Dr. Michael Ashburner and his colleagues, these 
puffs appear to encode regulatory proteins that both 
repress their own expression and induce the forma- 
tion of more than 100 secondary-response "late" 
puffs. Dr. Thummel's laboratory is studying this ge- 
netic regulatory hierarchy at the molecular level. 
By isolating and characterizing the ecdysone- 
inducible genes encoded within the early puff loci, 
and determining which genes they regulate and how 
they mediate this control, Dr. Thummel's laboratory 
hopes to clarify how ecdysone effects the develop- 
mental changes associated with metamorphosis. In a 
broader sense this project provides a model system 
for characterizing the role of steroid hormones in 
regulating gene expression as well as addressing the 
question of how regulatory hierarchies are con- 
trolled during development. 
Much of Dr. Thummel's research effort is focused 
on defining the regulation and function of early 
gene expression. Three early puff loci located at po- 
sitions 2B5, 74EF, and 75B in the polytene chromo- 
somes have been characterized at the molecular 
level. The genes corresponding to these puffs are 
designated the Broad- Complex (BR-C), E74, and 
E75, respectively. In addition, the ecdysone recep- 
tor, EcR, has been isolated and characterized in Dr. 
David Hogness's laboratory (Stanford University). 
These four genes share several features in common. 
First, they are unusually long for Drosophila, span- 
ning 60- 1 00 kb of DNA. Second, they contain multi- 
ple nested promoters that are directly activated by 
ecdysone. Finally, they all encode site-specific 
DNA-binding proteins, consistent with their pro- 
posed regulatory functions. Dr. Thummel's labora- 
tory has shown that the unusual lengths of these 
genes, combined with their induction by differ- 
ent threshold ecdysone concentrations, contrib- 
ute to their precise temporal regulation during 
development. 
The control of E74 transcription typifies the early 
genetic response to ecdysone. This gene encodes 
two transcripts, designated E74A and E74B, from 
unique promoters. E74A transcription is induced 
several orders of magnitude as a direct response 
to ecdysone and is subsequently repressed by 
ecdysone-induced proteins, as predicted for an early 
response. It is transcribed at a rate of ~ 1 . 1 kb/min. 
This rate measurement, combined with the 60-kb 
length of the E74A unit, accounts for most of the 1 -h 
delay seen between the time ecdysone activates the 
E74A promoter and the appearance of cytoplasmic 
E74A mRNA. Thus the unusual length of the E74A 
transcription unit has a regulatory function, acting 
as a timer that delays the synthesis of its encoded 
gene product. 
In contrast, E74B is designed to be expressed rap- 
idly upon hormonal stimulation. The E74B pro- 
moter is activated by an ~ 20-fold lower ecdysone 
concentration than that required for E74A induc- 
tion. In addition, the £745 transcription unit is one- 
third the length of E74A's, leading to only a 20-min 
transcriptional delay time. These two factors deter- 
mine that E74B expression will always precede that 
of E74A in response to an ecdysone pulse. Further- 
more, E74B transcription is repressed as E74A is 
induced, leading to an ecdysone-regulated switch in 
the expression of these two DNA-binding proteins. 
Similar mechanisms control the timing of E75, 
BR-C, and ii'c/? transcription. For example, the E75 
gene consists of three nested promoters that direct 
the synthesis of 20-, 50-, and 100-kb primary tran- 
scripts. At high ecdysone concentrations, the 
mRNAs derived from these transcripts appear in an 
order that is consistent with the lengths of their 
transcription units. Furthermore, the regulation of 
each early promoter, with one exception (E75C), 
can be accounted for by its being activated at one of 
two critical threshold ecdysone concentrations, sim- 
ilar to the two concentrations required for E74A and 
E74B induction. These activating ecdysone concen- 
trations are consistent with the temporal order of 
early gene induction during third instar larval devel- 
opment, suggesting that a gradual increase in ecdy- 
sone titer triggers the sequential activation of each 
early mRNA, leading up to the onset of metamorpho- 
sis. Thus the regulatory effects of ecdysone are not 
dependent solely upon its peak concentrations. 
Rather, the profile of the hormone pulse contains 
critical temporal information that is transduced into 
waves of DNA-binding proteins that could act in a 
combinatorial fashion to control the timing of sub- 
sequent steps in the regulatory hierarchy. 
Genetic studies have revealed that this precise se- 
quential induction of early gene expression is criti- 
cal for an appropriate regulatory response to the 
hormone. Dr. Thummel's laboratory, in collabora- 
tion with Dr. Greg Guild (University of Pennsyl- 
vania), has shown that mutations in the BR-C early 
gene result in reduced ecdysone induction of some 
E74 and E75 mRNAs. BR-C is expressed before these 
E74 and E75 target promoters are activated, sug- 
gesting that the 6J?-C proteins form part of the tran- 
scriptional machinery that is required for subse- 
quent E74 and E75 induction. Genetic studies of 
E74A in Dr. Thummel's laboratory have shown that 
the later induction of this mRNA, just preceding pu- 
parium formation, has no effect on early gene activ- 
ity but rather plays a key role in timing late gene 
induction. In the absence of E74A function, many 
late puffs are either reduced or absent. Dr. Thum- 
GENETICS 273 
