EMBRYONIC HEMOGLOBIN SYNTHESIS
117
(7 somites). Ribonuclease treatment did, however, somewhat potentiate
the inhibitory effects of another analog, 8-azaguanine.
The first inhibitor experiments on blastoderms utilized 8-azaguanine,
and the use of the analog, although apparently quite complex and far
from understood, continues to be very useful. O'Brien (1961) originally
showed that exposure of blastoderms younger than 7 somites, in vitro,
to 100 jug/ml prevented accumulation of detectable hemoglobin levels;
exposure after the 8-somite stage had little or no effect on the amount
of hemoglobin synthesis. The effect was irreversible after 1 hr of exposure, although exposure of young blastoderms to mixtures of guanine
and azaguanine showed guanine could prevent the effect of 8-azaguanine.
He concluded that 8-azaguanine was a rather specific inhibitor of the
initiation of hemoglobin synthesis prior to the 7-somite stage. At the
7-somite stage, the synthetic mechanism for hemoglobin production was
activated, but could not be maintained in the presence of 8-azaguanine;
and thereafter, Hb synthesis became refractory to the action of the
analog. The results obtained by O'Brien have been confirmed by Hell
(1964b) and Wilt (1962, 1965a). If low levels of 8-azaguanine are used,
the inhibitory effect of the analog prior to 7 somites is not as profound
and is partially reversible by subsequent culture in the presence of
guanine. Furthermore, very sensitive methods of hemoglobin detection
by colorimetric (Hell, 1964b) or immunochemical (Wilt, 1962) means
revealed small amounts of hemoglobin formation, even when 8-azaguanine is applied prior to the 7-somite stage. The mechanism of action of
8-azaguanine is an important consideration. O'Brien concluded on the
basis of the evidence available at that time that 8-azaguanine interfered
with "RNA associated mechanisms or templates for hemoglobin production" being elaborated at the 7-somite stage. He assumed that cells which
had not yet produced Hb had not reached the final stages of cytoplasmic
template formation, or in more recent terms, messenger RNA was lacking. Hell (1964b) has shown that the net accumulation of RNA and DNA
is severely blocked by 8-azaguanine, and protein synthesis is generally
reduced as much as hemoglobin formation. Furthermore, 8-azaguanine
affected protein synthesis in general, and hemoglobin synthesis in particular, to a similar extent when applied during the refractory period of
O'Brien. This inhibition was less profound (50-60%) than when it was
applied prior to the 7-somite stage (90% inhibition), but it is not an
absolute refractoriness to 8-azaguanine. Wilt showed (1966) that the
incorporation of 8-azaguanine-C
14
into acid insoluble material occurs
both before and after the 7-somite stage. The specific activity of incorporated 8-azaguanine (per microgram of RNA) in both 4- or 10-somite
blastoderms is variable, but often the specific activity is even higher
117
(7 somites). Ribonuclease treatment did, however, somewhat potentiate
the inhibitory effects of another analog, 8-azaguanine.
The first inhibitor experiments on blastoderms utilized 8-azaguanine,
and the use of the analog, although apparently quite complex and far
from understood, continues to be very useful. O'Brien (1961) originally
showed that exposure of blastoderms younger than 7 somites, in vitro,
to 100 jug/ml prevented accumulation of detectable hemoglobin levels;
exposure after the 8-somite stage had little or no effect on the amount
of hemoglobin synthesis. The effect was irreversible after 1 hr of exposure, although exposure of young blastoderms to mixtures of guanine
and azaguanine showed guanine could prevent the effect of 8-azaguanine.
He concluded that 8-azaguanine was a rather specific inhibitor of the
initiation of hemoglobin synthesis prior to the 7-somite stage. At the
7-somite stage, the synthetic mechanism for hemoglobin production was
activated, but could not be maintained in the presence of 8-azaguanine;
and thereafter, Hb synthesis became refractory to the action of the
analog. The results obtained by O'Brien have been confirmed by Hell
(1964b) and Wilt (1962, 1965a). If low levels of 8-azaguanine are used,
the inhibitory effect of the analog prior to 7 somites is not as profound
and is partially reversible by subsequent culture in the presence of
guanine. Furthermore, very sensitive methods of hemoglobin detection
by colorimetric (Hell, 1964b) or immunochemical (Wilt, 1962) means
revealed small amounts of hemoglobin formation, even when 8-azaguanine is applied prior to the 7-somite stage. The mechanism of action of
8-azaguanine is an important consideration. O'Brien concluded on the
basis of the evidence available at that time that 8-azaguanine interfered
with "RNA associated mechanisms or templates for hemoglobin production" being elaborated at the 7-somite stage. He assumed that cells which
had not yet produced Hb had not reached the final stages of cytoplasmic
template formation, or in more recent terms, messenger RNA was lacking. Hell (1964b) has shown that the net accumulation of RNA and DNA
is severely blocked by 8-azaguanine, and protein synthesis is generally
reduced as much as hemoglobin formation. Furthermore, 8-azaguanine
affected protein synthesis in general, and hemoglobin synthesis in particular, to a similar extent when applied during the refractory period of
O'Brien. This inhibition was less profound (50-60%) than when it was
applied prior to the 7-somite stage (90% inhibition), but it is not an
absolute refractoriness to 8-azaguanine. Wilt showed (1966) that the
incorporation of 8-azaguanine-C
14
into acid insoluble material occurs
both before and after the 7-somite stage. The specific activity of incorporated 8-azaguanine (per microgram of RNA) in both 4- or 10-somite
blastoderms is variable, but often the specific activity is even higher