Chromatin is the complex of DNA, histones and non-histone proteins that make up chromosomes and the region of dispersed chromosomes during the interphases and S phase of the cell cycle. It is found inside cell nuclei in eukaryotic cells. There are many important proteins in the nuclei which are involved in DNA replication, gene expression, DNA repair, etc. Core histones, linker histones and HMGs are the most important proteins in this group. In this thesis, a new, quick and efficient method is developed, to extract and purify proteins from cell nuclei, which is named "forward technology". By using the "forward technology", ultra-pure native core histone octamers, dimers and tetramers were extracted from chick erythrocytes. By using the pure histone dimers and tetramers, the complexes of NAP1-dimer and NAP1-tetramer were obtained in collaboration with others. Crystals of pure histone octamers with a higher resolution than before were produced (Chapter 2). A low KCI/phosphate wash of chick erythrocyte nuclei removed up to 1 g of proteins without nuclei lysis (cePNE1 proteins). The high KCI/phosphate soluble fraction of the cePNE1 is rich in HMG proteins, peptidyl-prolyl isomerases (FKBP3) and heatshock protein 70 which were fractionated by cation-exchange chromatography and anion-exchange chromatography (Chapter 3). Valuable (in terms of function) high-molecular-weight proteins were enriched, and another group of nucleoproteins called cePNE5 is fractionated by the "forward technology" from chick erythrocyte nuclei. It was confirmed that HMGB proteins prefer to bind to core histone H2A-H2B dimers (Chapter 4). It has proved possible to fractionate HMG-rich PNE1 proteins separate from a linker- histone rich nucleoprotein extract, separate from pure core histones (not as oetamers) from human tissue culture cells. The method has been applied to human leukocytes obtained from the National Blood Service. This enrichment of particular groups of proteins will be useful for proteomic studies (Chapter 5).
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:555846 |
| Date | January 2012 |
| Creators | Zhuang, Qin Qin |
| Publisher | Liverpool John Moores University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://researchonline.ljmu.ac.uk/6114/ |
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