HsRec2/Rad51L1, a protein influencing cell cycle progression, has protein kinase activity

Human Rec2/Rad51L1 is a member of the Rad51 family of proteins. Although recombinase activity, typical of this family, could not be established, its overexpression in mammalian cells has been shown to cause a delay in G1. Moreover, since hsRec2/Rad51L1 has been found to be induced by both ionizing and UV irradiation, it is likely that hsRec2/Rad51L1 is elevated following any DNA damage and causes a G1 delay to allow time for DNA repair to occur. Limited homology with catalytic domains X and XI of protein kinase A suggested that kemptide, an artificial substrate containing one phosphorylatable residue, a serine, might serve as a substrate for hsRec2/Rad51L1. Here, we report that hsRec2/Rad51L1 can phosphorylate kemptide, as well as myelin basic protein, p53, cyclin E, and cdk2, but not a peptide substrate containing tyrosine only. The finding that hsRec2/Rad51L1 exhibits protein kinase activity is a first step toward identifying a mechanism whereby this protein affects the cell cycle.     

Chromosomal mutations induced by triplex-forming oligonucleotides in mammalian cells.

Specific recognition of a region of duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation. Based on this, we have investigated the ability of the triplex-directed approach to induce mutations at a chromosomal locus in living cells. A mouse fibroblast cell line was constructed containing multiple chromosomal copies of the lambdasupFG1 vector carrying the supFG1 mutation-reporter gene. Cells were treated with specific (psoAG30) or control (psoSCR30) psoralen-conjugated TFOs in the presence and absence of UVA irradiation. The results demonstrated a 6- to 10-fold induction of supFG1 mutations in the psoAG30-treated cells as compared with psoSCR30-treated or untreated control cells. Interestingly, UVA irradiation had no effect onthe mutation frequencies induced by the psoralen-conjugated TFOs, suggesting a triplex-mediated but photoproduct-independent process of mutagenesis. Sequencing data were consistent with this finding since the expected T.A-->A.T transversions at the predicted psoralen crosslinking site were not detected. However, insertions and deletions were detected within the triplex binding site, indicating a TFO-specific induction of mutagenesis. This result demonstrates the ability of triplex-forming oligonucleotides to influence mutation frequencies at a specific site in a mammalian chromosome.     

RecA-mediated joint molecule formation between O-methylated RNA/DNA hairpins and single-stranded targets

Chimeric oligonucleotides consisting of one DNA strand paired with an O-methylated RNA strand interrupted by six DNA residues have been used in gene targeting experiments. Here we demonstrate that these hairpins can form a heteroduplex (or joint molecule) with single-stranded DNA targets in a reaction mediated by the E. coli RecA protein. One end of the double hairpin may unwind to form a 14-base-RecA filament which initiates the reaction. Chimeric oligonucleotides containing only O-methylated RNA residues on one strand or truncated hairpins lacking this 14-base segment did not participate in RecA-driven heteroduplex formation under these reaction conditions. The results presented here represent a first step in studying one facet of a strategy which uses O-methylated RNA residues as participants in homologous pairing events. Received: 2 June 1997 / Accepted: 29 September 1997     

Cardiac hypertrophy cannot proceed without initial suppression of protein synthesis

Stress was induced in rat hearts by heat shock and aortic banding and in cardiac cell cultures by heat shock. Proteins were labeled with [35S]-methionine, resolved by electrophoresis, and isotope incorporation measured. Determinations were made during and following stress. The immediate response was stress protein (SP) synthesis, cessation of normally occurring protein synthesis and reduced isotope incorporation. Following recovery, the prestress protein pattern returned, SP synthesis remained high and label incorporation doubled. Response of hearts to the two stresses were similar. Initial suppression may be an important precursor to accelerated protein synthesis seen in hypertrophy.     

Site-specific targeting of psoralen photoadducts with a triple helix-forming oligonucleotide: characterization of psoralen monoadduct and crosslink formation.

A polypurine tract in the supF gene of bacteriophage lambda (base pairs 167-176) was selected as the target for triple helix formation and targeted mutagenesis by an oligopurine (5'-AGGAAGGGGG-3') containing a chemically linked psoralen derivative (4'-hydroxymethyl-4,5',8-trimethylpsoralen) at its 5' terminus (psoAG10). The thymines at base pairs 166 and 167, a 5'ApT site, were targeted for photomodification. Exposure of the triple helical complex to long wavelength ultraviolet radiation led to the covalent binding of psoAG10 to the targeted region in the supF gene and to the induction of site-specific mutations. We report here experiments to characterize the photomodification of the targeted region of the supF gene in the context of triple helix formation. An electrophoretic mobility-shift assay showed that, at low radiation doses, monoadducts at base pair 166 were the major photoadducts. At higher doses the monoadducts were converted to crosslinks between base pairs 166 and 167. HPLC analysis of enzymatically hydrolyzed photoreaction mixtures was used to confirm the electrophoresis results. A strong strand preference for specific photoadduct formation was also detected.     

Kinetics of Intravenously Administered Lactose in Cows

Four adult Norwegian Red cows were employed in an experiment designed to study the kinetics of lactose. The cows were given 50 g or 60 g of lactose by rapid intravenous injection of a 10 % lactose solution. Blood samples were taken at different intervals after injection, and lactose concentrations in the samples determined by an enzymatic/spectrophotometric method. The mean half-time for lactose elimination was 85.7 min, and for distribution 8.4 min. The mean apparent volume of distribution was calculated to be 0.189 1/kg, and total body clearance 1.55 ml min−1 kg−1. There was evidence to suggest that lactose mainly is eliminated renally from its distribution volume by glomerular filtration in the cow.     

PQuad--a visual analysis platform for proteomic data exploration of microbial organisms

Summary: The visual Platform for Proteomics Peptide and Proteindata exploration (PQuad) is a multi-resolution environment thatvisually integrates genomic and proteomic data for prokaryoticsystems, overlays categorical annotation and compares differentialexpression experiments. PQuad requires Java 1.5 and has beentested to run across different operating systems. Availability: http://ncrr.pnl.gov/software Contact: bobbie-jo.webb-robertson{at}pnl.gov Associate Editor: Thomas Lengauer     

Disassembly and characterization of the nuclear pore complex-lamina fraction from bovine liver nuclei

The nuclear pore complex-lamina (PCL), composed of nuclear pore structures attached to fibrous lamina, was isolated from bovine liver nuclei. We found that the highly aggregated PCL was disrupted and 75% of the constituent polypeptides could be solubilized by extraction for 1 h with 2% deoxycholate (DOC) and 3% 2-mercaptoethanol. While some differential solubilization was observed at lower detergent concentrations, all PCL proteins were solubilized equally at 2% DOC. The reducing agent was necessary to achieve maximum dispersal of the PCL and to prevent aggregation of the solubilized proteins. No tightly bound phospholipid or Triton X-100 could be detected in these preparations. Rapid removal of DOC, by dialysis or gel filtration, resulted in aggregation and precipitation of the PCL proteins, but the detergent could be removed by centrifugation through sucrose gradients. The sedimentation profiles indicated that the three major polypeptides, lamins A, B, and C, each sedimented as a single peak with a shoulder of more rapidly sedimenting material, possibly higher oligomeric forms. The sedimentation coefficient of lamins B and C, in the presence and absence of detergent, was 4.5 S. In the presence of DOC, lamin A had a sedimentation coefficient of 5.6 S, but this value was decreased to 4.1 S, when DOC was omitted from the gradient. These studies suggested that lamins B and C do not interact with or bind DOC, while lamin A may bind appreciable amounts of the detergent. The Stokes radii of lamins A, B, and C were found by gel filtration to be 75, 75, and 70 A, respectively. The molecular weights and frictional ratios estimated from the sedimentation and gel filtration data indicated that the lamins are dimeric, rod-shaped molecules.     

Selenium and Zinc Concentrations in Kidney,Liver And Muscle of Cattle from Different Parts of Norway

Cattle slaughtered in four different parts of Norway have been examined with respect to selenium and zinc content in kidney, liver and muscle. Highest selenium concentrations were found in kidney and lowest in muscle. In spite of extensive use of standardized concentrates, geographic differences were detected with regard to selenium tissue levels, animals from the southeastern inland region having the lowest levels. According to other workers, this region has low-selenium humus soils, and selenium responsive diseases among young ruminants have been of considerable importance, especially when concentrates had not been given during winter feeding. The recorded tissue selenium levels are compared to other workers’ proposals for normal values. All animals examined in this study seem to be well within healthy limits. Kidney, liver and muscle from cattle are good sources of selenium with respect to human nutrition. As far as zinc concentrations are concerned, muscle has the highest and kidney the lowest levels. Geographic differences were found, and individuals from the midland and northern coastal regions have the highest zinc tissue levels. Cattle from the northern coastal region seems to have especially high zinc concentrations in the organs.