MOLECULAR BASIS OF METAMORPHOSIS IN DROSOPHIIA 
Carl S. Thummel, Ph.D., Assistant Investigator 
Dr. Thummel's laboratory is studying the molecu- 
lar basis of metamorphosis in Drosophila melano- 
gaster. Metamorphosis is triggered at the end of lar- 
val development by an increase in the titer of the 
steroid hormone ecdysone. In response to this sig- 
nal, the imaginal tissues grow and differentiate to 
form the adult fly, while most larval tissues are 
histolyzed. 
The ecdysone-receptor protein complex controls 
these developmental changes by triggering a com- 
plex genetic cascade. The eff^ect of ecdysone on 
gene expression can be visualized by observing 
changes in the puffing pattern of the giant polytene 
chromosomes in the larval salivary glands. A de- 
tailed study of this response has been carried out 
by Michael Ashburner and his colleagues. The hor- 
mone directly induces a small set of early puffs. 
One or more of these genes are required for repres- 
sion of the early genes and induction of a large set 
of more than 100 late genes. The late genes are 
thought to play a direct role in initiating metamor- 
phosis. By isolating and characterizing the early 
ecdysone-inducible genes and determining the 
mechanism of their induction, which genes they 
regulate, and how they mediate this control. Dr. 
Thummel's laboratory hopes to clarify how ecdy- 
sone effects the developmental changes associated 
with metamorphosis. In a broader sense this proj- 
ect provides a model system for characterizing the 
role of steroid hormones in regulating gene expres- 
sion, as well as addressing the question of how gene 
hierarchies are controlled during development. 
Current work in Dr. Thummel's laboratory is fo- 
cused on an early ecdysone-inducible gene, E74, 
which is located at position 74EF in the polytene 
chromosomes, within one of the largest early puffs 
described by Ashburner. This gene was isolated in 
collaboration with Ken Burtis and C. Weldon Jones in 
the laboratory of Dr. David Hogness at Stanford Uni- 
versity 
E74 consists of three transcription units that arise 
from unique promoters and share a common poly- 
adenylation site. The E74A promoter drives the syn- 
thesis of a 60 kb primary transcript that is spliced to 
form a 6 kb mRNA. Two internal promoters, located 
40 kb downstream from the E74A promoter, direct 
the synthesis of the E74B mRNAs, 4.7 and 5.0 kb in 
length. Similar complex giant genes have been 
found in Drosophila, although only in regulatory 
genes that are known to play a central role in de- 
velopment, such as the homeotic genes Antennape- 
dia and Ultrabithorax. Transcription of the E74A 
unit is induced several orders of magnitude as a di- 
rect response to ecdysone. Furthermore, the tem- 
poral profile of E74A mRNA accumulation parallels 
the 74EF puffing response in the polytene chromo- 
somes. E74B transcription, on the other hand, does 
not appear to be induced by ecdysone directly, al- 
though these RNAs are present at some of the same 
times during development as the E74A transcript. 
The E74A and E74B transcripts each contain 
unique 5' exons joined to a common set of three 3' 
exons. This sequence arrangement leads to the syn- 
thesis of two related E74 proteins, each with a 
unique amino-terminal domain joined to a com- 
mon carboxyl-terminal domain. The amino-terminal 
domains of the E74 proteins contain regions rich in 
acidic amino acids, whereas the carboxyl terminus 
is rich in basic amino acids. These domains are sep- 
arated by a "spacer" region of repetitive amino 
acids. The apposition of separate acidic and basic 
domains is reminiscent of yeast transcriptional ac- 
tivators. Comparison of the E74 proteins to the 
protein sequence database revealed that 84 ami- 
no acids near the carboxyl terminus are 50% identi- 
cal to the protein encoded by the ets-2 proto- 
oncogene. The function of ets-2 is unknown, 
although its activity is correlated with cellular pro- 
liferation during mouse development and ets-2 pro- 
tein is localized in the nucleus. 
Dr. Thummel's laboratory has begun to charac- 
terize the biochemical properties of the E74A pro- 
tein. Protein synthesized in vitro was used to scan a 
100 kb region encompassing the E74 gene for po- 
tential binding sites. A single site, 30 kb down- 
stream from the E74A promoter, was identified. 
This fragment was also bound by E74A protein that 
was prepared through overproduction in bacteria 
and found to contain two binding sites, as deter- 
mined by DNase I footprinting. Antibodies directed 
against the E74A protein stain salivary gland nuclei 
and reveal specific binding sites in the polytene chro- 
mosomes, indicating that E74A protein also binds 
DNA in vivo. The polytene chromosome stains 
should allow the identification of genes that are po- 
tentially regulated by E74A and thus direct future 
studies to the next level in the regulatory hierarchy. 
Ecdysone is a transducer of temporal information 
in Drosophila development. The precise timing of 
gene expression in response to ecdysone is evident 
Continued 
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