The Molecular Basis of Metamorphosis 
Carls. Thummel, Ph.D. — Assistant Investigator 
Dr. Thummel is also Assistant Professor of Human Genetics at the University of Utah School of Medicine. 
He obtained his undergraduate degree in biology from Colgate University and his Ph.D. degree in 
biochemistry, working with Robert Tjian, at the University of California, Berkeley. He received 
postdoctoral training in the laboratory of David Hogness at Stanford University. 
THE fruit fly Drosophila melanogaster pro- 
vides an ideal model system for studying the 
development of eukaryotes. Three-quarters of a 
century of biological, physiological, and genetic 
experiments, combined v^^ith recent intensive mo- 
lecular studies, has led to a greater understanding 
of its development than that of any other higher 
organism. 
Halfway through the fly's life cycle, a pulse of 
the steroid hormone ecdysone triggers a dramatic 
morphological transformation, from the rela- 
tively immobile feeding larva to a highly motile, 
reproductively active adult fly. We are studying 
the molecular basis of the ecdysone-induced reg- 
ulatory mechanisms that allow metamorphosis to 
proceed. 
When the larva begins to undergo metamorpho- 
sis, its salivary glands contain giant polytene 
chromosomes that can be visualized by light mi- 
croscopy. These 500-fold overreplicated, inter- 
phase chromosomes lie in register beside one an- 
other. A characteristic banding pattern along the 
length of the polytene chromosomes allows any 
gene of interest to be located precisely. Regions 
of the genome that are undergoing transcription 
are often represented by large areas of decon- 
densed chromatin that can be seen as puffs. Thus 
the transcriptional activity of specific genes at 
specific times can be followed in these chromo- 
somes by observing the appearance and disap- 
pearance of puff^s during development. 
Approximately 10 puffs can be distinguished 
when the salivary gland chromosomes first be- 
come large enough to see. These puff's remain 
until the end of the larval phase, when the burst 
of ecdysone triggers a dramatic change in the 
puffing pattern. Approximately six puffs are in- 
duced directly by the steroid hormone. These 
early ecdysone-inducible puffs appear to encode 
regulatory proteins that repress their own expres- 
sion and induce the formation of over 100 late 
puffs. This second wave of puffs is believed to 
encode the proteins responsible for initiating 
metamorphosis. 
By isolating and characterizing the ecdysone- 
inducible genes that lie within the early puffs, we 
hope to learn how these genes are induced by the 
hormone and how their encoded proteins might 
function in a regulatory capacity. In a broader 
sense, this project provides a model system for 
characterizing the role of steroid hormones in reg- 
ulating gene expression and for addressing the 
question of how gene hierarchies are controlled 
during development. 
Our studies have focused on E74, an ecdysone- 
inducible gene that is located within the large 
early puff at position 74EF in the polytene chro- 
mosomes. This unusually complex gene encodes 
three nested mRNAs that derive from unique start 
sites but share a common 3' end. The distal pro- 
moter directs the synthesis of a 60-kb primary 
transcript that is spliced to form the 6-kb E74A 
mRNA. Two other promoters, located 40 kb 
downstream from the E74A promoter, direct the 
synthesis of 4.8- and 5.1-kb E74B mRNAs. Al- 
though the E74A and E74B mRNAs are distinct 
from one another by virtue of their unique 5' 
exons, the majority of these mRNAs are identical, 
derived from a common set of three 3' exons. This 
nested arrangement of the E74 transcripts leads 
to the synthesis of two related E74 proteins that 
have unique amino-terminal domains joined to a 
common carboxyl-terminal domain. 
The sequence of the carboxyl terminus of the E74 
proteins is very similar to a portion of the protein 
encoded by the ets oncogene. This 85-amino acid 
ETS domain defines a family of proteins and has been 
shown to function as a site-specific DNA-binding do- 
main that recognizes a purine-rich DNA sequence. 
Studies of proteins related to oncogenes, such as 
E74, may help us learn more about how the normal 
counterparts of these disease genes function during 
development. 
By using antibody detection techniques to lo- 
calize the E74A protein bound to the giant poly- 
tene chromosomes, we have identified approxi- 
mately 70 binding sites, most of which 
correspond to late ecdysone-inducible puffs. 
Based on this observation, we predict that at least 
one function for the E74A protein is to activate 
late gene expression. In support of this predic- 
tion, many late puffs are either reduced or absent 
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