In Drosophila, the homeotic gene proboscipedia (pb) is required for the formation of the adult mouthparts. To determine the functional significance of putative pb regulatory DNA, we have performed an in vivo analysis of sequences upstream of and within pb using a series of minigenes. Additionally, we have initiated a dissection of pb's promoter and enhancer elements using lacZ reporter gene constructs. Our results establish that a conserved region located in the second intron is essential for proper formation of the adult mouthparts. A 0.5 kb fragment from this region was shown to direct lacZ expression in a pb pattern in both embryos and third instar labial discs when combined with a 600 bp pb basal promoter sequence. A 32 bp element contained within the 0.5 kb region functions as a labial disc enhancers for pb. Surprisingly, the conserved second intron pb enhancers do not function properly with a heterologous hsp70 promoter, suggesting that promoter-specific interactions occur at the pb locus. We also found redundant and cryptic enhancers in the large introns of pb that are not required for pb function. Finally, we demonstrate that the pb transcription unit does not require sequences upstream of −98 bp to provide pb function in the labial discs. Rather, pb's upstream DNA appears to contain negative regulatory DNA required for silencing PB accumulation in inappropriate domains of third instar imaginal discs. Thus, we have defined many of pb's cis-controlling sequences to an experimentally manageable size, thereby making this an attractive system for the discovery of transacting proteins and, consequently, for elucidating the mechanisms of homeotic gene regulation.

Biggin
M. D.
,
Tijian
R.
(
1989
)
Transcription factors and the control of Drosophila development.
TrendsGenet
5
,
377
383
Busturia
A.
,
Bienz
M.
(
1993
)
Silencersin Abdominal-B, a homeotic Drosophila gene.
EMBOJ
12
,
1415
1425
Castelli-Gair
J.
,
Muller
J.
,
Bienz
M.
(
1992
)
Function of an Ultrabithorax minigene in imaginal cells.
Development
114
,
877
886
Chouinard
S.
,
Kaufman
T. C.
(
1991
)
Control of expression of the homeotic labial (lab) locus of Drosophilamelanogaster: evidence for both positive and negative autogenous regulation.
Development
113
,
1267
1280
Cribbs
D. L.
,
Benassayag
C.
,
Randazzo
F. M.
,
Kaufman
T. C.
(
1995
)
Levels of homeotic protein function can determine developmental identity: evidence from low-level expression of the Drosophila homeotic gene proboscipedia under Hsp70 control.
EMBO J
14
,
767
778
Cribbs
D. L.
,
Pultz
M. A.
,
Johnson
D.
,
Mazzula
M.
,
Kaufman
T. C.
(
1992
)
Structural complexity and evolutionary conservation of the Drosophila homeotic gene proboscipedia.
EMBOJ
11
,
1437
1449
Duncan
I.
(
1987
)
The Bithorax Complex. Annu. Rev.
Genet
21
,
285
319
Eissenberg
J. C.
,
Elgin
S. C. R.
(
1991
)
Boundary function in the control of gene expression.
TrendsGenet
7
,
335
340
Huang
J.
,
Schwyter
D. H.
,
Shirokawa
J. M.
,
Courey
A. J.
(
1993
)
The interplay between multiple enhancer and silencer elements defines the pattern of decapentaplegic expression.
GenesDev
7
,
694
704
Kassis
J. A.
(
1990
)
Spatial and temporal control elements of the Drosophila engrailed gene.
GenesDev
4
,
433
443
Kaufman
T. C.
(
1978
)
Cytogenetic analysis of chromosome 3 in Drosophila melanogaster: Isolation and characterization of four new alleles of the proboscipedia (pb) locus.
Genetics
90
,
579
596
Levis
R.
,
Hazelrigg
T.
,
Rubin
G. M.
(
1985
)
Effects of genomic position on the expression of transduced copies of the white gene of Drosophila.
Science
229
,
558
561
Lewis
E. B.
(
1978
)
A gene complex controlling segmentation in Drosophila.
Nature
276
,
565
570
Mahaffey
J. W.
,
Kaufman
T. C.
(
1987
)
Distribution of the Sexcombs reduced gene products in Drosophilamelanogaster.
Genetics
117
,
51
60
Merrill
V. K. L.
,
Turner
F. R.
,
Kaufman
T. C.
(
1987
)
A genetic and developmental analysis of mutations in the Deformed locus in Drosophila melanogaster. Dev.
Biol
122
,
379
395
Muller
J.
,
Bienz
M.
(
1991
)
Long range repression conferring boundaries of Ultrabithorax expression in the Drosophila embryo.
EMBOJ
10
,
3147
3155
Parvin
J. D.
,
Timmers
H. T. M.
,
Sharp
P. A.
(
1992
)
Promoter specificity of basal transcription factors.
Cell
68
,
1135
1144
Pattatucci
A. M.
,
Kaufman
T. C.
(
1991
)
The homeotic gene Sexcombs reduced of Drosophilamelanogaster is differentially regulated in the embryonic and imaginal stages of development.
Genetics
129
,
443
461
Pultz
M. A.
,
Diederich
R. J.
,
Cribbs
D. L.
,
Kaufman
T. C.
(
1988
)
The proboscipedia locus of the Antennapedia Complex: a molecular and genetic analysis.
GenesDev
2
,
901
920
Randazzo
F. M.
,
Cribbs
D. L.
,
Kaufman
T. C.
(
1991
)
Rescue and regulation of proboscipedia: a homeotic gene of the Antennapedia Complex.
Development
113
,
257
271
Regulski
M.
,
Dessain
S.
,
McGinnis
N.
,
McGinnis
W.
(
1991
)
High affinity binding sites for the Deformed protein are required for the function of an autoregulatory enhancer of the Deformed gene.
GenesDev
5
,
278
286
Robertson
H. M.
,
Preston
C. R.
,
Phissis
R. W.
,
Johnson-Schlitz
D. M.
,
Benz
W. K.
,
Engels
W. R.
(
1988
)
A stable genomic source of P-element transposase in Drosophilamelanogaster.
Genetics
118
,
461
470
Scott
M. P.
,
Weiner
A. J.
,
Hazelrigg
T. I.
,
Polisky
B. A.
,
Pirrotta
V.
,
Scalenghe
F.
,
Kaufman
T. C.
(
1983
)
The molecular organization of the Antennapedia locus of Drosophila.
Cell
35
,
736
776
Smale
S. T.
,
Baltimore
D.
(
1989
)
The ‘initiator’ as a transcriptional control element.
Cell
57
,
103
113
Wilson
C.
,
Pearson
R. K.
,
Bellen
H. J.
,
O'Kane
C.
,
Grossniklaus
U.
,
Gehring
W. J.
(
1989
)
P-element-mediated enhancer detection: an efficient method for isolating and characterizing developmentally regulated genes in Drosophila.
GenesDev
3
,
1301
1313
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