The Molecular Basis of Metamorphosis 
ner and that its target sequence resembles that 
recognized by other ETS-domain proteins. By us- 
ing antibody detection techniques to localize the 
E74A protein bound to the giant polytene chro- 
mosomes, we are now moving to the next step in 
the regulatory hierarchy. A variety of binding 
sites have been identified in this manner, includ- 
ing both early and late ecdysone-induced puffs. 
These target genes will be used for biochemical 
studies to determine the function of E74A DNA 
binding. In addition, we plan to characterize 
some target genes to learn what role they might 
play in metamorphosis. 
The precise timing of the genetic response to 
ecdysone can be clearly seen in the pattern of 
pufifs that arise at the onset of metamorphosis. 
Our characterization of E74 transcription has 
provided insights into how timing can be built 
into a genetic regulatory hierarchy. Ecdysone di- 
rectly activates the E74A promoter, resulting in a 
dramatic induction of the 6-kb mRNA. This tran- 
script does not appear in the cytoplasm, how- 
ever, until 1 hour after promoter activation. This 
delay corresponds closely to the time it takes for 
RNA polymerase to traverse the 60-kb transcrip- 
tion unit, indicating that the length of the E74A 
unit functions as a timer to delay significantly the 
appearance of the encoded mRNA. The unusual 
length of the E74A primary transcript sets it apart 
from most transcription units in Drosophila, 
which are only slightly longer than the final pro- 
cessed mRNA. Ecdysone also directly activates 
the 20-kb E74B transcription unit. In agreement 
with its primary transcript length, mature E74B 
mRNA appears between 15 and 30 minutes after 
ecdysone addition. Thus the structure of the E74 
gene dictates an invariant order of appearance of 
its transcripts in response to ecdysone. The ear- 
lier appearance of E74B mRNA is enhanced by its 
activation at an approximately 25-fold lower ec- 
dysone concentration than E74A. E74B is further 
distinguished from E74A by its repression at a 
significantly higher ecdysone concentration than 
that required for its induction, close to the con- 
centration required for E74A activation. 
These regulatory properties lead to an ecdy- 
sone-induced switch in E74 expression, with an 
initial burst of E74B transcription followed by a 
burst of E74A transcription. E74A and E74B also 
show different patterns of transcription in four 
ecdysone target tissues. These studies provide a 
means to translate the profile of a steroid hor- 
mone pulse into different amounts and times of 
regulatory gene expression that, in turn, could 
direct different developmental responses in a 
temporally and spatially regulated manner. 
E74 provides a paradigm for our studies on the 
molecular basis of metamorphosis. We hope to 
use this gene to begin to understand how the hor- 
monal signal is transduced to result in specific 
morphogenetic changes. Our next step is to de- 
termine which genes are regulated by E74 and to 
characterize the products they encode, as well as 
their mode of regulation by E74. Our long-term 
goal is to isolate more early ecdysone-inducible 
genes to extend our understanding of this com- 
plex developmental process. 
448 
