Transcription Control During Early Drosophila Development 
region of the hb promoter, an Hb protein-bind- 
ing site potentially required for full anterior acti- 
vation. Mutations of this site are being tested in 
vivo for their ability to respond to hb function. In 
another region of the hb promoter, we have iden- 
tified a cluster of Hb-binding sites that may medi- 
ate the other instance of hb autoregulation at a 
later stage in development. 
Interestingly, these two examples of positive 
hb autoregulation contrast with the general re- 
pressive nature of hb. We have obtained in vivo 
evidence that, depending on the promoter con- 
text, hb can act as a positive or a negative regula- 
tor of gene expression. Finally, the hb promoter 
is also regulated by the neighboring gap gene 
Kriippel, for which two putative binding sites 
have been identified. This description and the ex- 
periments presented above may allow us to re- 
construct a functional hb promoter made exclu- 
sively of minimal response elements identified 
for the regulators of hb. 
DNA-binding Specificity 
of the Homeodomain 
Most developmental processes involve genes 
that encode a homeodomain (HD). The HD in- 
cludes a domain similar to the helix-turn-helix 
motif present in many prokaryotic DNA-binding 
transcriptional regulators. Our analysis of the 
function of the HD has led us to propose that the 
specificity changes among classes of HDs are due 
to the nature of a single amino acid at position 9 
of the recognition helix. This position is not criti- 
cal in the prokaryotic helix-turn-helix proteins, 
and indeed structural analysis of HD-DNA com- 
plexes recently showed that the HD-DNA interac- 
tion involves a new mode of recognition. In this 
mode, amino acid 9 fits in the major groove of 
DNA, in close contact with the base pairs being 
recognized. 
We have generalized the role of amino acid 9 to 
several classes of HDs. Using a powerful selection 
procedure from a library of random sequences, 
we have isolated specific sequences recognized 
by a set of mutant HD proteins carrying different 
residues at position 9. These sequences confirm 
our previous observations, but indicate that the 
different proteins interact with DNA in different 
modes. 
The paired Gene Encodes a Multifunctional 
Transcription Factor 
In addition to an HD, the paired gene product, 
the Prd protein, contains regions that are also 
found in other developmental genes. We have 
shown that the so-called Paired domain is a sec- 
ond DNA-binding domain in the Prd protein, 
which makes Prd a bifunctional transcription fac- 
tor. Although both the Paired domain and the HD 
can bind to DNA independently, they can also 
bind cooperatively to adjacent sites, but only 
when both domains are present in the same mole- 
cule. The cooperation between the two motifs 
may refine the functional specificity of genes 
containing highly related domains. 
We have undertaken an in vivo structure- 
function analysis of the product of prd. In this we 
are attempting to correlate the multiple DNA- 
binding functions of Prd defined in vitro with the 
multiple genetic functions of the prd gene, as a 
regulator of segmentation genes and of genes in 
the nervous system. Transgenic flies that carry the 
prd coding sequence driven by its own promoter 
rescue the lethal phenotype of prd" flies. Other 
lines carrying versions of the prd gene modified 
in regions encoding each of the subdomains of 
the Prd protein are now being tested for their 
ability to rescue some of the differential molecu- 
lar phenotypes of prd~ . 
From Segmentation to Organogenesis 
We recently discovered a gene that appears to 
be involved in the early steps of organogenesis of 
the salivary gland, but not in segmentation. This 
gene, highly homologous to prd, is first ex- 
pressed in a group of cells that represent the pro- 
genitor of the salivary gland placode, at a stage 
when no tissue differentiation has occurred. This 
expression is controlled by positional cues from 
the dorsoventral and anteroposterior determi- 
nants. Later, when the placode invaginates, other 
transcription factor-encoding genes are ex- 
pressed in the same tissue. Since the expression 
of this pr^Z-related gene precedes morphological 
events, it may provide an excellent model of tran- 
scriptional commitment to a particular differen- 
tiation pathway. We are now generating mutants 
to analyze the early expression of the putative 
target genes and the resulting morphological 
phenotype. 
Because the protein encoded by this gene has a 
Prd-like organization but a Prd domain from a di- 
vergent class, this molecule will also be very use- 
ful in dissecting the residues that determine the 
specificity of the Prd domain. In collaboration 
with John Kuriyan's group (HHMI, Rockefeller 
University) , we have undertaken a structural anal- 
ysis of the Prd domain and the HD present in Prd. 
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