lyzed in vivo mechanisms of transcriptional regula- 
tion by the homeotic proteins UBX (Ultrabithorax) 
and ABD-A (abdominal-A). The P2 promoter of the 
Antennapedia (Antp) gene was a candidate for tar- 
get, since its expression is repressed by Ubx and 
abd-A in abdominal segments. This promoter binds 
purified UBX and ABD-A proteins at 40 sites, each 
site showing a core TAAT sequence, within 2.1 kb of 
the P2 promoter transcription start site. The func- 
tional significance of these in vitro binding sites 
was tested by creating point mutations in the core 
binding sequences, introducing the mutated pro- 
moters, fused to a reporter gene, into the fly ge- 
nome, and examining their expression patterns in 
developing embryos. 
These studies have revealed that UBX and ABD-A 
proteins use two distinct mechanisms, depending 
on cell type, to regulate this target promoter. In neu- 
ronal cells of the embryonic ventral nervous system, 
a positive transcription factor, most likely ANTP 
protein itself, binds to the consensus TAAT sites and 
activates transcription from the P2 promoter. When 
UBX and ABD-A are present in these cells, they com- 
pete for the binding sites and replace the positive 
factor, thereby turning off the promoter. 
In tracheal cells, an entirely different mechanism 
is used to repress this promoter. In these cells, UBX 
and ABD-A bind to multiple sites within the pro- 
moter and inhibit a positive transcription factor that 
acts at a distance through an independent site(s). In 
the latter mechanism, what matters for repression is 
the total number of binding sites available for UBX 
and ABD-A (>15 are required), and not specific lo- 
cations of the binding sites. This suggests a nonspe- 
cific mechanism by which the homeotic proteins 
can make a promoter inaccessible to different posi- 
tive transcription factors. 
Phenotypic Suppression 
When UBX protein is ectopically expressed 
throughout the embryo via an inducible promoter, 
head and thoracic segments assume the UBX-speci- 
fied identity, that of the first abdominal (Al) seg- 
ment; however, no transformations of abdominal 
segments posterior to Al are observed. This resis- 
tance of more-posterior segments to transformation 
by ectopic UBX protein has become known as phe- 
notypic suppression. As an explanation for this phe- 
nomenon, others have proposed a functional hierar- 
chy model: when present in the same cells, the 
homeotic proteins ABD-A and ABD-B, normally ex- 
pressed in posterior abdominal segments, override 
the effect of ectopic UBX protein. Dr. Sakonju and 
his colleagues have tested this model by ectopically 
expressing ABD-B and UBX proteins, both individu- 
ally and simultaneously, throughout the embryo. 
Contrary to the expectations of the hierarchical 
model, the ABD-B-induced phenotype (the forma- 
tion of tail structures called filzkorper) does not 
prevail over the UBX-induced phenotype. Rather, 
predominance of each phenotype depends on the 
relative number of copies for inducible Ubx 
and Abd-B genes. Therefore, competition — rather 
than a functional hierarchy — explains phenotypic 
suppression. 
To explain why ectopic UBX competes poorly in 
abdominal segments, Dr. Sakonju and his colleagues 
have proposed and tested a model in which the 
identities of the abdominal segments become refrac- 
tory to transformation by ectopic UBX because they 
are determined earlier in development than those of 
the thoracic segments. In the test, UBX protein was 
ectopically expressed at different times during 
embryogenesis. When it is expressed very early 
in development, abdominal segments become 
transformed partially by ectopic UBX. However, ec- 
topic expression at later times does not affect the 
posterior segments. These results support their 
model that the identities of more-posterior seg- 
ments are determined earlier than those of thoracic 
segments. The laboratory is currently testing a hy- 
pothesis that the time of determination is related to 
the length of transcription units for various homeo- 
tic genes. 
Isolation of Candidate Genes 
That Are Targets of Homeotic Proteins 
Homeotic genes specify identities of body seg- 
ments by regulating expression of other genes. To 
identify genes that function downstream of homeo- 
tic genes. Dr. Sakonju and his colleagues have iso- 
lated a number of genes that respond to homeotic 
gene action. Two methods have been used to isolate 
such genes. 
In the first method, a high degree of DNA se- 
quence conservation allowed isolation of two 
members of a class of transcription factors called 
POU domain genes. In other organisms, POU do- 
main genes have been shown to participate in deter- 
mining identities of specific cell types. The expres- 
sion patterns of the two Drosophila POU genes 
suggest that they respond to positional cues pro- 
vided by homeotic genes. RNA in situ analyses show 
that they are expressed in a segment-specific fashion 
in the embryonic ventral nervous system: at high 
levels in thoracic segments but low levels in abdomi- 
nal segments. In the peripheral nervous system, they 
are also expressed in segment-specific patterns in 
neurons and their support cells. In mutant embryos 
that lack the activities of homeotic genes Ubx, abd- 
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