116
FRED H. WILT
synthesis at the 7-somite stage is independent of normal high molecular
weight RNA synthesis. This implies that the initiation of synthesis is
controlled at levels other than the genome, and this has been termed
"transitional level regulation."
The analog 5-fluorouracil (5-FU) has also been employed to derange
RNA metabolism, and has been used by Wilt (1965a, 1966) in an analysis
similar to that used with actinomycin. If blastoderms are exposed prior
to the head-process stage, 10
-4 M 5-FU prevents synthesis of hemoglobin,
but thereafter they became quite refractory to the analog. Unpublished
studies have shown that 5-FU is incorporated into all classes of RNA,
and it also inhibits incorporation of tritiated leucine into protein by
about 40%. Incorporation of guanine-H
3
into RNA is inhibited by 5-FU
by about 50%. The incorporation of 5-FU into RNA seems similar both
before and after the head-process stage. The inhibitory effects of 5-FU
prior to the head-process stage have been reversed by the addition of
a twofold excess of thymidine but not by uridine, indicating the primary
inhibitory effect is on the integrity of DNA synthesis.
Similar results have been obtained with the analog 5-bromodeoxyuridine (BUdR), an analog which is incorporated into DNA. The initiation of hemoglobin synthesis in blastoderms exposed prior to the headprocess stage, is prevented by 100 ju,g/ml of 5-BUdR, but blastoderms
thereafter become refractory. The incorporation of C
14
BUdR prior to
and after this transitional stage seems to be similar. The inhibitory
effects of BUdR are reversed by thymidine, pointing to an action on
DNA. Hell (1964b) exposed blastoderms (one somite or older) to BUdR
and found mitoses were reduced twofold and some pycnotic nuclei were
present. Inhibition of net protein synthesis occurred at all stages; hemoglobin synthesis was reduced to a somewhat less extent than synthesis
of other proteins. Both RNA and DNA accumulation was reduced, but
this became less marked when older blastoderms were exposed to BUdR.
Hell (1964b) has also performed similar experiments using X-irradiation. Subsequent normal mitoses were prevented by 2000 r and DNA
synthesis was inhibited. Protein and RNA synthesis were both inhibited
to some extent after irradiation. The earliest stage examined by Hell
was the 1-somite stage, and in spite of the profound effects produced by
irradiation, hemoglobin at 50% of normal levels was formed in the
cultures. Normal DNA synthesis and mitosis after the head-fold stage
is attained are clearly not essential for the initiation of substantial
amounts of hemoglobin synthesis. O'Brien (1961) has employed the
enzyme, ribonuclease (250 /Ag/ml), to examine its effects on initiation
of hemoglobin synthesis, and he found virtually no effect of the enzyme
on blastoderms just entering the stage of active hemoglobin synthesis
FRED H. WILT
synthesis at the 7-somite stage is independent of normal high molecular
weight RNA synthesis. This implies that the initiation of synthesis is
controlled at levels other than the genome, and this has been termed
"transitional level regulation."
The analog 5-fluorouracil (5-FU) has also been employed to derange
RNA metabolism, and has been used by Wilt (1965a, 1966) in an analysis
similar to that used with actinomycin. If blastoderms are exposed prior
to the head-process stage, 10
-4 M 5-FU prevents synthesis of hemoglobin,
but thereafter they became quite refractory to the analog. Unpublished
studies have shown that 5-FU is incorporated into all classes of RNA,
and it also inhibits incorporation of tritiated leucine into protein by
about 40%. Incorporation of guanine-H
3
into RNA is inhibited by 5-FU
by about 50%. The incorporation of 5-FU into RNA seems similar both
before and after the head-process stage. The inhibitory effects of 5-FU
prior to the head-process stage have been reversed by the addition of
a twofold excess of thymidine but not by uridine, indicating the primary
inhibitory effect is on the integrity of DNA synthesis.
Similar results have been obtained with the analog 5-bromodeoxyuridine (BUdR), an analog which is incorporated into DNA. The initiation of hemoglobin synthesis in blastoderms exposed prior to the headprocess stage, is prevented by 100 ju,g/ml of 5-BUdR, but blastoderms
thereafter become refractory. The incorporation of C
14
BUdR prior to
and after this transitional stage seems to be similar. The inhibitory
effects of BUdR are reversed by thymidine, pointing to an action on
DNA. Hell (1964b) exposed blastoderms (one somite or older) to BUdR
and found mitoses were reduced twofold and some pycnotic nuclei were
present. Inhibition of net protein synthesis occurred at all stages; hemoglobin synthesis was reduced to a somewhat less extent than synthesis
of other proteins. Both RNA and DNA accumulation was reduced, but
this became less marked when older blastoderms were exposed to BUdR.
Hell (1964b) has also performed similar experiments using X-irradiation. Subsequent normal mitoses were prevented by 2000 r and DNA
synthesis was inhibited. Protein and RNA synthesis were both inhibited
to some extent after irradiation. The earliest stage examined by Hell
was the 1-somite stage, and in spite of the profound effects produced by
irradiation, hemoglobin at 50% of normal levels was formed in the
cultures. Normal DNA synthesis and mitosis after the head-fold stage
is attained are clearly not essential for the initiation of substantial
amounts of hemoglobin synthesis. O'Brien (1961) has employed the
enzyme, ribonuclease (250 /Ag/ml), to examine its effects on initiation
of hemoglobin synthesis, and he found virtually no effect of the enzyme
on blastoderms just entering the stage of active hemoglobin synthesis