Abstract
The sedimentation of circular lambda DNA suggests that the molecular undergoes significant changes in shape and super-coiling as the NaC1 concentration increases. Closed circular lambda DNA, species I, isolated and purified from superinfected immune bacteria, sediments in sucrose gradients of low ionic strength at a rate 2.0 times faster than linear lambda DNA, species III. The addition of ethidium causes the sedimentation rate of species I DNA to decrease until enough dye is bound to remove 121 supercoils per molecule. At this point, species I co-sediments with nicked and nonsupercoiled species II. Futher additions of ethidium cause the sedimentation rate to increase until the relative rate of species I is again at least twice that of species III. This classical behavior is altered when NaC1 is present in the buffer. In 1.0 M NaC1 the changes in S are complex. Initially, species I sediments 1.55 times faster than species III. Titration with ethidium caused a decrease in S to an early minimum value, than an increase to a first maximum, followed by a decrease to the S of species II. At this point enough dye has intercalated to remove 208 superhelical turns. Further additions of dye introduce supercoils and cause S to increase again. In 0.1 to 0.4 M NaC1 the relative S of species I is 1.69 and 1.59, respectively. If titrated with ethidium, S first increases to a maximum value then decreases to the minimum rate when enough dye is bound to remove 158 and 183 supercoils, respectively. The results indicate an increase in the superhelix density from 0.026 turns per 10 base pairs in buffer alone to 0.045 in the same buffer with 1.0 M NaC1. If this change in superhelix density results from a concomitant change in the average rotation angle between base pairs in the Watson-Crick helix, the addition of 1.0 M NaC1 alters the rotation angle by 0.68 degrees per base pair.
Highlights
When the free ethidium in the gradient was less than 0.1 fig per ml, its concentration in the fractions after sedimentation was measured in one-half of each fraction by adding a saturating amount of E. coli or salmon sperm DNA
Comments on Purijkation of Supercoiled X DNA--For these studies we developed an improved method for the purification of circular X DNA from superinfected lysogenic bacteria
70is the apparent number of superhelical turns originally present in the molecule, and (rois the corresponding superhelix density calculated as described in the preceding paper [3]
Summary
The sedimentation of circular X DNA suggests that the molecule undergoes significant changes in shape and supercoiling as the NaCl concentration increases. The addition of ethidium causes the sedimentation rate of species I DNA to decrease until enough dye is bound to remove 121 supercoils per molecule. S first increases to a maximum value decreases to the minimum rate when enough dye is bound to remove 158 and 183 supercoils, respectively. The sedimentation rate of purified circular X [3H]DNA is measured relative to a linear X [V]DNA marker as a function of bound ethidium and (thereby the number of supercoils) at four NaCl concentrations, 0.0, 0.1, 0.4, and 1.0 M in 0.01 M Tris-HCl plus 10 mM sodium-EDTA buffer.
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