Regulation of Gene Expression in Steroid Hormone Biosynthesis 
subsequently defined two mouse 1 1 jS-hydroxy- 
lase genes located on chromosome 1 5 . Although 
the overall structures of the two genes are quite 
similar, selected regions diverge significantly, 
suggesting that the proteins encoded by these 
two genes might have different activities. More- 
over, the 5'-flanking regions of the two genes di- 
verge significantly, suggesting that their regula- 
tion may differ. 
To assess the enzymatic activities of the pro- 
teins produced by these two genes, we analyzed 
their activities following gene transfer into COS- 
7 cells, which normally do not make any steroid- 
metabolizing enzymes. One of the two proteins, 
designated 1 l/S-hydroxylase, was able to produce 
glucocorticoids but could not make mineralocor- 
ticoids. In contrast, the other protein, designated 
aldosterone synthase, performed all of the reac- 
tions needed to make aldosterone. Thus there are 
significant differences in the enzymatic activities 
of the two mouse 1 1 |S-hydroxylase homologues. 
To see where these two proteins are made in 
the adrenal cortex, we used the technique of in 
situ hybridization to examine sites of expression 
in the sections of the mouse adrenal gland. The 
aldosterone synthase protein is only present in 
the outer zone of the cortex, where mineralo- 
corticoids are made. In contrast, the 11(8- 
hydroxylase protein is only present in the sites of 
glucocorticoid production. These results docu- 
ment an exquisite coupling between enzymatic 
activities and sites of expression. The ability to 
maintain this segregation of the two proteins is 
undoubtedly an important part of the adrenal 
gland's ability to regulate separately the produc- 
tion of mineralocorticoids and glucocorticoids. 
Based on these results, we plan to use these 
promoter regions in transgenic mice to target 
gene expression to specific cortical zones. Ini- 
tially we are using the two promoters to direct the 
expression of renin, a gene previously shown to 
cause genetic hypertension when expressed in 
multiple tissues of transgenic animals. If success- 
ful, these experiments will validate the zone-spe- 
cific expression of the two promoter regions and 
will establish that adrenal expression of renin is 
relevant to this hypertensive state. 
We will also express the aldosterone synthase 
protein in the inner, glucocorticoid-producing 
zone. To this end, we have prepared a hybrid 
gene with the inner-zone-specific promoter 
driving expression of the aldosterone synthase 
gene. Based on the gene transfer experiments de- 
scribed above, we anticipate that this hybrid gene 
will synthesize large amounts of mineralocorti- 
coids, thereby creating a genetic form of hyper- 
tension. Moreover, treatment with glucocorti- 
coids should alleviate the hypertension by 
suppressing the expression of the hybrid gene in 
the inner zone. Recent studies strongly suggest 
that just such a mechanism is responsible for a 
subset of human patients with a glucocorticoid- 
remediable hypertension. 
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