Visualization of a Spinach Plastid Transcriptionally Active DNA-Protein Complex in a Highly Condensed Structure

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Abstract
A transcriptionally active DNA-protein complex isolated from spinach S. oleracea plastids is visualized by EM in different conditions. This structure, after glutaraldehyde fixation, is highly condensed. DNA is supertwisted with proteins bound to it producing a beaded substructure. When glutaraldehyde fixation is omitted this structure is less condensed and DNA fibrils project from a proteinous central body. The DNA-protein complex can be separated into 2 populations by CsCl centrifugation: 1 with a buoyant density of 1.570 g/cm3 and the other of 1.610 g/cc3. By visualization of these 2 populations, proteins are either firmly bound to DNA in the central body, or more loosely bound to the DNA fibrils. These latter proteins could play a role in enzymic functions and/or in the supercoiling of DNA. The DNA from the DNA-protein complex possesses all fragments that belong to pure circular chloroplast DNA hydrolyzed by 2 restriction enzymes: BamHI and EcoRI. Some molecules in a supercondensed form with a beaded substructure probably contain entire chloroplast DNA molecules. A hydrolysis test with microccocal nuclease gives no indication of the presence of nucleosome-like structures. Thirty-six polypeptides with MW ranging from 12,000 to 180,000 are present in the complex, and 7 of them are highly soluble in 0.4 N H2SO4; their MW range from 14,000 to 46,000 as shown by 2-dimensional gel electrophoresis. No linolenic acid can be detected in the preparation, indicating the absence of chloroplast membranes.