ABSTRACT
The RepA protein from bacteriophage P1 binds DNA to initiate replication. RepA covers one face of the DNA and the binding site has a completely conserved T that directly faces RepA from the minor groove at position +7. Although all four bases can be distinguished through contacts in the major groove of B-form DNA, contacts in the minor groove cannot easily distinguish between A and T bases. Therefore the 100% conservation at this position cannot be accounted for by direct contacts approaching into the minor groove of B-form DNA. RepA binding sites with modified base pairs at position +7 were used to investigate contacts with RepA. The data show that RepA contacts the N3 proton of T at position +7 and that the T=A hydrogen bonds are already broken in the DNA before RepA binds. To accommodate the N3 proton contact the T(+7 )/A(+7)((')) base pair must be distorted. One possibility is that T(+7) is flipped out of the helix. The energetics of the contact allows RepA to distinguish between all four bases, accounting for the observed high sequence conservation. After protein binding, base pair distortion or base flipping could initiate DNA melting as the second step in DNA replication.
Subject(s)
DNA Helicases , DNA Replication , DNA/chemistry , DNA/metabolism , Proteins/metabolism , Replication Origin , Thymine/metabolism , Trans-Activators , Base Pairing , Base Sequence , Binding Sites , Conserved Sequence , DNA-Binding Proteins/metabolism , Electrophoretic Mobility Shift Assay , Hydrogen Bonding , Models, Genetic , Nucleic Acid Conformation , Protein Binding , ProtonsABSTRACT
Mice with combined lymphotoxin-alpha (LTalpha) and tumor necrosis factor (TNF) deficiencies show defects in the structure of peripheral lymphoid organs such as spleen, lymph nodes, and gut-associated lymphoid tissues. To identify genes associated with this defective phenotype in spleen, we applied a gene profiling approach, including subtractive cloning and gene array hybridizations, to mice with combined TNF/LT deficiency. The differentially expressed genes identified by these techniques was then evaluated by Northern blot analysis for splenic expression in knockout mice with single LTalpha or single TNF deficiency. Most of the genes detected in this analysis are directly or indirectly associated with disrupted LT and not TNF signaling.
Subject(s)
Gene Expression Profiling , Lymphoid Tissue/pathology , Lymphotoxin-alpha/genetics , Spleen/metabolism , Tumor Necrosis Factor-alpha/deficiency , Animals , Cell Adhesion Molecules/biosynthesis , Cell Adhesion Molecules/genetics , Chemokines/biosynthesis , Chemokines/genetics , DNA, Complementary/genetics , Expressed Sequence Tags , Gene Expression Profiling/methods , Gene Expression Regulation , Group II Phospholipases A2 , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Neutrophils/metabolism , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Organ Specificity , Pancreas/enzymology , Phenotype , Phospholipases A/biosynthesis , Phospholipases A/genetics , Phospholipases A/isolation & purification , Receptors, Chemokine/biosynthesis , Receptors, Chemokine/genetics , Specific Pathogen-Free Organisms , Spleen/pathology , Subtraction Technique , Tumor Necrosis Factor-alpha/geneticsABSTRACT
The divalent cation binding properties of human prothymosin alpha, an abundant nuclear protein involved in cell proliferation, were evaluated. By using prothymosin alpha retardation on a weak cation chelating resin charged with various divalent cations, specific binding of Zn2+ ions by prothymosin alpha was observed. This finding was further confirmed by the equilibrium dialysis analysis which demonstrated that, within the micromolar range of Zn2+ concentrations, prothymosin alpha could bind up to three zinc ions in the presence of 100 mM NaCl and up to 13 zinc ions in the absence of NaCl. Equilibrium dialysis analysis also revealed that prothymosin alpha could bind Ca2+, although the parameters of Ca2+ binding by prothymosin alpha were less pronounced than those of Zn2+ binding in terms of the number of metal ions bound, the KD values, and the resistance of the bound metal ions to 100 mM NaCl. The effects of Zn2+ and Ca2+ on the interaction of prothymosin alpha with its putative partners, Rev of HIV type 1 and histone H1, were examined. We demonstrated that Rev binds prothymosin alpha, and that prothymosin alpha binding to Rev but not to histone H1 was significantly enhanced in the presence of zinc and calcium ions. Our data suggest that the modes of prothymosin alpha interaction with Rev and histone H1 are distinct and that the observed zinc and calcium-binding properties of prothymosin alpha might be functionally relevant.
Subject(s)
Cations , Protein Precursors/metabolism , Thymosin/analogs & derivatives , Calcium/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Gene Products, rev/metabolism , HIV-1/metabolism , Histones/metabolism , Humans , Kinetics , Magnesium/metabolism , Plasmids/metabolism , Protein Binding , Recombinant Fusion Proteins/metabolism , Thymosin/metabolism , Time Factors , Zinc/metabolism , rev Gene Products, Human Immunodeficiency VirusABSTRACT
Lymphotoxin (LT) deficient mice have profound defects in the splenic microarchitecture associated with defective expression on certain gene products, including chemokines. By using subtraction cloning of splenic cDNA from wild-type and LT alpha or TNF/LT alpha double deficient mice we isolated a novel murine gene encoding a secretory type phospholipase A2, called SPLASH. The two major alternative transcripts of SPLASH gene are predominantly expressed in lymphoid tissues, such as spleen and lymph nodes. SPLASH maps to the distal part of chromosome 4, to which several cancer-related loci have been also mapped.
