RESUMO
The age distribution and incidence of loss of heterozygosity (LOH) of 1p and 19q was analyzed in 85 oligodendroglial tumors WHO II and III. The peak of tumor manifestation was in the age group of 35 to 55 years. There was no association between age at diagnosis and LOH incidence. We conclude that the prognostic effect of age on survival is not mediated by LOH 1p/19q.
Assuntos
Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 1/genética , Perda de Heterozigosidade/genética , Oligodendroglioma/diagnóstico , Oligodendroglioma/genética , Adulto , Idade de Início , Envelhecimento/fisiologia , Neoplasias Encefálicas/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Oligodendroglioma/patologiaRESUMO
The integration host factor (IHF) of Escherichia coli is a major nucleoid-associated protein that binds to specific sites on DNA. Using gel retardation and competition experiments we have estimated that in vitro IHF binds specific sites 1000-10,000 times more tightly than non-specific, chromosomal DNA. We have analyzed the in vitro and in vivo interaction of IHF with three specific binding sites using UV laser footprinting. Because there is a strict correspondence between the intensity of the footprinting signal and the occupancy of a site, we can correlate in vitro association constants with in vivo site occupancy. From the fractional occupancy of various ihf sites in vivo, we then estimate the amount of free IHF in the cell. Exponentially growing cells contain only about 0.7 nM of free IHF, a value 20-fold smaller than the one previously deduced from DMS footprinting. As a consequence low affinity sites are only partially occupied and strong binding sites reach semi-saturation. In stationary phase the concentration of free IHF in the cell increases about sevenfold. These results show that only a very small fraction of total IHF is free in solution. Given the affinity of IHF for non-specific DNA our data imply that a large part of chromosomal DNA is accessible to IHF, and that IHF is a major contributor to chromosomal DNA condensation. The in vivo UV-laser footprinting method is of general interest, because it allows the measurement and the comparison of DNA-protein interactions in vitro and in vivo.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Pegada de DNA , Primers do DNA/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Fatores Hospedeiros de Integração , Cinética , Lasers , Dados de Sequência Molecular , Raios UltravioletaRESUMO
The histone-like protein integration host factor (IHF) of Escherichia coli binds to specific binding sites on the chromosome or on mobile genetic elements, and is involved in many cellular processes. We have analyzed the interaction of IHF with five different binding sites in vitro and in vivo using UV laser footprinting, a technique that probes the immediate environment and conformation of a segment of DNA. Using this generally applicable technique we can directly compare the binding modes and interaction strengths of a DNA binding protein in its physiological environment within the cell to measurements performed in vitro. We conclude that the interactions between IHF and its specific binding sites are identical in vitro and in vivo. The footprinting signal is consistent with the model of IHF-binding to DNA proposed by Yang and Nash (1989). The occupancy of binding sites varies with the concentration of IHF in the cell and allows to estimate the concentration of free IHF protein in the cell.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sítios de Ligação , Sequência Consenso , Primers do DNA/química , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Desoxirribonucleoproteínas/química , Fatores Hospedeiros de Integração , Dados de Sequência Molecular , Sequências Reguladoras de Ácido NucleicoRESUMO
The histone-like protein integration host factor (IHF) of Escherichia coli binds to specific binding sites on the chromosome or on mobile genetic elements, and is involved in many cellular processes. We have analyzed the interaction of IHF with five different binding sites in vitro and in vivo using UV laser footprinting, a technique that probes the immediate environment and conformation of a segment of DNA. Using this generally applicable technique we can directly compare the binding modes and interaction strengths of a DNA binding protein in its physiological environment within the cell to measurements performed in vitro. We conclude that the interactions between IHF and its specific binding sites are identical in vitro and in vivo. The footprinting signal is consistent with the model of IHF-binding to DNA proposed by Yang and Nash (1989). The occupancy of binding sites varies with the concentration of IHF in the cell and allows to estimate the concentration of free IHF protein in the cell.
