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ALBERTO MONROY AND RACHELE MAGGIO
synchronously dividing eggs, from fertilization to the 8-cell stage, it
could be shown that synthesis of DNA measured by the incorporation of
either thymidine, deoxyuridine or cytidine, immediately follows each
cleavage. The same was observed for the incorporation of cytidine into
RNA.
The experiments of Nemer on the utilization of cytidine and thymidine
for DNA synthesis demonstrate that an endogenous reservoir of similar
compounds must be present in the unfertilized egg. Furthermore, the
fact that incorporation of deoxyuridine into DNA was observed before
the first cleavage, indicates that the conversion of this compound into
thymidylic acid may take place quite early in development.
In order to find out how long the endogenous DNA precursors can
continue to meet the requirements for DNA synthesis, the embryos were
exposed to 5-fluorodeoxyuridine, a compound known to block the
conversion of 5-deoxyuridylic acid (dUMP) to 5-thymidylic acid (dTMP)The lethal action of this agent on the development of sand-dollar
embryos had already been studied by Karnofsky and Bäsch (1960), who
found that when administered to the eggs it blocked development at the
early blastula stage. Thymidine and thymine were able to release the
inhibition to a great extent. The toxic effect of the compound is
considerably lower when it is given to blastulae. Karnofsky and
Bevelander (1958) demonstrated that the two glutamine antagonists.
6-diazo-5-oxo-L-norleucine (DON) and azaserine, blocked the development of the embryos and that their effect could be counteracted by the
addition of hypoxanthine and inosine. Since sand-dollar embryos can
develop for a longer period in the presence of DON than in the presence
of 5-fluorodeoxyuridine, the authors concluded that the sand-dollar
embryo has a larger reserve of purine than of pyrimidine precursors.
The action of 5-bromouracil is also very important. No toxic effects
appear until the early blastula stage, but the embryos die at a more
advanced stage of development. This compound is probably incorporated
into the newly formed DNA (see review by Matthews, 1958). As
Karnofsky and Bäsch have clearly stated, 'the 5-fluorodeoxyuridine and
5-bromodeoxyuridine appear to inhibit the formation and alter the
structures of DNA respectively, distinctive effects which may provide a
means for studying the role of DNA in embryonic development'.
In his experiments Nemer (1962) found that, when given to the eggs
immediately after fertilization, 5-fluorodeoxyuracil stopped development at the 8-cell stage. If, however, smaller amounts of thymidine,
5-methyldeoxycytidine or 5-iododeoxyuridine were added at the same
time, development proceeded until the blastula stage, whereas deoxyuridine and deoxycytidine had no effect. From this the author concluded
that the supply of thymidine in the egg is just enough to support
ALBERTO MONROY AND RACHELE MAGGIO
synchronously dividing eggs, from fertilization to the 8-cell stage, it
could be shown that synthesis of DNA measured by the incorporation of
either thymidine, deoxyuridine or cytidine, immediately follows each
cleavage. The same was observed for the incorporation of cytidine into
RNA.
The experiments of Nemer on the utilization of cytidine and thymidine
for DNA synthesis demonstrate that an endogenous reservoir of similar
compounds must be present in the unfertilized egg. Furthermore, the
fact that incorporation of deoxyuridine into DNA was observed before
the first cleavage, indicates that the conversion of this compound into
thymidylic acid may take place quite early in development.
In order to find out how long the endogenous DNA precursors can
continue to meet the requirements for DNA synthesis, the embryos were
exposed to 5-fluorodeoxyuridine, a compound known to block the
conversion of 5-deoxyuridylic acid (dUMP) to 5-thymidylic acid (dTMP)The lethal action of this agent on the development of sand-dollar
embryos had already been studied by Karnofsky and Bäsch (1960), who
found that when administered to the eggs it blocked development at the
early blastula stage. Thymidine and thymine were able to release the
inhibition to a great extent. The toxic effect of the compound is
considerably lower when it is given to blastulae. Karnofsky and
Bevelander (1958) demonstrated that the two glutamine antagonists.
6-diazo-5-oxo-L-norleucine (DON) and azaserine, blocked the development of the embryos and that their effect could be counteracted by the
addition of hypoxanthine and inosine. Since sand-dollar embryos can
develop for a longer period in the presence of DON than in the presence
of 5-fluorodeoxyuridine, the authors concluded that the sand-dollar
embryo has a larger reserve of purine than of pyrimidine precursors.
The action of 5-bromouracil is also very important. No toxic effects
appear until the early blastula stage, but the embryos die at a more
advanced stage of development. This compound is probably incorporated
into the newly formed DNA (see review by Matthews, 1958). As
Karnofsky and Bäsch have clearly stated, 'the 5-fluorodeoxyuridine and
5-bromodeoxyuridine appear to inhibit the formation and alter the
structures of DNA respectively, distinctive effects which may provide a
means for studying the role of DNA in embryonic development'.
In his experiments Nemer (1962) found that, when given to the eggs
immediately after fertilization, 5-fluorodeoxyuracil stopped development at the 8-cell stage. If, however, smaller amounts of thymidine,
5-methyldeoxycytidine or 5-iododeoxyuridine were added at the same
time, development proceeded until the blastula stage, whereas deoxyuridine and deoxycytidine had no effect. From this the author concluded
that the supply of thymidine in the egg is just enough to support