Membrane Association of the Deoxyribonucleic Acid Growing-Point Region in Mycoplasma gallisepticum

Abstract

Newly replicated deoxyribonucleic acid (DNA) in Mycoplasma gallisepticum A5969 is membrane associated. Cells pulse-labeled 1 to 3 min with 3H-thymidine are lysed by a freeze-thaw procedure. After brief sonic treatment to shear the DNA, differential centrifugation gives a cell fraction (P2) that is enriched sevenfold for pulse-labeled DNA. P2 contains 80% of the total adenosine triphosphatase activity, 65% of the total cholesterol, and morphologically intact terminal bleb structures. Three to four minutes are needed to fully label the DNA growing-point region, whereas 7 to 8 min are required to "chase" 50% of the 3H-labeled DNA. This pulse-chase removes 80 to 85% of the nascent DNA from the P2 fraction.on July 31, 2020 by guest

Partial Purification of a Membrane-Associated Deoxyribonucleic Acid Complex from Mycoplasma gallisepticum

“…Biochemical assays. Protein determinations and measurements of adenosine triphosphatase activity were done as described by Quinlan and Maniloff (11). Isolation of P2 fraction.…”

“…Cells were harvested in the mid to late logarithmic phase of growth. The P2 subcellular fraction was isolated by differential centrifugation of a cell homogenate obtained by freezethawing and low-frequency sonic treatment (20 to 40 s with a low-frequency probe) as described previously (11). P2 was the pellet of subcellular material obtained after 20 min of centrifugation at 40,000 x g (a previous step removed material sedimenting at 4,000 x g for 5 min), suspended in either Na buffer [0.2 M tris (hydroxymethyl) aminomethane and 0.15 M NaCl, adjusted to pH 7.6 at 4 C] or TSM buffer [0.01 M tris (hydroxymethyl) aminomethane-hydrochloride, 0.005 M sodium succinate, and 0.01 M MgCl2 adjusted to pH 7.6 at 4 C and then diluted 1:10 with distilled water].…”

“…Electron microscopy. Samples for electron microscopy were centrifuged (45,000 x g for 30 min), and the pellets were prepared for electron microscopy (glutaraldehyde and osmium tetroxide fixation and Epon embedding) as described previously (11). The thin sections were stained with uranyl acetate (6) and lead citrate (14) and examined with a Zeiss EM9 S-2 electron microscope.…”

“…Previous studies (11) have shown that a M. gallisepticum subcellular fraction, designated P2, can be isolated by differential centrifugation of a cell homogenate obtained by repeated freeze-thaw cycles and brief sonic treatment. This P2 fraction contains the membraneassociated deoxyribonucleic acid (DNA) replication complex.…”

Wall-Less Prokaryotes of Plants

“…In most species of mollicutes, DNA replication and cell division are not closely coordinated; however, in Mycoplasma gallisepticum cell division by binary fission is synchronous with DNA replication (132). In the case of S. citri, the elongation observed around the constriction zone may play a role in the segregation of DNA strands in the two newly formed elementary helices.…”