thyroid liver mRNA. This pool is now being subdi- 
vided to identify the cDNA that codes for the func- 
tional type I deiodinase enzyme. 
The other major focus of Dr. Larsen's activities 
has been the study of T^ regulation of gene expres- 
sion. This project has involved Dr. Ronald Koenig 
(University of Michigan) and Dr. Gregory Brent. It 
has been performed in collaboration with Dr. David 
Moore (Massachusetts General Hospital). 
To identify the sequences in rGH that are critical 
for Tj stimulation, a functional assay has been used 
in which rGH promoter/chloramphenicol acetyl- 
transferase (CAT) constructs are transfected into pi- 
tuitary tumor cells. Dr. Larsen's previous studies 
had demonstrated the necessity for sequences be- 
tween -189 and -172 in the rGH promoter to con- 
fer complete T^ responsiveness. The sensitivity and 
precision of this analysis were limited by the only 
two- to threefold T^ induction of the constructs 
containing rGH T^RE (T^ response element). How- 
ever, cotransfection of a (3 T^ receptor-expressing 
plasmid with the rGH constructs led to a sixfold 
amplification of the T^ response. This demon- 
strated that the T^ receptor is rate limiting in these 
GH4C1 cells. The greater induction allowed the 
recognition of the importance of an additional se- 
quence between -172 and -167, relative to the 
start site, which also contributed to the T^ re- 
sponse. A single G to T mutation at nucleotide 167 
of the -191 to -162 rGH oligonucleotide caused a 
striking fourfold increase in the T^ induction of an 
amputated rGH promoter. This up-mutant T^RE 
was used as the basis for an evaluation of the other 
portions of the rGH T^RE. Three domains were 
identified in this region of the rGH gene, spaced 
—10 nucleotides apart, all of which were required 
for an optimal T^ response. Cotransfection of ex- 
cess receptor did not restore a full response of a 
TjRE with a single mutation in any one of the three 
critical areas. On the basis of these results, a series 
PUBLICATIONS 
of TjRE "half sites" that have the sequence 
AGGT(C/A)A were defined in the rGH promoter. 
These are oriented as two direct repeats followed 
by an inverted repeat, the A, B, and C domains. 
Three such elements are present in the highly T^- 
inducible rGH and a-myosin heavy-chain promot- 
ers. In the a-glycoprotein and (3-TSH (thyroid-stim- 
ulating hormone) genes, only two direct repeats of 
this half site are found. This may have relevance as 
to how Tj induces repression, as opposed to stim- 
ulation, of the transcription of these genes. Dr. 
Larsen's group had demonstrated earlier that T^ 
does not induce the human growth hormone 
(hGH) promoter but that the bovine growth 
hormone (bGH) promoter is T^ responsive. The lat- 
ter contains sequences quite similar to the palin- 
dromic B and C domains of rGH, but the former 
does not. 
The receptor cotransfection system was also used 
to demonstrate that a non-T^-binding variant of the 
ttj-receptor, called a^, cloned by Dr. William W 
Chin's group (HHMI, Harvard Medical School), can 
interfere with the expression of T^-dependent pro- 
moters in JEG (cotransfected with receptor) or 
GH4C1 cells. This protein is formed by alternate 
splicing of the a^-gene transcript and binds DNA 
but not T^. This interference indicated the possibil- 
ity of a T^-response-limiting function for the espe- 
cially high levels of the a^-protein in the central 
nervous system. It acts as a dominant negative mu- 
tation. Nuclear T^ receptors in this unique tissue 
are nearly saturated in the euthyroid state, due to 
the type II deiodinase activity discussed earlier. The 
presence of the a^-protein could serve as an alter- 
nate regulatory mechanism to regulate the T^ re- 
sponse. 
Dr. Larsen is also Professor of Medicine at Har- 
vard Medical School and Senior Physician at the 
Brigham and Women's Hospital. 
Books and Chapters of Books 
Larsen, PR. 1989. Regulation of thyroid hormone metabolism. In Iodine and the Brain (Delong, G.R., Rob- 
bins, J., and Condliffe, PG., Eds.). New York: Plenum, pp 5-18. 
Refetoff, S., and Larsen, PR. 1989. Transport, cellular uptake, and metabolism of thyroid hormone. In Endo- 
crinology (DeGroot, L.J., Ed.). Philadelphia: Saunders, pp 541-561. 
Continued 
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