The Molecular Basis of Metamorphosis 
in the polytene chromosomes of mutant animals 
that do not express E74A protein. Our long-term 
goal is to identify some of these late genes in 
order to determine what role they might play 
during metamorphosis and whether they are di- 
rectly regulated by E74A. 
The precise timing of the genetic response to 
efcdysone can be clearly seen in the pattern of 
puffs 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, however, does not appear in the cyto- 
plasm until one hour after promoter activation. 
The delay corresponds quite closely to the time it 
takes for RNA polymerase to traverse the 60-kb 
transcription unit, indicating that the length of 
the .£"74^1 unit functions as a timer to delay signifi- 
cantly the appearance of the encoded mRNA. The 
unusual length of the E74A primary transcript 
sets it apart from most transcription units in Dro- 
sopfoila, which are only slightly longer than the 
final processed mRNA. 
Ecdysone also directly activates the 20-kb 
E74B transcription unit. In agreement with its 
primary transcript length, mature E74B mRNA 
appears between 1 5 and 30 minutes after ecdy- 
sone addition. Thus the structure of the E74 gene 
dictates an invariant order of appearance of its 
transcripts in response to ecdysone. 
The earlier appearance of E74B mRNA is en- 
hanced by its activation at an approximately 25- 
fold lower ecdysone concentration than E74A. 
E74B is further distinguished from E74A by its 
repression at a significantly higher ecdysone con- 
centration than that required for its induction, 
close to the concentration required for E74A ac- 
tivation. These regulatory properties lead to an 
ecdysone-induced switch in E74 expression, 
with an initial burst of E74B transcription fol- 
lowed by a burst of E74A transcription. These 
studies provide a means to translate the profile of 
a steroid hormone pulse into different amounts 
and times of regulatory gene expression that, in 
turn, could direct different developmental re- 
sponses in a temporally and spatially regulated 
manner. 
To date, E74 has provided a valuable paradigm 
for our molecular characterization of the ecdy- 
sone regulatory hierarchy. We have recently be- 
gun to extend our studies to include three other 
early genes, all of which, like E74, are unusually 
long and encode multiple DNA-binding proteins. 
Characterization of the temporal regulation of 
these early genes, like that described above for 
E74, has confirmed that both promoter sensitiv- 
ity to ecdysone and primary transcript length con- 
tribute to the timing of early gene activation. 
In addition, these studies have allowed us to 
divide the early transcription units into two 
classes. One class of transcripts is activated by a 
low ecdysone concentration, like E74B, and ap- 
pears to play earlier roles in the regulatory hierar- 
chy. This class includes transcripts encoding the 
ecdysone receptor, needed for initiating the ge- 
netic response to the hormone. The second class, 
typified by E74A, is activated by a higher hor- 
mone concentration and appears to play a later 
role in the hierarchy. 
In addition, analysis of the effects of early gene 
mutations on both early and late gene expression 
should provide valuable clues regarding func- 
tional regulatory interactions within the hierar- 
chy. The predictions that arise from these genetic 
studies can then be tested at the molecular level. 
An additional long-term goal v/ill be to isolate 
more early and late ecdysone-inducible genes to 
extend our understanding of this complex devel- 
opmental process. 
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