The Enzymes Causing Thyroid Hormone Activation 
as the starting source for preparing a DNA library 
for this purpose. 
The preparation of cDNA from the liver of the 
hyperthyroid rat was size selected because stud- 
ies showed that it was approximately 2,000 nu- 
cleotides in length. Many cDNA clones of this size 
were separated into pools of approximately 
2^000 each. Then the normal process that is 
carried out in the cell was reproduced in the test 
tube. Messenger RNA was synthesized from each 
of the pools of these cDNA clones, injected into 
Xenopus oocytes, and the oocytes were analyzed 
three days later for the deiodinase activity. 
One pool produced a significant quantity of 
deiodinase and was therefore subdivided further. 
Pools of approximately 100 cDNAs were again 
studied by transcribing mRNA, and this was in- 
jected in turn into the Xenopus. By this tech- 
nique a positive pool of about 100 of these 
clones was isolated. These were diluted and 
grown up as single colonies in a matrix arrange- 
ment. The RNA was prepared from pools of these 
clones from rows and columns, and an intersec- 
tion was identified that corresponded to the 
clone that coded for the deiodinase. As antici- 
pated, the clone was approximately 2,100 nu- 
cleotides in base pairs in length. Surprisingly, 
there was a group of three nucleotides, TGA (re- 
ferred to as a codon), in the clone that corre- 
sponded to the signal for the protein synthesis 
machinery to stop. This codon, however, did not 
stop the synthesis of protein for this particular 
mRNA. We were able to show that in the case of 
this protein the stop codon TGA (UGA in the 
mRNA) coded for an extremely rare amino acid, 
selenocysteine. This amino acid is similar to the 
more common amino acid cysteine, except that 
selenium replaces the sulfur atom. Selenium is in 
the same category in the periodic table of chemi- 
cal elements as is sulfur, but its chemistry differs 
from that of sulfur in a number of important re- 
spects. Only one other enzyme previously identi- 
fied in animals and plants contains a selenocys- 
teine. The role of this enzyme — glutathione 
peroxidase — is to reduce the peroxide that accu- 
mulates in various cells and damages cell 
membranes. 
In the deiodinase, the selenium atom donates 
the electron that permits the removal of iodine 
from the thyroxine molecule to activate it. The 
chemical nature of the reaction — reduction of 
substrate and oxidation of the enzyme — is well 
known in cellular biochemistry. We were able to 
prove that this amino acid containing selenium is 
essential for the normal deiodination process, 
which explains the recent observation that a defi- 
ciency of selenium (which is present as a trace 
element in the eanh's crust) causes hypothyroid- 
ism in experimental animals. We conclude that 
selenium is required for normal thyroid hormone 
action. 
Our current research is directed at understand- 
ing why this particular mRNA is able to permit 
the suppression of the normal stop codon and 
therefore produce this unique enzyme. We are 
also investigating the biochemistry of the normal 
and mutated deiodinase molecule, so that we may 
understand more about its physiological function 
and deiodination mechanism. 
Our laboratory continues to investigate the 
function of the nuclear protein to which T, binds 
and the DNA sequences that recognize it. With 
these two eflforts, we hope to have a complete 
picture of how thyroid hormone produces its ef- 
fects, beginning with the prohormone thyroxine 
and ending with the actual molecular mechanism 
by which thyroid hormone-responsive genes are 
regulated. 
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