The Sex combs reduced locus 
Scr establishes the identity of the labial and pro- 
thoracic segments and is expressed primarily in the 
ectoderm. Dr. Kaufman and his colleagues are using 
Scr clones to investigate the molecular basis of Scr 
regulation through the identification of positive and 
negative cis-acting control elements. To localize 
these elements, the 80-kb upstream regulatory re- 
gion and transcribed portions of the gene have been 
divided into 18 overlapping subfragments. These 
were subcloned into P-element vectors containing 
either an hsplO-lacZ or Scr-lacZ chimera, and fu- 
sion gene expression was assayed in transformant 
embryos. 
The largest of the 5cr-/acZ fusions contains 2.3 kb 
of upstream sequence as well as the first exon and 
intron of the gene. This construct is expressed over a 
domain that includes the intercalary segment anteri- 
orly and the third thoracic segment posteriorly. Ad- 
dition of a specific fragment normally located more 
than 30 kb upstream serves to truncate this ectopic 
pattern to one more reflective of the normal Scr 
pattern. 
In addition, several DNA fragments can be shown 
to direct patterns of fusion gene expression, de- 
pending on whether they are upstream of the heter- 
ologous hsplO or homologous Scr promoter. This 
suggests that there may be promoter-specific in- 
teractions between regulatory elements in this 
interval. 
Genetic evidence suggests that Scr regulation 
during larval development is sensitive to homo- 
logue pairing. Three DNA fragments from the Scr 
regulatory interval have been isolated that, when in 
a P-element vector containing a white minigene, in- 
duce the mosaic repression of white expression in 
the eye. This repression is either enhanced or only 
observed when transformant lines are made homo- 
zygous. This result suggests that these DNA frag- 
ments contain pairing-sensitive negative regulatory 
elements and likely house the genomic sequences 
that are responsible for the observed "transvection" 
at the Scr locus. The mosaic white expression ap- 
pears to be set during the first larval instar and main- 
tained until the eye is pigmented in the pupal stage. 
Moreover, the degree of mosaicism depends on 
the dosage of Polycomb-group and trithorax-group 
gene products. This interaction has been shown to 
depend on the paired state of the transgene. There- 
fore it would appear that the pairing-sensitive regu- 
latory elements present in these DNA fragments in- 
teract with genes necessary for the maintenance of 
the normal patterns of homeotic gene expression 
and that the identified fragments are likely relevant 
to normal 5cr regulation. (This work is supported by 
a grant from the National Institutes of Health.) 
Dr. Maureen Gorman has augmented these analy- 
ses by constructing a set of Scr minigenes. These 
behave like the reporter constructs described 
above, in that they exhibit ectopic patterns of ex- 
pression. It was surprising, therefore, when stable 
transformed lines were recovered, since expression 
of the Scr protein product outside its normal do- 
main should have deleterious effects. Surviving 
transgenic adults, however, do show homeotic 
transformations of the thorax and head capsule that 
mimic the phenotype of animals bearing heat-shock 
Scr constructs subjected to specific heat-shock re- 
gimes. 
The proboscipedia locus 
During embryogenesis, pb is expressed in cells of 
the mesoderm, maxillary lobes, labial lobes, and 
ventral nerve cord. In imaginal tissues, pb accumu- 
lates in cells of the CNS and the labial discs. Null 
mutations of pb that result in the transformation of 
labial palps to first thoracic legs were rescued with a 
pb minigene constructed from genomic sequences 
that lie within the genetically defined limits of the 
locus. Reporter gene constructs containing the first 
exon and upstream regions from the minigene do 
not accumulate /3-galactosidase in a pb pattern. How- 
ever, when a 1.6-kb region from intron 2 is placed 
within the 7.3 bp of upstream sequence, the vector 
is competent to direct jS-galactosidase in a pb pat- 
tern in both embryos and third instar larvae. 
The expression patterns of a series of minigene 
deletion constructs were examined to further the 
dissection of pb regulatory sequences. Two internal 
deletion lines were made that remove sequences 
within the intron 2 region. Additionally two 5' dele- 
tion lines were created. One removes sequences up- 
stream of 1 .6 kb; a second deletes all sequences ex- 
cept 90 bp upstream of the transcription start site. 
The two 5' deletion minigene lines rescue the pb 
homeotic phenotype and accumulate wild-type lev- 
els of pb protein in the labial discs. Therefore se- 
quences upstream of —90 bp are apparently not 
needed to activate pb in the correct spatial pattern 
in this imaginal tissue. However, both 5' deletion 
minigenes additionally exhibit ectopic pb accumu- 
lation in the eye-antennal disc, suggesting that the 
sequences deleted are important for repressing 
pb in regions outside the wild-type domain. The 
— 90-bp 5' deletion line additionally directs pb pro- 
tein ectopically in the leg discs. Therefore the re- 
gion between -1 .6 kb and -90 bp seems to be re- 
quired to repress pb in the legs. 
208 
