Homeotic Gene Action in Drosophila 
from a large population of molecules that in- 
cludes all possible sequence combinations. 
This method has been used to determine and 
compare the optimal binding sites for two closely 
related homeodomain peptides encoded by the 
Drosophila homeotic genes Ultrabithorax 
(Obx) and Deformed {Dfd). The optimal sites 
for these proteins hold in common a core se- 
quence, TAAT, that is very important for binding. 
Additional bases to either side of the TAAT core 
are also recognized, but these base preferences 
dilfer between the two proteins. Comparative 
studies indicate 5- to 10-fold relative preferences 
of Dfd and Ubx homeodomains for their own op- 
timal sites. Additional studies are under way to 
determine which of the amino acid sequence dif- 
ferences between Ubx and Dfd are responsible 
for the differences in DNA recognition. Addi- 
tional insight into the nature of the critical molec- 
ular interactions should come from a crystallo- 
graphic study in collaboration with Carl Pabo 
(HHMI) and Neil Clarke, at the Johns Hopkins 
University, of a Ubx homeodomain complex with 
its optimal binding site. 
In vivo studies with the full-length proteins 
indicate that their DNA sequence preferences are 
similar to those of the homeodomain peptides. 
The full-length C/fojtr protein, however, unlike the 
f/fcjc homeodomain peptide, binds cooperatively 
to multiple sites. This cooperativity can increase 
complex stability from less than a minute for a 
single isolated site to several hours for four or 
more sites. 
The most striking property of these coopera- 
tive interactions is that they can occur between 
sites separated by a variety of distances, ranging 
from a single helical turn of the DNA to more than 
20 turns. The long-range interactions involve a 
mechanism in which the intervening DNA is 
curved to permit contact between proteins 
bound at the base of a resulting DNA loop. The 
affinity for binding of full-length Ubx protein is 
therefore a function of the number of sites pres- 
ent within a DNA fragment, and Ubx proteins ap- 
pear to act as integrators of the number and affin- 
ity of core binding site sequences. 
With regard to differential gene regulation, 
these results suggest the following: first, differ- 
ences in DNA sequence recognition account for 
at least some of the biological specificity of ho- 
meotic genes; second, the molecular basis for dis- 
crimination between homeotic protein-binding 
sites may be interactions with bases flanking a 
common TAAT core; third, large differential af- 
finities for a DNA region might be generated by 
cooperative binding of homeotic proteins to col- 
lections of core sites that individually show only 
small differences in affinity. 
Identification of Ubx Regulatory Targets 
We have used a method similar to the one for 
determination of optimal DNA binding sites to 
select and clone DNA fragments containing Ubx 
binding sites from total Drosophila genomic 
DNA. We expect that genes regulated by Z/fejc will 
be located near such binding sites. We have also 
identified sequences from Drosophila cultured 
cell lines that are differentially expressed in the 
presence and absence of Ubx protein. The se- 
quences isolated by these procedures are being 
analyzed to determine how they function in the 
embryo. 
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