Biomedical Engineering Reference
In-Depth Information
some 100 nm in diameter, which are comprised circumferentially wrapped DNA. In Figure
1.43, we present a number of examples of toroidal-shaped condensed linear
-174
RFII:
Xho
I-digested plasmid DNAs (5386 bp) that were condensed by the biological triva-
lent cation spermidine
3
. The structures were visualized by TEM after preparation by the
freeze-fracture, deep-etch technique to yield Pt-shadowed replicas of the hydrated frozen
toroidal DNA specimens. In these images, individual DNA segments, highlighted with
arrows, can be observed wrapping around the toroidal condensates. Using stereoscopic
techniques, we were also able to map the
z
-axis topology of individual DNA strands wrap-
ping circumferentially in different toroidal DNA condensates (137). As Figure 1.44 shows,
with this technique we could map the relative
z
-axis heights of interesting DNA strand fea-
tures on the torus surface as well as features on the ice substrate surface.
Different toroidal DNA systems of varying length and topological state (calf thymus
DNA, bacteriophage
-174 bacteriophage linear and circular DNAs) were also
studied using nonspecific deoxyribonuclease enzyme cleavage of the DNAs (138-141).
These studies invariably showed an arithmetic length series of fragments generated by
digestion of spermidine
3
-DNA condensates from different organisms. Figure 1.45 pres-
ents data on the DNA fragment sizes resulting from different micrococcal nuclease diges-
tion times for spermidine
3
DNA,
X-174 RFII:
Xho
I-digested plasmid DNAs.
Following digestion, the fragment sizes were obtained relative to molecular weight stan-
dards by electrophoresis on agarose gels. Extrapolation of each fragment's digestion time
to 0 provides a biochemical estimate, immediately prior to digestion, of the arithmetic
fragment sizes corresponding to integer multiples of DNA length wrapping around the
toroidal DNA condensates (~800 bp: monomer; 1600 bp: dimer; and 2400 bp: trimer). The
extrapolated monomer DNA length around 800 bp is exactly the length needed to wrap
once around the average toroidal circumference measured from the TEM images we pre-
sented for these DNA toroidal condensates in Figure 1.43. To account for these digestion
data, we proposed a general model for how an arithmetic series of DNA fragments can be
generated in these experiments (134,138). By applying a topological argument to the
micrococcal nuclease cleavage study results of the linear and circular DNA-spermidine
3
condensed linear
FIGURE 1.44
Stereoscopic measurements performed on a toroidal sper-
midine
3
-calf thymus DNA condensate using two 10° tilted
TEM images for the stereoscopic measurements as
described in and reprinted from Ruben, G.C., Marx, K.A.
(1984). Parallax Measurements on Stereomicrographs of
Hydrated Single Molecules: Their Accuracy and Precision at
High Magnification.
J. Microsc. Res. Tech.
1:373-385. With
permission of Wiley-Liss, Inc., a subsidiary of John Wiley &
Sons, Inc.
200Å
