RESUMO
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.
Assuntos
DNA Helicases , Replicação do DNA , DNA/química , DNA/metabolismo , Proteínas/metabolismo , Origem de Replicação , Timina/metabolismo , Transativadores , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Ligação de Hidrogênio , Modelos Genéticos , Conformação de Ácido Nucleico , Ligação Proteica , PrótonsRESUMO
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.
Assuntos
Perfilação da Expressão Gênica , Tecido Linfoide/patologia , Linfotoxina-alfa/genética , Baço/metabolismo , Fator de Necrose Tumoral alfa/deficiência , Animais , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Quimiocinas/biossíntese , Quimiocinas/genética , DNA Complementar/genética , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Fosfolipases A2 do Grupo II , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Pâncreas/enzimologia , Fenótipo , Fosfolipases A/biossíntese , Fosfolipases A/genética , Fosfolipases A/isolamento & purificação , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/genética , Organismos Livres de Patógenos Específicos , Baço/patologia , Técnica de Subtração , Fator de Necrose Tumoral alfa/genéticaRESUMO
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.
Assuntos
Cátions , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Produtos do Gene rev/metabolismo , HIV-1/metabolismo , Histonas/metabolismo , Humanos , Cinética , Magnésio/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Timosina/metabolismo , Fatores de Tempo , Zinco/metabolismo , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
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.
