ABSTRACT
The radial compression properties of single DNA molecules have been studied using vibrating scanning polarization force microscopy. By imaging DNA molecules at different vibration amplitude set-point values, we obtain the correlations between radially applied force and DNA compression, from which the radial compressive elasticity can be deduced. The estimated elastic modulus is approximately 20-70 MPa under small external forces (<0.4 nN) and increases to approximately 100-200 MPa for large loads.
Subject(s)
DNA/chemistry , DNA/ultrastructure , Micromanipulation/methods , Microscopy, Atomic Force/methods , Microscopy, Polarization/methods , Models, Chemical , Models, Molecular , Compressive Strength , Computer Simulation , DNA/analysis , Elasticity , Image Interpretation, Computer-Assisted/methods , Nucleic Acid Conformation , Stress, Mechanical , VibrationABSTRACT
Recently, the isolation and biochemical analysis of DNA at the single-molecule level has been recognized as very important for genetic research and clinical analysis. A unique technique for the positioning, dissection, and isolation of single DNA molecules using atomic force microscopy (AFM) has been demonstrated. Full-length genome DNA molecules were first deposited and stretched by a modified "molecular combing" technique onto a 3-aminopropyl triethoxysilane-coated mica substrate. A single DNA fragment was dissected from one of those genome DNA strands with the AFM tip at the desired position, and then isolated (or picked up) after a special operation called "kneading". All the operations including imaging, dissection, and isolation could be carried out with one tip. The isolated DNA fragment on the AFM tip could be successfully amplified by single-molecule PCR.
Subject(s)
DNA/analysis , DNA/isolation & purification , Nanotechnology/methods , Polymerase Chain Reaction/methods , Microscopy, Atomic Force , Plasmids/analysis , Plasmids/isolation & purificationABSTRACT
Using atomic force microscopy (AFM), the dynamic process of the in vitro nucleosome reconstitution followed by slow dilution from high salt to low salt was visualized. Data showed that the histone octamers were dissociated from DNA at 1M NaCl. When the salt concentration was slowly reduced to 650 mM and 300 mM, the core histones bound to the naked DNA gradually. Once the salt concentration was reduced to 50 mM the classic "beads-on-a-string" structure was clearly visualized. Furthermore, using the technique of the in vitro reconstitution of nucleosome, the mono- and di- nucleosomes were assembled in vitro with both HS2core (-10681 to -10970 bp) and NCR2 (-372 to -194 bp) DNA sequences in the 5 flanking sequence of human b-globin gene. Data revealed that HMG 1/2 and HMG14/17 proteins binding to both DNA sequences are changeable following the assembly and disassembly of nucleosomes. We suggest that the changeable binding patterns of HMG 14/17 and HMG1/2 proteins with these regulatory elements may be critical in the process of nucleosome assembly, recruitment of chromatin-modifying activities, and the regulation of human b-globin gene expression.
Subject(s)
High Mobility Group Proteins/metabolism , Nucleosomes/metabolism , Animals , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Chickens , Chromatin/metabolism , Chromatin/ultrastructure , DNA/metabolism , DNA/ultrastructure , Gene Expression Regulation , Globins/genetics , HMGB1 Protein/metabolism , HMGB2 Protein/metabolism , HMGN1 Protein/metabolism , HMGN2 Protein/metabolism , Histones/metabolism , Histones/ultrastructure , Humans , In Vitro Techniques , Microscopy, Atomic Force/methods , Nucleosomes/ultrastructure , Protein BindingABSTRACT
Chromium (III) enhanced the sensitivities of diamine silver staining of four proteins between 6- and 50-fold over that of the Coomassie Brilliant Blue (CBB)-chromium modified thiosulfate-silver staining method (Zhou et al. Biotechnology Letters, 2002, 24: 1561-1567). Using six dsDNA fragments, the detection limits of this new method was 10 to 30 pg per band, being 10- to 25-fold more sensitive than previous methods.
Subject(s)
Chromium , DNA/analysis , Diamines , Electrophoresis, Polyacrylamide Gel/methods , Proteins/analysis , Rosaniline Dyes , Silver , Staining and Labeling/methods , DNA/chemistry , Proteins/chemistry , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
This study revealed that the content of protein S29 in ribosomes of cancer cell line A549 was distinctly low (equivalent to about 30% of that of 2BS). The conclusion was acquired based on the ratios of spot volume of ribosomal protein S29 to that of several other ribosomal proteins (S29/L37a, S29/L38, S29/S27 and S29/S28) in the same gel plate. The possible biological roles of ribosomal protein S29 in malignant transformation and translation regulation are briefly discussed.
