Estructura y propiedades de las placas de cromatina de los cromosomas metafásicos: Estudio mediante técnicas de microscopía TEM, AFM y Espectroscopia de Fuerza Atómica

Abstract

In our laboratory the study of the chromosomes in the presence of high concentrations of divalent ions, has led us to report the chromatin plate-like structure for the first time as a fundamental element of the metaphase chromosome. In the experimental part of this Ph.D. Thesis (5 years), it has been analysed extensively this plate-like structures. The exhaustive TEM study of this planar structures obtained with different preparation procedures and the different performed controls, suggest that the plates are not an artifact of the sample preparation of this technique. The atomic force microscopy (AFM) has led us to image and investigate the mechanical properties of the plates in aqueous solution. The plates are thin (∼6.5 nm; determined by TEM and AFM) but compact and resistant to the penetration by the AFM tip: Their Young's modulus is ∼0.2 GPa and the stress required for surface penetration is ∼0.3 GPa (5-20 mM Mg2+. It has been applied a real time denaturation study (with AFM) and friction force measurements at the nanoscale (Nanotribology) to analyse the structural properties of the plates in aqueous solution at room temperature. The results show that at high concentrations of NaCl and EDTA, and extensive digestion with protease and nuclease enzymes cause plate denaturation. Nanotribology studies show that native plates under structuring conditions (5 mM Mg2+ have a relatively high friction coefficient (μ∼0.3), which is markedly reduced when high concentrations of NaCl or EDTA are added (μ∼0.1). Protease digestion increases the friction coefficient (μ∼0.5), but the highest friction is observed when DNA is cleaved by micrococcal nuclease (μ∼0.9), indicating that DNA is the main structural element of plates. This results suggest that the native chromosomes are formed by staked plates, that are composed by a flexible and mechanically resistant two-dimensional network of DNA and proteins.

Date of Award	15 Sept 2010
Original language	Undefined/Unknown
Supervisor	Joan Ramon Daban Balaña (Director)