MOLECULAR ANALYSIS OF DEVELOPMENT IN DROSOPHILA MELANOGASTER 
Shigeru Sakonju, Ph.D., Assistant Investigator 
Dr. Sakonju's laboratory is interested in under- 
standing how homeotic gene products determine 
the fate of individual cells in body segments of Dro- 
sophila melanogaster. Much of the work has fo- 
cused on abd-A and Abd-B, two homeotic genes in 
the bithorax complex that specify the identity of ab- 
dominal segments. Analysis of the abd-A and Abd-B 
gene structures has provided insights into the mo- 
lecular functions of these genes. This knowledge is 
being used to investigate the molecular mecha- 
nisms by which the homeotic proteins regulate the 
expression of their target genes. 
L Molecular Analysis of the abd-A Gene. 
The abd-A gene is required for the identity of 
parasegment (PS) 7 through PS 13 (corresponding 
to the posterior part of the first through the ante- 
rior part of the eighth abdominal segments). The 
complete gene structure of abd-A has been deter- 
mined. The gene encodes a 4.8 kb transcript de- 
rived from six exons spread over 23 kb of genomic 
DNA. Transcription of abd-A initiates from a clus- 
ter of three sites separated by a few bases. The 
abd-A transcripts contain a single long open read- 
ing frame (ORF). Translation of the ORF yields a 
36 kDa protein containing a highly conserved 
homeodomain and a cluster of glutamine residues, 
the so-called opa or M repeats. Unlike Ubx and 
Abd-B (the other two genes within the bithorax 
complex), multiple transcripts are not produced by 
alternative splicing of the abd-A transcripts. Thus 
the diverse genetic functions attributed to abd-A 
must be carried out by the sole product of this 
gene. 
II. Regulation of Transcription by the abd-A Gene 
Product. 
Homeotic gene products are assumed to function 
by binding to DNA sequences and regulating the 
expression of target genes. This assumption is de- 
rived from in vitro DNA-binding studies and from 
experiments using tissue culture cells. One goal of 
Dr. Sakonju's laboratory is to test if this assumption 
is correct in vivo during Drosophila development. 
Genetic analysis indicates that Antennapedia 
(Antp) gene expression is negatively regulated by 
abd-A in the abdominal segments of Drosophila. 
Furthermore, Dr. Anne Boulet and Dr. Matthew P 
Scott (HHMI, University of Colorado, Boulder) have 
shown that the repression by abd-A is mediated 
through 1.4 kb of DNA upstream from the Antp P2 
promoter. Hence this promoter was chosen as a po- 
tential in vitro and in vivo target site for the abd-A 
gene product. The abd-A protein was overpro- 
duced and purified from bacteria. This protein was 
first shown to immunoprecipitate a restriction frag- 
ment located ~900 bp upstream of the Antp P2 
start site. The precise location of DNA binding was 
then determined, using a footprint analysis. The 
binding site includes two stretches of the consen- 
sus sequence derived from other homeodomain- 
associated proteins. To demonstrate that this bind- 
ing site is essential for in vivo repression by abd-A, 
it is being deleted from the Antp P2 upstream DNA. 
The DNA will then be fused to (3-galactosidase, in- 
troduced using the P element, and tested to see if 
this construct escapes the repression in the abdom- 
inal segments of the wild-type Drosophila. 
III. Effects of iab-4 Mutations on the Gonad 
Development. 
Lesions in the iab-4 region of the abd-A domain 
are thought to disrupt cis-regulatory regions re- 
quired for proper regulation of the abd-A tran- 
script. The iab-4 mutants show a transformation of 
the fourth abdominal segment toward the third. In 
addition, they are sterile due to a loss of gonads. 
To define the specific step during the gonad forma- 
tion that is affected by the abd-A mutation, 
Dr. Sakonju's laboratory (in collaboration with Dr. 
Janos Szabad) has characterized the gonadal defect 
in more detail. Pole cell transplantation experi- 
ments and antibody staining of pole cells in both 
wild-type and mutant embryos indicate that 1) the 
defect is somatic, not germline, dependent, and 
2) in iab-4 mutant embryos the pole cells migrate 
normally, but the gonadal sheath that surrounds 
the pole cells is never formed. Genetic mosaic 
analysis is being carried out to determine which 
type of cells (mesodermal, ectodermal, or both) 
must be mutant to produce the mutant gonadal 
phenotype. 
lY Molecular Analysis of the Abd-B Gene. 
Genetic analysis has revealed two separable func- 
tions within the Abd-B gene: 1) the morphogenetic 
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