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 Medical Center. He 
obtained his undergraduate degree in biology from Colgate University. He received his Ph.D. degree in 
biochemistry at the University of California, Berkeley, where he worked with Robert Tjian. 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 higher eukaryotes. Three- 
quarters of a century of biological, physiological, 
and genetic experiments, combined with recent 
intensive molecular studies, have 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 a relatively 
immobile feeding larva to a highly motile, repro- 
ductively active adult fly. We are studying the 
molecular basis of the ecdysone-induced regula- 
tory mechanisms that allow metamorphosis to 
proceed. 
When the larva begins to undergo metamorpho- 
sis, its salivary glands contain giant polytene 
chromosomes, which can be visualized by light 
microscopy. These 500-fold overreplicated, in- 
terphase chromosomes lie in register beside one 
another. A characteristic banding pattern along 
the length of the polytene chromosomes allows 
any gene of interest to be located precisely. Re- 
gions of the genome that are being actively tran- 
scribed are often represented by large regions of 
decondensed chromatin that can be seen as puff's 
in these chromosomes. Thus the transcriptional 
activity of specific genes at specific times can be 
followed 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 puffs remain 
until the end of larval development, when the 
burst of ecdysone triggers a dramatic change in 
the puffing pattern coincident with the onset of 
metamorphosis. 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 more than 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, as well as addressing the 
question of how gene hierarchies are controlled 
during development. 
Our current studies center around E74, an ec- 
dysone-inducible gene that is located within the 
large early puff at position 74EF in the polytene 
chromosomes. This unusually complex gene en- 
codes three nested mRNAs that derive from 
unique start sites but share a common 3' end. The 
distal promoter 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 
amino-terminal domains are rich in acidic amino 
acids, whereas the common carboxyl-terminal 
domain is rich in basic amino acids. This charge 
distribution is reminiscent of yeast transcrip- 
tional activators and thus consistent with the no- 
tion that E74 may encode regulatory proteins. In 
addition, the sequence of the carboxyl terminus 
of the E74 proteins is very similar to a portion of 
the protein encoded by the els oncogene. This 
85-amino acid ETS domain defines a family of 
proteins and has been shown to function as a site- 
specific DNA-binding domain that recognizes a 
purine-rich DNA sequence. Studies of oncogene- 
related proteins, such as E74, may help us learn 
more about how the normal counterparts of these 
disease genes function during development. 
Biochemical analysis of the E74A protein has 
revealed that it binds DNA in a site-specific man- 
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