124
HERMAN DENIS
and increasing amounts of nonradioactive RNA (competitor RNA). If
the reference RNA and the competitor RNA are the same, the latter will
progressively replace the former in the hybrid, and an apparent decrease
in the percentage of binding will ensue (Hoyer et al., 1964). If the
RNA's under comparison have no nucleotide sequence in common, the
addition of increasing amounts of competitor RNA will have no influence
on the hybridization of the reference RNA with DNA. The curve obtained by diluting a given amount of labeled RNA by increasing amounts
of identical, nonlabeled RNA should be linear in a double logarithmic
plot and drop with a 45° angle, provided that all the complementary sites
in DNA are saturated by the reference RNA (Denis, 1967). If the
amount of labeled RNA used is insufficient to occupy all the complementary sites of DNA, more RNA becomes hybridized with DNA
when increasing amounts of competitor RNA are added. In nonsaturating
conditions, two contradictory factors are thus acting. On the one hand,
the isotope dilution produced by the competitor RNA results in an apparent reduction in the percentage of hybridization; on the other hand,
the addition of increasing amounts of RNA tends to increase the total
amount of RNA (labeled and nonlabeled) that binds to DNA. As a result, the slope of the dilution curve will be lower than 45°. If the amount
of competitor RNA added becomes sufficient, the complementary sites
of DNA will eventually reach saturation. At this point, the slope of the
dilution curve will increase and become equal to 45°. In all the competition experiments carried out with embryonic RNA, the amount of
reference RNA used was equal or inferior to the amount of trapped DNA
present in the incubating mixture. This amount of RNA does not suffice
to saturate all the complementary sites in DNA. This explains why the
slope of the dilution curve is always lower than 45°.
C. Competition between Labeled RNA from Stage 42
(Differentiated Tadpoles) and Nonlabeled RNA from Other Stages
In a first experiment (Fig. 5), RNA from differentiated tadpoles
(stage 42) exposed to
1 4
C0 2 for 1 hour, was made to compete with nonlabeled RNA either from the same stage or from earlier stages. When
stage 42 RNA is used as competitor RNA, a dilution curve is obtained
that is linear when plotted logarithmically (Fig. 5f). Ribonucleic acid
from cleaving eggs does not interfere at all with the hybridization between DNA and stage 42 RNA (Fig. 5a). On the contrary, RNA from
gastrulae and from later embryos is increasingly competitive versus RNA
from differentiated tadpoles (Fig. 5b-e). None of the nucleotide sequences
HERMAN DENIS
and increasing amounts of nonradioactive RNA (competitor RNA). If
the reference RNA and the competitor RNA are the same, the latter will
progressively replace the former in the hybrid, and an apparent decrease
in the percentage of binding will ensue (Hoyer et al., 1964). If the
RNA's under comparison have no nucleotide sequence in common, the
addition of increasing amounts of competitor RNA will have no influence
on the hybridization of the reference RNA with DNA. The curve obtained by diluting a given amount of labeled RNA by increasing amounts
of identical, nonlabeled RNA should be linear in a double logarithmic
plot and drop with a 45° angle, provided that all the complementary sites
in DNA are saturated by the reference RNA (Denis, 1967). If the
amount of labeled RNA used is insufficient to occupy all the complementary sites of DNA, more RNA becomes hybridized with DNA
when increasing amounts of competitor RNA are added. In nonsaturating
conditions, two contradictory factors are thus acting. On the one hand,
the isotope dilution produced by the competitor RNA results in an apparent reduction in the percentage of hybridization; on the other hand,
the addition of increasing amounts of RNA tends to increase the total
amount of RNA (labeled and nonlabeled) that binds to DNA. As a result, the slope of the dilution curve will be lower than 45°. If the amount
of competitor RNA added becomes sufficient, the complementary sites
of DNA will eventually reach saturation. At this point, the slope of the
dilution curve will increase and become equal to 45°. In all the competition experiments carried out with embryonic RNA, the amount of
reference RNA used was equal or inferior to the amount of trapped DNA
present in the incubating mixture. This amount of RNA does not suffice
to saturate all the complementary sites in DNA. This explains why the
slope of the dilution curve is always lower than 45°.
C. Competition between Labeled RNA from Stage 42
(Differentiated Tadpoles) and Nonlabeled RNA from Other Stages
In a first experiment (Fig. 5), RNA from differentiated tadpoles
(stage 42) exposed to
1 4
C0 2 for 1 hour, was made to compete with nonlabeled RNA either from the same stage or from earlier stages. When
stage 42 RNA is used as competitor RNA, a dilution curve is obtained
that is linear when plotted logarithmically (Fig. 5f). Ribonucleic acid
from cleaving eggs does not interfere at all with the hybridization between DNA and stage 42 RNA (Fig. 5a). On the contrary, RNA from
gastrulae and from later embryos is increasingly competitive versus RNA
from differentiated tadpoles (Fig. 5b-e). None of the nucleotide sequences