Structural Determinants of Human a-Globin Gene Expression 
man transgenes contain the necessary informa- 
tion for appropriate developmental control and 
2) the factors responsible for developmental 
switching in mouse red cells have been suffi- 
ciently conserved during evolution to substitute 
in the control over the human transgenes. By gen- 
erating transgenic mice that carry only the human 
a-globin gene in the absence of the f-globin gene, 
we have further demonstrated that the appro- 
priate expression of this gene is not dependent on 
competition between the two developmentally 
specific genes. By introducing into the mouse ge- 
nome more limited gene fragments, as well as 
genes with specific alterations, and by studying 
their pattern of developmental expression in the 
transgenic model system, we should be able to 
define the signal (s) critical to globin gene 
switching. 
Selective stabilization of globin mRNA is essen- 
tial to its accumulation in terminally differentiat- 
ing red cells to 95 percent of total cellular mRNA. 
We are attempting to define the structural basis 
for the stability of a-globin mRNA through inves- 
tigation of an a-thalassemia mutation in which 
this stability is lost: a Constant Spring (aCS) . This 
mutation, the most common cause of nondele- 
tional a-thalassemia in Southeast Asia, is a CAA for 
UAA substitution at the normal termination codon 
of the a2-globin gene. As a result of this single- 
base substitution, the ribosome translates into the 
normally untranslated 3' region and destabilizes 
the mRNA. 
To study the basis for this destabilization in de- 
tail, we have established an experimental system 
that reproduces the selective instability of aCS 
mRNA in tissue culture cells. Remarkably, we 
find that the instability of the aCS mRNA relative 
to normal a-globin mRNA observed in the patient 
is recapitulated faithfully when the a- and aCS- 
globin genes are expressed in an erythroid tissue 
culture cell line. In contrast, we find that the 
mRNAs from these two genes are expressed at 
equivalent levels when expressed in nonery- 
throid cell lines. These data suggest that the sta- 
bility of globin mRNA may depend on interaction 
with one or more erythroid-specific factors. 
By specifically altering the structure of the a- 
globin genes prior to expression, we have demon- 
strated that the region critical to the stability of 
the a-globin mRNA is located in a segment of the 
3'-nontranslated region just past the translation 
termination codon. This determinant can be de- 
stabilized by the translating ribosome if it is al- 
lowed to enter this region, as is the case in the 
aCS mutation, or by site-specific alterations in 
the primary sequence of this segment. These data 
suggest that it should now be possible to map the 
limits of this determinant and subsequently iden- 
tify critical cellular factors mediating this 
response. 
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