RNA-directed DNA methylation mediated by DRD1 and Pol IVb: a versatile pathway for transcriptional gene silencing in plants

RNA-directed DNA methylation, which is one of several RNAi-mediated pathways in the nucleus, has been highly elaborated in the plant kingdom. RNA-directed DNA methylation requires for the most part conventional DNA methyltransferases, histone modifying enzymes and RNAi proteins; however, several novel, plant-specific proteins that are essential for this process have been identified recently. DRD1 (defective in RNA-directed DNA methylation) is a putative SWI2/SNF2-like chromatin remodelling protein; DRD2 and DRD3 (renamed NRPD2a and NRPD1b, respectively) are subunits of Pol IVb, a putative RNA polymerase found only in plants. Interestingly, DRD1 and Pol IVb appear to be required not only for RNA-directed de novo methylation, but also for full erasure of methylation when the RNA trigger is withdrawn. These proteins thus have the potential to facilitate dynamic regulation of DNA methylation. Prominent targets of RNA-directed DNA methylation in the Arabidopsis thaliana genome include retrotransposon long terminal repeats (LTRs), which have bidirectional promoter/enhancer activities, and other types of intergenic transposons and repeats. Intergenic solitary LTRs that are targeted for reversible methylation by the DRD1/Pol IVb pathway can potentially act as switches or rheostats for neighboring plant genes. The resulting alterations in gene expression patterns may promote physiological flexibility and adaptation to the environment.     

The pR plasmid: a tool for discriminating between DNA lesions induced by different types of cytotoxic agents in cultured mammalian cells

The LA-D cells, obtained by cotransformation of LTA mouse cells (tk- aprt-) with pR plasmid and with tk gene as selective marker, are significantly more resistant to UV light and 4-nitroquinoline-N-1-oxide than LTA control cells. In this work, we report that the LA-D cells exhibit different degrees of response to various DNA-damaging agents: wild-type survival to mitomycin, increased sensitivity to bleomycin, cis-diamminedichloroplatinum and N-methyl-N'-nitro-N-nitrosoguanidine. The pR plasmid could, therefore, play an important role in the DNA-repair mechanisms that modulate the cytotoxic effect of the DNA-inhibitory agents. The possible interactions between pR plasmid products and the different repair enzymes involved are discussed.     

PREDICTOR: a web-based tool for the prediction of atomic structure from sequence for double helical DNA with up to 150 base pairs

We announce the release of a web-based tool for DNA structure prediction (PREDICTOR) which allows the calculation of atomic structures of double-helical DNA with up to 150 Watson-Crick base pairs (http://farwer.staff.shef.ac.uk/ PREDICTOR). The semi-empirical method uses computational chemistry to extrapolate knowledge of sequence-dependent DNA structure contained in the X-ray crystal structure database. The properties of the base stacking interactions are treated theoretically, and an empirical model is used to add the conformational constraints imposed by the backbone. For DNA oligomers in the X-ray crystal structure database that were not used for parameterisation of the model, the method distinguishes A and B form DNA reasonably reliably, and the final structures are accurate to 2 ? rmsd. Simulation of a 150mer and a 494mer with experimentally confirmed bending clearly reproduces the bending whereas the predicted structure of a random 150mer does not show any curvature. Calculation times are 90 seconds for an octamer and 7 minutes for a 30mer.     

Molecular dissection of a quantitative trait locus: a phenylalanine-to-tyrosine substitution in the transmembrane domain of the bovine growth hormone receptor is associated with a major effect on milk yield and composition

We herein report on our efforts to improve the mapping resolution of a QTL with major effect on milk yield and composition that was previously mapped to bovine chromosome 20. By using a denser chromosome 20 marker map and by exploiting linkage disequilibrium using two distinct approaches, we provide strong evidence that a chromosome segment including the gene coding for the growth hormone receptor accounts for at least part of the chromosome 20 QTL effect. By sequencing individuals with known QTL genotype, we identify an F to Y substitution in the transmembrane domain of the growth hormone receptor gene that is associated with a strong effect on milk yield and composition in the general population.     

Seed selection for grassland restoration: competitive effect of a dominant grass is mediated by seed source and nutrient availability

Cultivars are selected for advantageous traits, which may enable them to have a greater competitive effect (CE), particularly under high nutrient conditions. Sites with high nutrient availability may favor the development of dominant grasses rather than subordinate species, especially in nutrient‐limited systems, such as calcareous grasslands. The aim of this experiment was to determine whether seed source influences the CE of a dominant grass on a subordinate species, and whether this relationship is mediated by nutrient availability. A greenhouse experiment with three nutrient levels was established; Dianthus carthusianorum was chosen as the subordinate “phytometer” to detect variation in the CE of different Festuca rubra seed sources. The grass species was sourced from 13 cultivar and 12 commercially propagated, but not selected, wild sources. When CE was calculated from biomass, propagated wild seed sources of F. rubra had a greater CE on the subordinate species than cultivars for medium and high nutrient levels. Based on phytometer height, propagated wild seed sources of F. rubra had a greater CE under all three nutrient levels. Our findings do not support the general notion that cultivars are more competitive than wild genotypes. Thus, the cultivar F. rubra may facilitate the establishment of other species during grassland restoration, particularly under elevated nutrient conditions.     

Monocyte proliferation induced by modified serum is associated with endogenous M-CSF production: evidence for involvement of a signalling pathway via scavenger receptors

It was found that human serum stored for 2 months at 4 degrees C (modified serum) induced monocyte proliferation and simultaneous macrophage colony stimulating factor (M-CSF) production by these cells in vitro. Cell number, estimated by DNA content, doubled after 10 days in culture in the presence of modified serum, while it decreased in culture with freshly thawed control serum. As the addition of more than 2.5 ng/ml of recombinant M-CSF significantly supported monocyte survival/proliferation, cells were cultured for 10 days in medium supplemented with control serum, and endogenous M-CSF production was investigated by enzyme-linked immunosorbent assay. M-CSF concentration in the supernatants was 15-30 ng/ml after 10 day in culture with modified serum, a level that might be sufficient for monocyte proliferation. The modified serum induced M-CSF from freshly isolated monocytes, while M-CSF was hardly detected in cultures supplemented with control serum. Assay for peroxidized lipid and agarose gel electrophoresis demonstrated that the modified serum contained more oxidized low density lipoproteins (LDL) than the control serum. Ligands of scavenger receptors, which are receptors for oxidized LDL, such as dextran sulphate, polyinosinic acid, heparin and acetylated LDL also significantly induced M-CSF production from human monocytes, although this was at levels below 2 ng/ml. These results indicate that serum modified by oxidation stimulates monocytes to produce M-CSF resulting in their proliferation, and that signalling via scavenger receptors is one of the mechanisms responsible for this induction of M-CSF.     

Two sisters with a distal deletion at the Xq26/Xq27 interface: DNA studies indicate that the gene locus for factor IX is present

Summary Two sisters with premature menopause and a small deletion of the long arm of one of their X chromosomes [del (X)(pterq26.3:)] were investigated with polymorphic DNA probes near the breakpoint. The deleted chromosome retained the factor IX (F9) locus and the loci DXS51 (52A) and DXS100 (pX45h), which are proximal to F9. However, the factor VIII (F8) locus was not present, nor were two loci tightly linked to this locus, DXS52 (St14) and DXS15 (DX13) This deletion refines the location of the F9 locus to Xq26 or to the interface Xq26/Xq27, thus placing it more proximally than has been previously reported. The DNA obtained from these patients should be valuable in the mapping of future probes derived from this region of the X chromosome.     

The DNA sequence of the nuclear gene coding for the 17-kd subunit VI of the yeast ubiquinol-cytochrome c reductase: a protein with an extremely high content of acidic amino acids

We have determined the DNA sequence of the nuclear gene coding for the 17-kd subunit VI of the ubiquinol-cytochrome c reductase. The reading frame found encodes a putative polypeptide of 17 394 daltons. This protein is highly unusual: 38% of its residues are acidic and 14% are basic amino acids. The most notable feature in the protein sequence is a stretch of 25 consecutive acidic amino acids. The polypeptide has homology with the 9-kd so-called 'hinge' protein of beef-heart complex III, which also has a cluster of acidic residues. Acidic amino acids are likely to be essential for the function of these proteins, since their degree of conservation is higher than that of other residues.     

DNA ligase I is recruited to sites of DNA replication by an interaction with proliferating cell nuclear antigen: identification of a common targeting mechanism for the assembly of replication factories.

In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of approximately 20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS). This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues. N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro. Moreover, the RFTS of DNA ligase I and of the RF-C large subunit is necessary and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21(CIP1/WAF1), we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories.     

Cutting edge: a potent adjuvant effect of ligand to receptor activator of NF-kappa B gene for inducing antigen-specific CD8+ T cell response by DNA and viral vector vaccination

The ligand to receptor activator of NF-kappaB (RANK-L)/RANK interaction has been implicated in CD40 ligand/CD40-independent T cell priming by dendritic cells. In this report, we show that the coadministration of the RANK-L gene with a Trypanosoma cruzi gene markedly enhances the induction of Trypanosoma Ag-specific CD8(+) T cells and improves the DNA vaccine efficacy. A similarly potent adjuvant effect of the RANK-L gene on the induction of Ag-specific CD8(+) T cells was also observed when recombinant influenza virus expressing murine malaria Ag was used as an immunogen. In contrast, the coadministration of the CD40L gene was not effective in these systems. Our results demonstrated, for the first time, the potent immunostimulatory effect of the RANK-L gene to improve the CD8(+) T cell-mediated immunity against infectious agents.     

Induction of iron regulatory protein 1 RNA-binding activity by nitric oxide is associated with a concomitant increase in the labile iron pool: implications for DNA damage

Iron regulatory protein 1 (IRP1) is a bifunctional [4Fe-4S] protein that controls iron homeostasis. Switching off its function from an aconitase to an apo-IRP1 interacting with iron-responsive element-containing mRNAs depends on the reduced availability of iron in labile iron pool (LIP). Although the modulation of IRP1 by nitric oxide has been characterized, its impact on LIP remains unknown. Here, we show that inhibition of IRP1 aconitase activity and induction of its IRE-binding activity during exposure of L5178Y mouse lymphoma cells to NO are associated with an increase in LIP levels. Removal of NO resulted in a reverse regulation of IRP1 activities accompanied by a decrease of LIP. The increased iron burden in LIP caused by NO exacerbated hydrogen peroxide-induced genotoxicity in L5178Y cells. We demonstrate that the increase in LIP levels in response to chronic but not burst exposure of L5178Y cells to NO is associated with alterations in the expression of proteins involved in iron metabolism.     

Evidence for genetic relationship between RNA and DNA viruses from the sequence homology of a putative polymerase gene of bluetongue virus with that of vaccinia virus: conservation of RNA polymerase genes from diverse species.

The nucleotide sequence of segment 1 of the double stranded RNA genome of bluetongue virus serotype 10 (BTV-10), encoding the largest viral core protein, VP1, has been determined. Linear sequence analysis of the predicted amino acid sequence of the 149-K Da protein, a putative component of the viral RNA-directed RNA polymerase, revealed extensive homology with the vaccinia virus 147K Da DNA-directed RNA polymerase subunit. Similar homologies were detected between the VP1 polypeptide and the beta chain subunit of Escherichia coli and common tobacco chloroplast RNA polymerases, yeast RNA polymerase II and III and fruit fly polymerase II.