ROLE OF mRNA IN EMBRYONIC DEVELOPMENT
123
synthesize any rRNA but only sRNA and mRNA (Brown and Gurdon,
1964). When the mutant embryos are exposed to
1 4
C0 2 , not only sRNA
becomes labeled, but also a class of RNA that sediments throughout the
sucrose gradient (Fig. 4; Brown and Gurdon, 1966). This heterogeneous
RNA cannot be other than mRNA.
III. Types of Messenger RNA Synthesized
during Embryonic Development
A. Principle of the Hybridization Experiments
The sucrose gradient analysis of RNA tells us the time at which
heterogeneous (messenger) RNA synthesis begins, and provides some information with respect to the amount of RNA present at each stage.
This technique does not, however, enable us to decide whether the mRNA
present at a given stage is transcribed on the same or on different DNA
sites compared with the mRNA present at other stages. To answer this
question, it is necessary to compare the nucleotide sequences of the
mRNA's present at different times. The hybridization technique between DNA and RNA was used in an attempt to describe the changes in
gene activity that occur during embryonic development (Denis, 1966a,b;
1967). This method is based on the ability of RNA to form a hybrid
molecule with the DNA region on which it has been transcribed (Hall
and Spiegelman, 1961).
The technique adopted for studying the types of mRNA synthesized
during development was that of Bolton and MacCarthy (1962). Denatured DNA from Xenopus was immobilized in an inert matrix of agar
and incubated at 60°C with labeled RNA extracted from embryos by
the procedure of Brown and Littna (1964a). At the end of the incubation period, the DNA-agar was washed free of the nonhybridized RNA.
It was then heated at 75 °C in order to break the hydrogen bonds between
DNA and complementary RNA. This treatment releases the hydridized
RNA. The proportion of labeled RNA present in the incubation mixture
that binds to DNA (percentage of hydridization) depends on many
factors. Among these, the most important are the labeling time of RNA
(see Figs. 12 and 13) and the relative proportions of RNA and DNA-agar
in the incubation mixture (see Fig. 11).
B. Principle of the Competition Experiments
The most instructive experiments consisted in incubating a fixed
amount of DNA with a given amount of labeled RNA (reference RNA)
123
synthesize any rRNA but only sRNA and mRNA (Brown and Gurdon,
1964). When the mutant embryos are exposed to
1 4
C0 2 , not only sRNA
becomes labeled, but also a class of RNA that sediments throughout the
sucrose gradient (Fig. 4; Brown and Gurdon, 1966). This heterogeneous
RNA cannot be other than mRNA.
III. Types of Messenger RNA Synthesized
during Embryonic Development
A. Principle of the Hybridization Experiments
The sucrose gradient analysis of RNA tells us the time at which
heterogeneous (messenger) RNA synthesis begins, and provides some information with respect to the amount of RNA present at each stage.
This technique does not, however, enable us to decide whether the mRNA
present at a given stage is transcribed on the same or on different DNA
sites compared with the mRNA present at other stages. To answer this
question, it is necessary to compare the nucleotide sequences of the
mRNA's present at different times. The hybridization technique between DNA and RNA was used in an attempt to describe the changes in
gene activity that occur during embryonic development (Denis, 1966a,b;
1967). This method is based on the ability of RNA to form a hybrid
molecule with the DNA region on which it has been transcribed (Hall
and Spiegelman, 1961).
The technique adopted for studying the types of mRNA synthesized
during development was that of Bolton and MacCarthy (1962). Denatured DNA from Xenopus was immobilized in an inert matrix of agar
and incubated at 60°C with labeled RNA extracted from embryos by
the procedure of Brown and Littna (1964a). At the end of the incubation period, the DNA-agar was washed free of the nonhybridized RNA.
It was then heated at 75 °C in order to break the hydrogen bonds between
DNA and complementary RNA. This treatment releases the hydridized
RNA. The proportion of labeled RNA present in the incubation mixture
that binds to DNA (percentage of hydridization) depends on many
factors. Among these, the most important are the labeling time of RNA
(see Figs. 12 and 13) and the relative proportions of RNA and DNA-agar
in the incubation mixture (see Fig. 11).
B. Principle of the Competition Experiments
The most instructive experiments consisted in incubating a fixed
amount of DNA with a given amount of labeled RNA (reference RNA)