Biology Reference
In-Depth Information
216
explanation can consider the linearized molecules as representations of the
conventional double helical DNA fiber structure. On the other hand, the
intact supercoils may consist of two novel separable complementary single
strand DNA circles, which are hydrogen-bonded to each other, representing
the four-stranded DNA fiber structure suggested before. During agraose gel
electrophoresis, they may separate due to the electrostatic repulsion of the
negatively charged phosphate groups on their backbones. However, for
most plasmid molecules, the molecular weights of the two complementary
strands are very close to each other. Thus, even if they have separated, the
two complementary single stranded DNA circles would appear as a single
band. New experimental methods would be required to study how they can
be distinguished.
Effect of mRNA
Many of the plasmid molecules carry drug resistant markers, i.e. they direct
the synthesis of enzymes which can degrade certain antibiotics. Thus, if the
plasmid molecules are purified under conditions so that the mRNA
molecules remain intact, these mRNA molecules will bind to one of the two
complementary strands of DNA selectively. Since RNA-DNA hybridization
is more stable than DNA-DNA hybridization, it will be possible to select
conditions for agarose gel electrophoresis such that the mRNA molecules
are still associated with one of the DNA strands. The molecular weight of
that DNA strand together with the mRNA is thus larger than the other DNA
strand alone. Furthermore, the configuration of the former may also be
different.
Experimentally, the two complementary intact single strand DNA circles of
plasmid molecules, in the presence of mRNA molecules binding selectively
to one of them, can separate into two bands on agarose gel electrophoresis
(Wu and Wu, 1996). Each of the bands can then be concentrated with the
glass bead method, and their nucleotide sequences determined. For pUC19,
one of the smaller plasmids, their sequences are indeed complementary to
each other. This experiment must be repeated for other plasmid molecules
of larger molecular weights, in order to establish that the complementary
strands of all plasmids are indeed separable regardless of their lengths.
Agarose gel electrophoresis may have a length limit of about 50 kb for such
studies, as in the case of cosmid molecules.
