RESUMO
We have previously shown the existence of two DNA-binding sites on the globular domain of H5 (termed GH5), both of which are required for nucleosome organisation, as judged by the protection of a 166 bp chromatosome intermediate during micrococcal nuclease digestion of chromatin. This supports a model in which GH5 contacts two duplexes on the nucleosome. However, studies of a nucleosome assembled on the 5 S rRNA gene have argued against the requirement for two DNA-binding sites for chromatosome protection, which has implications for the role of linker histones. We have used this proposed difference in the requirement for a second site on the globular domain in the two models as a means of investigating whether bulk and reconstituted 5 S nucleosomes are indeed fundamentally different. GH5 protects a 166 bp chromatosome in both "bulk" and 5 S systems, and in both cases protection is abolished when all four basic residues in site II are replaced by alanine. Binding to four-way DNA junctions, which present a pair of juxtaposed duplexes, is also abolished. Single mutations of the basic residues did not abolish chromatosome protection in either system, or binding to four-way junctions, suggesting that the residues function as a cluster. Both bulk and 5 S nucleosomes thus require a functional second DNA-binding site on GH5 in order to bind properly to the nucleosome. This is likely to reflect a similar mode of binding in each case, in which two DNA duplexes are contacted in the nucleosome. There is no indication from these experiments that linker histones bind fundamentally differently to 5 S and bulk nucleosomes.
Assuntos
DNA/química , DNA/metabolismo , Histonas/química , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Galinhas , Dicroísmo Circular , DNA/genética , Pegada de DNA , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histonas/genética , Nuclease do Micrococo/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Conformação de Ácido Nucleico , Nucleossomos/genética , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , RNA Ribossômico 5S/genética , Alinhamento de Sequência , Especificidade por Substrato , Termodinâmica , XenopusRESUMO
BACKGROUND: The crisis in health care brings a new focus to defining successful outcomes of medical treatments. The surgical literature has been criticized for not assessing functional outcomes in addition to technical success. METHODS: We evaluated the functional outcomes of limb salvage surgery over 3 years in 38 patients 65 years of age and older with limb-threatening ischemia. The RAND-36-Item Health Survey 1.0 was used as a health assessment tool. RESULTS: In spite of an 80% limb salvage rate, only 58% of patients survived 3 years and only 25% survived with the index limb and were able to walk. The RAND scores of patients whose limbs were amputated did not significantly differ from those of patients whose surgery was successful. CONCLUSION: Functional outcome goals need to be better defined for patients who need limb salvage vascular operations to enhance the quality of care given these patients and to be in concert with emerging health policy.