant, heterotrophic, and microbial respiration. Differential responses of primary production and respiration to climate change or ecosystem fertilization have the potential to cause significant interrannual to decadal imbalances in terrestrial C storage and release. Rates of carbon storage and release in recently disturbed ecosystems can be much larger than rates in more mature ecosystems. Changes in disturbance frequency and regime resulting from future climate change may be more important than equilibrium responses in determining the carbon balance of terrestrial ecosystems.

The age of the universe from nuclear chronometers

An overview is presented of the current situation regarding radioactive dating of the matter of which our Galaxy is comprised. A firm lower bound on the age from nuclear chronometers of ≈9–10 Gyr is entirely consistent with age determinations from globular clusters and white dwarf cooling histories. The reasonable assumption of an approximately uniform nucleosynthesis rate yields an age for the Galaxy of 12.8 ± 3 Gyr, which again is consistent with current determinations from other methods.

Effects of Short- and Long-Term Elevated CO2 on the Expression of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Genes and Carbohydrate Accumulation in Leaves of Arabidopsis thaliana (L.) Heynh.1

To investigate the proposed molecular characteristics of sugar-mediated repression of photosynthetic genes during plant acclimation to elevated CO2, we examined the relationship between the accumulation and metabolism of nonstructural carbohydrates and changes in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) gene expression in leaves of Arabidopsis thaliana exposed to elevated CO2. Long-term growth of Arabidopsis at high CO2 (1000 μL L−1) resulted in a 2-fold increase in nonstructural carbohydrates, a large decrease in the expression of Rubisco protein and in the transcript of rbcL, the gene encoding the large subunit of Rubisco (approximately 35–40%), and an even greater decline in mRNA of rbcS, the gene encoding the small subunit (approximately 60%). This differential response of protein and mRNAs suggests that transcriptional/posttranscriptional processes and protein turnover may determine the final amount of leaf Rubisco protein at high CO2. Analysis of mRNA levels of individual rbcS genes indicated that reduction in total rbcS transcripts was caused by decreased expression of all four rbcS genes. Short-term transfer of Arabidopsis plants grown at ambient CO2 to high CO2 resulted in a decrease in total rbcS mRNA by d 6, whereas Rubisco content and rbcL mRNA decreased by d 9. Transfer to high CO2 reduced the maximum expression level of the primary rbcS genes (1A and, particularly, 3B) by limiting their normal pattern of accumulation through the night period. The decreased nighttime levels of rbcS mRNA were associated with a nocturnal increase in leaf hexoses. We suggest that prolonged nighttime hexose metabolism resulting from exposure to elevated CO2 affects rbcS transcript accumulation and, ultimately, the level of Rubisco protein.

Structure and conformational changes of DNA topoisomerase II visualized by electron microscopy.

Type II DNA topoisomerases, which create a transient gate in duplex DNA and transfer a second duplex DNA through this gate, are essential for topological transformations of DNA in prokaryotic and eukaryotic cells and are of interest not only from a mechanistic perspective but also because they are targets of agents for anticancer and antimicrobial chemotherapy. Here we describe the structure of the molecule of human topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] as seen by scanning transmission electron microscopy. A globular approximately 90-angstrom diameter core is connected by linkers to two approximately 50-angstrom domains, which were shown by comparison with genetically truncated Saccharomyces cerevisiae topoisomerase II to contain the N-terminal region of the approximately 170-kDa subunits and that are seen in different orientations. When the ATP-binding site is occupied by a nonhydrolyzable ATP analog, a quite different structure is seen that results from a major conformational change and consists of two domains approximately 90 angstrom and approximately 60 angstrom in diameter connected by a linker, and in which the N-terminal domains have interacted. About two-thirds of the molecules show an approximately 25 A tunnel in the apical part of the large domain, and the remainder contain an internal cavity approximately 30 A wide in the large domain close to the linker region. We propose that structural rearrangements lead to this displacement of an internal tunnel. The tunnel is likely to represent the channel through which one DNA duplex, after capture in the clamp formed by the N-terminal domains, is transferred across the interface between the enzyme's subunits. These images are consistent with biochemical observations and provide a structural basis for understanding the reaction of topoisomerase II.

Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach.

Advances in screening technologies allowing the identification of growth factor receptors solely by virtue of DNA or protein sequence comparison call for novel methods to isolate corresponding ligand growth factors. The EPH-like receptor tyrosine kinase (RTK) HEK (human EPH-like kinase) was identified previously as a membrane antigen on the LK63 human pre-B-cell line and overexpression in leukemic specimens and cell lines suggested a role in oncogenesis. We developed a biosensor-based approach using the immobilized HEK receptor exodomain to detect and monitor purification of the HEK ligand. A protein purification protocol, which included HEK affinity chromatography, achieved a 1.8 X 10(6)-fold purification of an approximately 23-kDa protein from human placental conditioned medium. Analysis of specific sHEK (soluble extracellular domain of HEK) ligand interactions in the first and final purification steps suggested a ligand concentration of 40 pM in the source material and a Kd of 2-3 nM. Since the purified ligand was N-terminally blocked, we generated tryptic peptides and N-terminal amino acid sequence analysis of 7 tryptic fragments of the S-pyridylethylated protein unequivocally matched the sequence for AL-1, a recently reported ligand for the related EPH-like RTK REK7 (Winslow, J.W., Moran, P., Valverde, J., Shih, A., Yuan, J.Q., Wong, S.C., Tsai, S.P., Goddard, A., Henzel, W.J., Hefti, F., Beck, K.D., & Caras, I.W. (1995) Neuron 14, 973-981). Our findings demonstrate the application of biosensor technology in ligand purification and show that AL-1, as has been found for other ligands of the EPH-like RTK family, binds more than one receptor.

Isolation of an oxygen-sensitive FNR protein of Escherichia coli: interaction at activator and repressor sites of FNR-controlled genes.

The Escherichia coli fnr gene product, FNR, is a DNA binding protein that regulates a large family of genes involved in cellular respiration and carbon metabolism during conditions of anaerobic cell growth. FNR is believed to contain a redox/O2-sensitive element for detecting the anaerobic state. To investigate this process, a fnr mutant that encodes an altered FNR protein with three amino acid substitutions in the N-terminal domain was constructed by site-directed mutagenesis. In vivo, the mutant behaved like a wild-type strain under anaerobic conditions but had a 14-fold elevated level of transcriptional activation of a reporter gene during aerobic cell growth. The altered fur gene was overexpressed in E. coli and the resultant FNR protein was purified to near homogeneity by using anaerobic chromatography procedures. An in vitro Rsa I restriction site protection assay was developed that allowed for the assessment of oxygen-dependent DNA binding of the mutant FNR protein. The FNR protein was purified as a monomer of M(r) 28,000 that contained nonheme iron at 2.05 +/- 0.34 mol of Fe per FNR monomer. In vitro DNase I protection studies were performed to establish the locations of the FNR-binding sites at the narG, narK, dmsA, and hemA promoters that are regulated by either activation or repression of their transcription. The sizes of the DNA footprints are consistent with the binding of two monomers of FNR that protect the symmetrical FNR-recognition sequence TTGAT-nnnnATCAA. Exposure of the FNR protein or protein-DNA complex to air for even short periods of time (approximately 5 min) led to the complete loss of DNA protection at a consensus FNR recognition site. A model whereby the FNR protein exists in the cell as a monomer that assembles on the DNA under anaerobic conditions to form a dimer is discussed.

UME6 is a central component of a developmental regulatory switch controlling meiosis-specific gene expression.

The UME6 gene of Saccharomyces cerevisiae was identified as a mitotic repressor of early meiosis-specific gene expression. It encodes a Zn2Cys6 DNA-binding protein which binds to URS1, a promoter element needed for both mitotic repression and meiotic induction of early meiotic genes. This paper demonstrates that a complete deletion of UME6 causes not only vegetative derepression of early meiotic genes during vegetative growth but also a significant reduction in induction of meiosis-specific genes, accompanied by a severe defect in meiotic progression. After initiating premeiotic DNA synthesis the vast majority of cells (approximately 85%) become arrested in prophase and fail to execute recombination; a minority of cells (approximately 15%) complete recombination and meiosis I, and half of these form asci. Quantitative analysis of the same early meiotic transcripts that are vegetatively derepressed in the ume6 mutant, SPO11, SPO13, IME2, and SPO1, indicates a low level of induction in meiosis above their vegetative derepressed levels. In addition, the expression of later meiotic transcripts, SPS2 and DIT1, is significantly delayed and reduced. The expression pattern of early meiotic genes in ume6-deleted cells is strikingly similar to that of early meiotic genes with promoter mutations in URS1. These results support the view that UME6 and URS1 are part of a developmental switch that controls both vegetative repression and meiotic induction of meiosis-specific genes.

Reversible phosphorylation controls the activity of cyclosome-associated cyclin-ubiquitin ligase.

Cyclin B/cdc2 is responsible both for driving cells into mitosis and for activating the ubiquitin-dependent degradation of mitotic cyclins near the end of mitosis, an event required for the completion of mitosis and entry into interphase of the next cell cycle. Previous work with cell-free extracts of rapidly dividing clam embryos has identified two specific components required for the ubiquitination of mitotic cyclins: E2-C, a cyclin-selective ubiquitin carrier protein that is constitutively active during the cell cycle, and E3-C, a cyclin-selective ubiquitin ligase that purifies as part of a approximately 1500-kDa complex, termed the cyclosome, and which is active only near the end of mitosis. Here, we have separated the cyclosome from its ultimate upstream activator, cdc2. The mitotic, active form of the cyclosome can be inactivated by incubation with a partially purified, endogenous okadaic acid-sensitive phosphatase; addition of cdc2 restores activity to the cyclosome after a lag that reproduces that seen previously in intact cells and in crude extracts. These results demonstrate that activity of cyclin-ubiquitin ligase is controlled by reversible phosphorylation of the cyclosome complex.

Identification of endothelin 1 and big endothelin 1 in secretory vesicles isolated from bovine aortic endothelial cells.

Vesicles containing endothelin 1 (ET-1) were isolated from bovine aortic endothelial cells (BAECs) by fractionation of homogenates on sucrose density gradients by ultracentrifugation. The vesicles were localized at the 1.0/1.2 M sucrose interface using a specific anti-ET-1-(16-21) RIA. Identification of ET-1 and big ET-1 in this fraction was confirmed by HPLC analysis combined with RIA. Morphological examination of the ET-1-enriched fraction by electron microscopy identified clusters of vesicles approximately 100 nm in diameter. Immunostaining of ultrathin cryosections prepared from the vesicle fraction for ET-1 or big ET-1 showed clusters of 15-nm gold particles attached to or within vesicles. Immunofluorescence staining of whole BAECs using a specific ET-1-(16-21) IgG purified by affinity chromatography revealed punctate granulation of the cell cytoplasm viewed under light microscopy. This distinct pattern of staining was shown by confocal light microscopy to be intracellular. Immunofluorescence staining of whole cells with a polyclonal antiserum for big ET-1-(22-39) showed a defined perinuclear localization of precursor molecule. Hence, several different approaches have demonstrated that ET-1 and big ET-1 are localized within intracellular vesicles in BAECs, suggesting that these subcellular compartments are an important site for processing of big ET-1 by endothelin-converting enzyme.

Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen fixation in angiosperms.

Of the approximately 380 families of angiosperms, representatives of only 10 are known to form symbiotic associations with nitrogen-fixing bacteria in root nodules. The morphologically based classification schemes proposed by taxonomists suggest that many of these 10 families of plants are only distantly related, engendering the hypothesis that the capacity to fix nitrogen evolved independently several, if not many, times. This has in turn influenced attitudes toward the likelihood of transferring genes responsible for symbiotic nitrogen fixation to crop species lacking this ability. Phylogenetic analysis of DNA sequences for the chloroplast gene rbcL indicates, however, that representatives of all 10 families with nitrogen-fixing symbioses occur together, with several families lacking this association, in a single clade. This study therefore indicates that only one lineage of closely related taxa achieved the underlying genetic architecture necessary for symbiotic nitrogen fixation in root nodules.

United Kingdom, 1826 (Image 1 of 3) (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Cary's six sheet map of England and Wales, with part of Scotland : on which are carefully laid down all the direct and principal cross roads, the course of the rivers, and navigable canals, cities, market and borough towns, the principal villages, parks, &c., compiled from the most recent surveys. It was published by J. Cary in 1826. Scale [ca. 1:510,000]. This layer is image 1 of 3 total images of the three sheet source map, representing the northern portion of the map. Covers portions of England, Scotland, North Ireland, and Isle of Man.The image inside the map neatline is georeferenced to the surface of the earth and fit to the 'British National Grid' coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as drainage, cities and other human settlements, mail roads, turnpike roads, bye roads, territorial and administrative boundaries, parks, shoreline features, and more. Relief shown by hachures.This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.

United Kingdom, 1826 (Image 2 of 3) (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Cary's six sheet map of England and Wales, with part of Scotland : on which are carefully laid down all the direct and principal cross roads, the course of the rivers, and navigable canals, cities, market and borough towns, the principal villages, parks, &c., compiled from the most recent surveys. It was published by J. Cary in 1826. Scale [ca. 1:510,000]. This layer is image 2 of 3 total images of the three sheet source map, representing the southern portion of the map. Covers portions of England and Wales.The image inside the map neatline is georeferenced to the surface of the earth and fit to the 'British National Grid' coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as drainage, cities and other human settlements, mail roads, turnpike roads, bye roads, territorial and administrative boundaries, parks, shoreline features, and more. Relief shown by hachures.This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.