Biology Reference
In-Depth Information
Determination of the X, the Copy Number of Mutant
Sufficient to Block Individual Reaction
DNA-packaging activity of multiple copies of pRNA may be dis-
rupted by the incorporation of only one mutant pRNA molecule into
the group.
107,131
In a series of predicted curves generated using the
binomial distribution,
Z
was held constant at 12 while
X
varied from
one to 12, as can be seen by the 12 separate curves (Fig. 8). The value
of
p
varied from zero (0%) to one (100%) by increments of .1 (10%)
for each predicted curve. These predicted curves assume that mutant
and wild type have equal procapsid binding affinities and thus can
compete equally for binding sites, and that the binding of mutant and
wild type is irreversible. When
X
increases, the efficiency of inhibition
by mutant decreases. The curve of observed empirical data could then
be superimposed onto the hypothetical curves to find a match, and
thus the
X
can be deduced from the overlapped curve.
One problem in using the binomial distribution method is the
large number of theoretical curves that will be produced, and the rel-
atively little difference between curves with similar
X
and
Z
values (i.e.
Z
1 for another curve).
However, most of the curves will be quite different from the empiri-
cal curves in shape and slope and can be immediately excluded. This
will leave a relatively small number of curves to choose from. Such an
approach, coupled with another method such as slopes of log/log
plot curves, may produce accurate stoichiometry of pRNA.
107,125,133
In Fig. 8, predicted curves generated using Eq. (1) under condi-
tions of
Z
=
10,
X
=
1 for one curve and
Z
=
11,
X
=
12 while
X
varies from one to 12 indicate that inhibition
of phi29 as a function of % mutant pRNA becomes greater as the
number of pRNAs required to inhibit DNA packaging (
X
) decreases.
When the empirical data of inhibition of phage assembly by mutant
pRNA P8/P4 were superimposed onto the predicted curves, the
slope and magnitude of inhibition observed did not match any of the
predicted curves. Attention should be paid to compare the slope of
the curve rather than the height of the curve. Slope is influenced by
the ratio of mutant and wild type, while the height of the curve is
influenced by the absolute total concentration of mutant plus wild
=
