Whole rock and Cr-rich spinel geochemistry of ODP Legs 152 and 163 and DSDP Hole 38-338 samples

Coring during Ocean Drilling Program and Deep Sea Drilling Project Legs 163, 152, 104, 81, and 38 recovered sequences of altered basalt from North Atlantic seaward-dipping reflector sequences (SDRS) erupted during the initial rifting of Greenland from northern Europe and likely associated with excessive mantle temperatures caused by an impacting mantle plume head. Cr-rich spinel is found abundantly as inclusions and groundmass crystals within the olivine-rich lavas of Hole 917A (Leg 152) cored into the Southeast Greenland SDRS, but only rarely as inclusions within plagioclase in the lavas of the Vøring Plateau SDRS, and it is absent from other cored SDRS lavas from the Rockall Plateau and Southeast Greenland. Eruptive melt compositions determined from inferred, thermodynamically-defined, spinel-melt exchange equilibria indicate that the most primitive melts represented by Hole 917A basalts have Mg/(Mg + Fe2+) at least as high as 0.70 and approach near-primary mantle melt compositions. In contrast, Cr-rich spinels from Hole 338 (Leg 38) lavas on the Vøring Plateau SDRS give evidence for melt with Mg/(Mg + Fe2+) only as high as 0.64. This study underlines that primitive melts similar to those from Hole 917A comprise only a small fraction of the eruptive North Atlantic SDRS melts, and that most SDRS basalts were, in fact, too evolved to have precipitated Cr-rich spinel, with true melt Mg/(Mg + Fe2+) likely below 0.60. The evolved nature of the SDRS basalts implies large amounts of fractionation at the base of the crust or deep within it, consistent with seismic results that indicate an abnormally thick Layer 3 underlying the SDRS.

Whole rock and elements concentrations from IODP Hole 343-C0019E

The microstructures, mineralogy and chemistry of four representative samples collected from cores extracted from the Japan Trench during Integrated Ocean Drilling Project Expedition 343, the Japan Trench Fast Drilling Project (JFAST) have been studied using optical microscopy, TEM, SEM, XRF, XRD and microprobe analyses. The samples provide a transect from relatively undeformed marine sediments in the hanging wall, to the undeformed footwall material, crossing the thrust interface between the Pacific and North American plate, where the fault slipped during the March 2011 Tohoku-Oki earthquake. Our preliminary results suggest that the low strength of JFAST fault gouge material is caused by the high amount of clay minerals (~ 60% smectite, ~ 14 illite). Clay minerals in the décollement (gouge) sample are partly replaced by newly formed manganese oxide, which precipitated from hydrothermal fluids. Dauphine twins were found in quartz grains of the décollement sample suggesting local high stress possible during seismic loading. Other microstructures cannot be assigned unambiguously to co-seismic or a-seismic faulting processes. The observed scaly clay fabric is consistent with observations in many other plate-boundary fault zones. Significant grain size reduction was found in the fault (decollement) zone sample. But a change in lithology of the fault material cannot be ruled out. Microstructures typical for a-seismic deformation like dissolution-precipitation features (e.g. dissolved grain boundaries, mineral alteration) occur in all JFAST core samples, but more frequently in the décollement sample.

Developmental dissociation of thymic dendritic cell and thymocyte lineages revealed in growth factor receptor mutant mice

Thymocytes and thymic dendritic cell (DC) lineages develop simultaneously and may originate from a common intrathymic progenitor. Mice deficient for two growth factor receptor molecules [c-kit and the common cytokine receptor γ chain (γc)] lack all thymocytes including T cell progenitors. Despite this lack of pro-T cells, thymic DC compartments were identified in c-kit−γc− mice. Thus, c-kit- and γc-mediated signals are not essential to generate thymic DCs. In addition, pro-T cells do not appear to be obligatory progenitors of thymic DCs, because DC development is dissociated from the generation of thymocytes in these mice. Thymic DCs in c-kit−γc− mice are phenotypically and functionally normal. In contrast to wild-type mice, however, thymic DCs in c-kit−γc− and, notably, in RAG-2-deficient mice are CD8αneg/low, indicating that CD8α expression on thymic DCs is not independent of thymocytes developing beyond the “RAG-block.”

Antigen-driven CD4+ T cell and HIV-1 dynamics: Residual viral replication under highly active antiretroviral therapy

Antigen-induced stimulation of the immune system can generate heterogeneity in CD4+ T cell division rates capable of explaining the temporal patterns seen in the decay of HIV-1 plasma RNA levels during highly active antiretroviral therapy. Posttreatment increases in peripheral CD4+ T cell counts are consistent with a mathematical model in which host cell redistribution between lymph nodes and peripheral blood is a function of viral burden. Model fits to patient data suggest that, although therapy reduces HIV replication below replacement levels, substantial residual replication continues. This residual replication has important consequences for long-term therapy and the evolution of drug resistance and represents a challenge for future treatment strategies.

Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF–RecO–RecR complex in DNA repair and recombination

The products of the recF, recO, and recR genes are thought to interact and assist RecA in the utilization of single-stranded DNA precomplexed with single-stranded DNA binding protein (Ssb) during synapsis. Using immunoprecipitation, size-exclusion chromatography, and Ssb protein affinity chromatography in the absence of any nucleotide cofactors, we have obtained the following results: (i) RecF interacts with RecO, (ii) RecF interacts with RecR in the presence of RecO to form a complex consisting of RecF, RecO, and RecR (RecF–RecO–RecR); (iii) RecF interacts with Ssb protein in the presence of RecO. These data suggested that RecO mediates the interactions of RecF protein with RecR and with Ssb proteins. Incubation of RecF, RecO, RecR, and Ssb proteins resulted in the formation of RecF–RecO–Ssb complexes; i.e., RecR was excluded. Preincubation of RecF, RecO, and RecR proteins prior to addition of Ssb protein resulted in the formation of complexes consisting of RecF, RecO, RecR, and Ssb proteins. These data suggest that one role of RecF is to stabilize the interaction of RecR with RecO in the presence of Ssb protein. Finally, we found that interactions of RecF with RecO are lost in the presence of ATP. We discuss these results to explain how the RecF–RecO–RecR complex functions as an anti-Ssb factor.

DNA annealing by Rad52 Protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA

Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA–ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA–Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA–ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.

Comparing protein–ligand interactions in solution and single crystals by Raman spectroscopy

By using a Raman microscope, we show that it is possible to probe the conformational states in protein crystals and crystal fragments under growth conditions (in hanging drops). The flavin cofactor in the enzyme para-hydroxybenzoate hydroxylase can assume two conformations: buried in the protein matrix (“in”) or essentially solvent-exposed (“out”). By using Raman difference spectroscopy, we previously have identified characteristic flavin marker bands for the in and out conformers in the solution phase. Now we show that the flavin Raman bands can be used to probe these conformational states in crystals, permitting a comparison between solution and crystal environments. The in or out marker bands are similar for the respective conformers in the crystal and in solution; however, significant differences do exist, showing that the environments for the flavin's isoalloxazine ring are not identical in the two phases. Moreover, the Raman-band widths of the flavin modes are narrower for both in and out conformers in the crystals, indicating that the flavin exists in a more limited range of closely related conformational states in the crystal than in solution. In general, the ability to compare detailed Raman data for complexes in crystals and solution provides a means of bridging crystallographic and solution studies.

An initial ATP-independent step in the unwinding of a herpes simplex virus type I origin of replication by a complex of the viral origin-binding protein and single-strand DNA-binding protein

Using a spectrophotometric assay that measures the hyperchromicity that accompanies the unwinding of a DNA duplex, we have identified an ATP-independent step in the unwinding of a herpes simplex virus type 1 (HSV-1) origin of replication, Oris, by a complex of the HSV-1 origin binding protein (UL9 protein) and the HSV-1 single-strand DNA binding protein (ICP8). The sequence unwound is the 18-bp A + T-rich segment that links the two high-affinity UL9 protein binding sites, boxes I and II of Oris. P1 nuclease sensitivity of Oris and single-strand DNA-dependent ATPase measurements of the UL9 protein indicate that, at 37°C, the A + T-rich segment is sufficiently single stranded to permit the binding of ICP8. Binding of the UL9 protein to boxes I and II then results in the formation of the UL9 protein–ICP8 complex, that can, in the absence of ATP, promote unwinding of the A + T-rich segment. On addition of ATP, the helicase activity of the UL9 protein–ICP8 complex can unwind boxes I and II, permitting access of the replication machinery to the Oris sequences.

The human XPG gene: gene architecture, alternative splicing and single nucleotide polymorphisms

Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP–Cockayne syndrome complex. While the XPG cDNA sequence was known, determination of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Analysis of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amount of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in association with multiple alternatively spliced isoforms of XPG mRNA.