83
Molecular and Cellular Endocrinology, 71 (1990) 83-91 Elsevier Scientific Publishers Ireland, Ltd.
MOLCEL
02294
Structural
analysis of human thyroid hormone
receptor p gene
Akihiro Sakurai, Akira Nakai and Leslie J. DeGroot The Thyroid Study Unit, Department
of Medicine,The University of Chicago, Chicago, IL 60637, U.S.A.
(Received 29 November 1989; accepted 13 March 1990)
Key worak
Thyroid hormone receptor; Exon/intron
organization: mRNA
Thyroid hormone receptors (TR) are ligand-dependent, DNA-binding, truns-acting transcriptional factors belonging to the &A-related steroid/thyroid hormone receptor superfamily. To better understand the structural and functional characteristics of TRs, we isolated the gene encoding human TRPl (hTRP1). The coding region of hTRP1 is split into at least eight exons. Each exon well correlates with functional domains of hTRP1 protein, and the exon/intron organization is highly conserved when compared with the chicken c-erbA gene which encodes an a-type chicken TR. We demonstrate that hTRP has at least two mRNA forms having different lengths of the 3’ untranslated region. We also note several nucleotide corrections of hTRP1 cDNA sequence.
Introduction Thyroid hormones play important roles in growth, development, and metabolism of vertebrates (Oppenheimer et al., 1983). These effects are mediated via specific thyroid hormone receptors which are the cellular homologues of v-erbA (Sap et al., 1986; Weinberger et al., 1986). The existence of two thyroid hormone receptors (TRs; Tra and TRP) and their subtypes is known (Mitsuhashi et al., 1988; Nakai et al., 1988a, b; Hodin et al., 1989; Lazar et al., 1989a; Miyajima et al., 1989). In the human, genes encoding TRa (hTRcY) and TRP (hTR/?) have been localized to
Address for correspondence: Leslie J. DeGroot, M.D., Thyroid Study Unit - Box 138, The University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, U.S.A. This study was supported by a United States Public Health Service grant DK13377. 0303-7207/90/$03.50
chromosome 17q11.2-21 (Sheer et al., 1985) and 3~21-25 (Gareau et al., 1988) respectively. TRcx~ and TRPl are biologically active, while TRa2 does not bind thyroid hormone and may act as an inhibitor of thyroid hormone action (Koenig et al., 1989; Lazar et al., 1989b; Nakai et al., submitted). Although it has not yet been cloned in human, another functional thyroid hormone receptor, hTRP2, may also exist. The significance of the existence of multiple receptor forms is not yet clarified. Generalized Resistance to Thyroid Hormone (GRTH) is a syndrome characterized by elevated circulation levels of thyroid hormone and impaired response to thyroid hormone in the presence of an overall eumetabolic state (Refetoff et al., 1967; Refetoff, 1982). Tight linking of hTRP and GRTH has been reported (Usala et al., 1988) and recently significant point mutations were found in the hTRP gene in members of families with GRTH (Sakurai et al., 1989b; Usala et al.,
0 1990 Elsevier Scientific Publishers Ireland, Ltd.
84
1990). These results indicate that normal expression of the P-receptor is critical for metabolism. The importance of hTR/3 encouraged us to analyze the structure of the hTR/? gene and to compare it to chicken c-erbA, which encodes an a-type TR (Sap et al., 1986).
denatured by the standard method and transferred to a nylon membrane (Zetabind; CUNO). Membrane was hybridized with a 32P-labeled cDNA probe (pheA4 plus pheA12, specific activity, 1.0 X lo9 cpm/pg DNA). After hybridization, membrane was washed according to the manufacturer’s instruction prior to autoradiography.
Materials and methods cDNA probes hTRj3 cDNA clones (pheA4 and pheA12) were generously provided by Dr. Cary Weinberger (Weinberger et al., 1986). pheA4 contains the entire 5’ untranslated region but lacks the carboxy terminal coding region. pheA12 contains the whole coding region but lacks the 5’ untranslated region. Screening the libraries Two human genomic libraries (XC and XE) were screened using a nick-translated hTR/I cDNA probe (pheA12). XC was constructed using human fetal liver DNA that was partially digested with AluI/HaeIII and inserted into the EcoRI site of the hCharon 4A vector using EcoRI linkers (Lawn et al., 1978). XE was constructed from partial MboI-digested human placenta DNA cloned into the BarnHI site of AEMBL-3 vector, and was kindly provided by Dr. Carol Westbrook of the University of Chicago. Sequencing of clones Exons containing EcoRI fragments were restriction enzyme-mapped following subcloning into M13mp18 or M13mp19 vector, and sequenced by the dideoxy chain termination method (Sanger et al., 1980). For sequencing of hTRj3 cDNA and DNA fragments amplified by the polymerase chain reaction (PCR), the same method was applied. To subclone PCR products into Ml3 vector, fragments were collected from low-melting temperature agarose gel (Sea Plaque Agarose; FMC) and both ends were polished using T4 DNA polymerase before ligation. Southern hybridization High molecular weight DNA from peripheral blood leukocytes. was digested with EcoRI overnight by electrophoresis in a 0.8% agarose
was extracted DNA (10 pg) and separated gel. DNA was
Polymerase chain reaction (PCR) Two oligonucleotides were synthesized. Primer A, 5’-TGGCAACAGATTTGGTGCTG-3’ is complementary to the sequence of hTRP cDNA from nucleotide 806 to 825. Primer B, 5’AAAGAGCTAGGCAATGGAAT-3’ is complementary to nucleotides of the hTRP gene 55 base pairs (bp) downstream from the reported polyadenylation site. For the synthesis of antisense cDNA, 20 pg of total RNA from human brain and 100 fmol of primer B were hybridized, followed by reverse transcription using avian myeloma virus reverse transcriptase in the presence of 0.5 mM of each dNTP. Synthesized antisense cDNA was then amplified by PCR technique using Taq polymerase (Saiki et al., 1988). 100 pmol of each of primers A and B were used. PCR was performed using a Perkin-Elmer/Cetus thermocycler for 40 cycles. Each cycle included 1 min at 94 o C for denaturation, 2 min at 50 o C for hybridization and 3 min at 65OC for extension. The method of amplification and cloning of hTR/? cDNA from one family with GRTH was already described (Sakurai et al., 1989b). Results Exon / intron organization of hTRP1 To evaluate the structure of the gene and its evolutionary relation to other hTR genes, we attempted to isolate genomic clones for the coding region of hTRP1. Screening of two genomic libraries (XC and XE) identified several positive clones. These clones were aligned by restriction mapping. Although we cannot indicate the total size of hTR/?l gene because some clones are not overlapping, the coding region of hTR/?l gene was found to span a minimum length of 60 kb (Fig. 1A). This is much longer than the hTRa gene, in which the coding region spans about 15 kb (Nakai, unpublished data). The coding region of the p
85
gene is split into at least eight exons. Nucleotide sequences of exon-intron junction are shown in Table 1. As shown in Fig. lA, no exons exist in the same EcoRI fragment, and therefore each exon (B to H) correlates with one Southern fragment (Fig. 1B). Although the gene structure corresponding to region A is not yet characterized, the
3’ B
A
C
F
DE
G
H
1Okb f
ChTRP $1 ChTRa 2{
,~ChTR~4
,XChTR1331
L
,XChTRO 5,
,hEhTRaf,
9EcoRI T&m
HI 7
8,
polymorphic
A
-23.1
H D
-
9.4 6.6
Barn HI
-
4.4
-
2.3 2.0
-
1.35
F A
E
Site of BamHI restriction fragment length polymorphism (RFLP)
Our clone XChTRP4 has a 2.8 kb Exon G-containing BamHI fragment followed by a 2.5 kb intro& BarnHI fragment in the adjacent downstream sequence, while hChTR/35 has a 5.3 kb Exon G-containing BarnHI fragment at the corresponding position. The sizes of these polymorphic fragments agree with a reported polymorphism which is tightly linked to Generalized Resistance to Thyroid Hormone (GRTH) (Usala et al., 1988). By restriction mapping, this polymorphic BamHI site was determined to exist about 550 bp downstream of Exon G (Fig. 1A).
1 Eson
W)
B
existence of three Southern fragments (25, 5.4, and 2.8 kb) corresponding to region A indicates this region is split at least into three exons since there are no EcoRI sites in the cDNA of this region.
-
1.08
-
0.87
Fig. 1. Physical map of the hTRj?l gene. (A) The positions of the exons are shown as filled boxes, and designated as B to H. The positions of EcoRI and BarnHI restriction sites are indicated schematically. The positions of the genomic clones are shown below the chromosome map. (B) Southern blot analysis of genomic DNA digested with EcoRI. Exons corresponding to each fragment are indicated. Positions of DNA size markers are also indicated.
Comparison of exon structure
Fig. 2A shows the compa~son of exon/intron boundaries of hTR@l and chicken c-erbA (Zahraoui and Cuny, 1987). In both genes, as noted for chicken progesterone receptor (Huckaby et al., 1987), human estrogen receptor (Ponglikitmongkol et al., 1988) and human androgen receptor (Kuiper et al., 1989), each exon correlates well with protein functional domains. For example, all DNA binding domains are split into two exons and each exon contains one zinc-finger motif. It should be noted that the exon/intron ~rangement of hTRfl from Exon C through Exon F is almost identical to that of the corresponding region in chicken c-erbA with one exception. Exon e of chicken c-erbA is one nucleotide shorter than Exon C of hTRP1. Sequences near these exon/intron junctions are shown in Fig. 2B. Strict conservation of exon/intron orga~zation is evident. No similarities are found in the intronic sequences or the size of introns. Mdtiplicity CDNA
of 3’-untranslated
region
in hTRP
The previously reported hTRP cDNA (Weinberger et al., 1986) has a very short 3’-untranslated region (3’-UT) of only 30 bp and lacks a typical polyadenylation signal. On the other hand, the reported cDNA of rat (r) TRP has a 3’-UT 3
86 TABLE
1
EXON/INTRON
ORGANIZATION
OF hTR/zI
The positions at which introns interrupt the mRNA are indicated. Exon sequences are in uppercase, and intron sequences are in lowercase. Codon numbers (odd numbers only) are shown under the amino acid sequence. The estimated sixes of the introns are noted. Exon
Exon
Intron
....aaccctttag
AA
AAT GGC .... B
Met Tbr
Glu
Glu
Aan
1
3
3
A .. .. An;
AC4 G
B .... ‘IGI AAA G Cys
Lys
gtaaccagat....
>12 kb
.. ..tccttcacag
Gly
5
GG ‘I-AC ATC .... C Gly
89
Cys
121
Asp
....tttcatatag
TIC
Gly
Pbe
239
125
gtaagtagat....
2.9 Lb
....cccctccccag
VaI
173
173
gtaaggcttc....
>I5 kb
TGT GAG
CKA GAA
....tcctccttag
Pm
. F
Glu 243
gtgaggatgt....
5.7 kb
ClG CCA .... G
....ttcttttcag
Glu
Leu
289
Pro
291
G ....‘l-CT ‘IC4 G Ser
Leu 175
241
Cys
D
GIG GIG .... E
ClG
Ser
azr
‘IG
Leu
Pbe
5.4 kb
Leu
AAA TIC Pbe
Ile
Leu
Lys
F .... TlT
.
123
171
E
gtaangccc
Lys
D .. . ACA GAT T Wr
Try 91
C .... G3Z ‘IGC AAG Gly
Gly
gtgagtacgc....
4.2 kb
....ttccccgcag
Asp
375
kb in length which does not reach to the polyadenylation site (Koenig et al., 1988; Murray et al., 1988). This discrepancy between hTRP and rTRj3 implies the possibility that hTRj3 may also have a long 3’-UT form. When rTRP cDNA and the hTRP gene were compared, high homology was found between two sequences downstream of the termination codon (Fig. 3A). To confirm that the sequences downstream of the reported polyadenylation site in hTRj3 are also transcribed as a
AT
G3Z CCG
Asp
Arg
. H
Pro
377
‘long form’ of 3’-UT, PCR was attempted using an oligonucleotide which is complementary to the sequence downstream of the poly A site (primer B, Fig. 3A and B). For the upstream primer, we used primer A which is complementary to nucleotides 806 to 825 (Fig. 3B). As expected, a single amplified fragment 937 bp in length was obtained (Fig. 3C). Sequencing of this fragment (Fig. 30) confirmed that the sequences downstream of poly A are transcribed; that is, hTRP1 has at least two
a7
A
262
101 148
206
147
259
,272
G
H
(bp)
hTR/3 1 A
190 c.c-
erb A
B
55
202
CD
E
63 100148
206
F
147
(bp) ---
- -
--a
bcdef
9
h
6 hTRj3 1
cc-erb
C
B
A
--ACAG MMT-----------------AAAG --ThrG
A
GGTAC--MG
D GGT---GATT
E G F B TGGTG----CTG CCA---GAG CTG-----TCAG ATCGC--
QUASI-----------------L~SG lyTry--Lys Gly---AspL
cuVal----La, Pro---Cl"
b c d a f --CTCT GCCC--ACTCG GTGG--TCAGG GTAC--AAG GGC---GACC
h g TGGTG----CTG CCC---GAG
--ThrAr
ghp--SerGl
yTyr--Lys Gly---AspL
Icu-----BerA
spArg--
euVal----Lea h-o---Glu
Fig. 2. Exon/intron organization of hTR/31 and chicken (c) c-erbA. (A) The positions of the exon/intron junctions are indicated by filled wedges above the schematic cDNA structures of hTR/?l and c.c-erbA. Coding regions are indicated by open boxes. Solid lines indicate 5’- and 3’untranslated regions. Each exon is designated alphabetically as shown below the figure. The lengths of exons are also shown. Functional domain assignments of hTR/31 are according to Evans (1988). DNA, DNA binding domain; T3/T4, ligand binding domain. (B) Amino acid and nucleotide sequence of the two genes near the exon/intron junctions are shown. A to H and b to h correspond to those of Fig. 2 A.
mRNA forms. This result is compatible with the multiplicity of hTRB1 transcripts we previously reported (Sakurai et al., 1989a). Correction of hTRj31 cDNA sequence During this study, we found a number of nucleotide differences between our genomic clones and the hTRB1 cDNA originally reported (Weinberger et al., 1986). Three of these changes alter the amino acid sequence [Pro(CCG) at codon 238 to Arg(CGG), Ile(ATA) at codon 332 to Thr(ACA), and Leu(CTC) at codon 446 to Phe(TTC)]. Initially we assumed these differences showed the existence of normal polymorphism, and we sequenced several genomic clones and cDNAs from normal subjects and patients with GRTH (Sakurai et al., 1989b). They all have identical sequences and we confirmed these changes by sequencing the original cDNA clones, pheA4
and pheA12 (Fig. 4A). These misreadings could be caused by uncertainty of the Maxam-Gilbert method (Maxam and Gilbert, 1977) since 12 out of 14 changes are G to A(A to G) or C to T(T to C). These corrections are schematically summarized in Fig. 4B. Until now, we have not found any clones which have a nucleotide in these positions other than the one we report. Discussion We here demonstrated strict conservation of the TR gene during evolution. Although the amino acid sequences are not the same, the exon/intron organization of the DNA binding and following domains of hTRP and c.c-erbA are very similar and the sizes of the corresponding exons are almost identical. The exon/intron organization of c.c-erbA in its thyroid hormone binding domain
88
A
hutnanGnne
C
bp
b
13531078872~ 603Primer ~TR~~CDNA
rTRB
cDNA
---+
A+
IDNAI
lo-1
I
T3n4
AAAAn j E---7 + Primer B
,,L..-___
Fig. 3. Existence of ‘long form’ of hTRP mRNA. (A) Nucleotide sequences of three clones; hTRj3 cDNA as reported by Weinberger et al. (1986), hTR/I gene and rat(r) TR/3 cDNA are aligned beginning at their termination codons. The nucleotide sequence number follow those of the original report (Weinberger et al., 1986). Asterisks under the nucleotide sequences of hTR@ cDNA and rTRj3 cDNA indicate nucleotide differences from the hTR@ gene. Position and sequence of primer B used for PCR are also shown. (B) hTR/31 cDNA and rTRP1 cDNA are shown schematically. Arrows indicate the position of primers A and B, and their direction of elongation. (C) Agarose gel electrophoresis of PCR product. The amplified DNA fragment was eleetrophoresed in a 1% agarose gel, and stained with ethidium bromide. HueIII-digested PbiX 174 (200 ng) was used for the size marker. (D) Sequences of PCR product (‘LONG’ 3’-UT) and pheAl2 (‘SHORT’ 3’-UT) around the reported ~lyadenylation site are shown. Asterisks indicate nucleotide differences between the reported sequence and actual (shown in picture) sequence (see Fig. 4).
(Sap et al., 1986) has not been reported, but it is highly likely its structure is similar (or identical) to hTR/3. hTRol is a counterpart c.c-e&A in human (Nakai et al., 1988), and its gene structure also has an identical pattern to hTRP in its DNA binding domain and following domains (Nakai, unpublished data). This conservation gives evidence to the concept that both hTR genes are derived from a common ancestral. In the DNA binding domain, two zinc-finger motifs are separately encoded in Exons C and D, as in other e&A-related nuclear
receptor genes. Xenopus transcription factor IIIA has nine finger motifs and many of them are encoded by distinct exons (Tso et al., 1986) which implies these finger domains have duplicated and evolved as separate units. It is not certain if this is also the case in the nuclear receptor family since the two ‘fingers’ of nuclear receptors are not highly homologous and likely have different functions in DNA binding. Green et al. demonstrated by finger swapping experiments that the first zinc-finger determines target gene specificity, while the sec-
89
D 3 “LONG” 3’-UT
“SHORT” 3’-UT
i C G A
GATC
3’
r
A A
A
C T T
:
C T T C* C T T
5’
5’ Fig. 3 (continued).
ond finger is supposed to stabilize the DNA-protein interaction (Green and Chambon, 1987; Green et al., 1988). A pituitary specific TRP isoform, TRP2, has been isolated from rat pituitary (Hodin et al., 1989) and differs in sequence at its 5’ terminus from TRPl. It may be derived from the same gene by alternative splicing, and its expression may be controlled by a pituitary specific promoter. In comparison to the structure of the hTR/3 gene sequence, rat (r)TRPl (Koenig et al., 1988; Murray et al., 1988) and rTR/32 cDNA sequences coincide beginning at a position four nucleotides before the 3’ end of Exon B in hTR& In the rTRP gene, an exon/intron junction between Exons B and C may exist four nucleotides upstream of the position in hTR/3, and divergent amino termini
may be encoded by separate exons. Alternatively the exon/intron junction in rTRB may exist at the same position as in the human gene, in which case the exon encoding rTR/32 would contain the four nucleotide sequence ‘AAAG’ at its 3’ end, identical to the 3’ end of Exon B (See Fig. 2B). The possibility that the adjacent intron upstream of Exon C encodes the amino terminal of TRP2 is less likely since no sequence homology is found between rTRP2 cDNA and the intron. RFLP studies are useful for the analysis of hereditary abnormalities. BumHI and Hind111 RFLP for the hTRP gene locus have been reported (Bale et al., 1988; Gareau et al., 1988; Wyllie et al., 1988). Usala et al. reported tight linkage between GRTH and hTRP BamHI and EcoRV RFLP in three unrelated affected families (Usala et al., 1988, 1990). Our finding that a polymorphic BamHI site exists with the hTR/3 gene helps explain these observations. We previously reported the multiplicity of hTRP mRNAs (Sakurai et al., 1989a). According to the present study, we speculate that the short transcript has a short 3’-UT and the long mRNA has a long 3’-UT. The 6 bp motif ‘ATAATI which is 3 bp upstream of the reported polyadenylation site or ‘ATTAGA’ at the position of the termination codon could function as an incomplete polyadenylation signal, since these motifs do not exist in rTR/3 and no short transcript has been reported for rTR/3 (Murray et al., 1988). The significance of the expression of multiple mRNA forms is not known, but it is of interest that only the 2.0 kb short mRNA form was observed in HeLa and MCF-7 cultured cell lines (Weinberger et al., 1986). This might suggest that the long and short mRNA forms have different regulatory mechanisms and different physiological roles. One allele encoding hTR/3 is deleted in small cell lung cancer (Drabkin et al., 1988; Leduc et al., 1989), and loss of the long form of hTR/3 transcripts without gene deletion has recently been reported in colon cancer (Markowitz et al., 1989). Although the expression of the short mRNA form in colon cancer was not analyzed, these findings indicate a strong correlation between the expression of the long form of hTR/3 mRNA and malignant transformation of the cell. The mechanisms of the loss of hTR/3 mRNA expression and the possible role
90 1011 1 321 281 CGGCGGGGATCAAC......ACCTATAACCCCCA......CAGCTTGGGACA......ACCGAAATTTCTGCCAGA
A
...
.. ..CAGCCTGGGAC A .... ..ACGGAAATTCCTGgtaag ...
Genomic
.. ..ACCTATGACTCCC A ......CAGCCTGGGACA ......ACGGAAATTCCTGCCAGA ...
GRTH
.. ..ACCTATGACTCCCA......CAGCCTGGGACA......ACGGAAATTCCTGCCAGA
Normal
A4,A12
CGGCGGGGATTAAC......ACCTATGACTCCCA......CAGCePGGGACA......ACGGAAATTCCTGCCAGA * * * * * *
1251
1291
1311
... ...
1351
. ..CCCGGAAAGTG A ......GTGATACGGGGC .... ..AAATGGGGGTCT......CTAGGCATGTCT ...... Genomic
. ..CCCAGAAAGTG
A ......
GTGACACGGGGC ....
..AAATGGGGGTCT......CTGGGCATGTCT
......
GRTH
. ..CCCAGAAAGTG
A ......
GTGACACGGGGC ....
..AAATGGGzGTCT......CTGGGCATGTCT
......
Normal
. ..CCCAGAAAGTG
A ......
GTGACACGGGGC ....
..AAATGGGGGTC
T ....
..CTGGGCATGTCT
......
A4,A12
. ..CCCAGAAAGTG *
A ....
..AAATGGGGGTC
T ....
..CTGGGCATGTCT *
......
..GTGACACGGGG *
C ....
1631 . ..AACTCCTCCCCCCTTTGTTCCTGG
1601 . . . . . .ATTCCTTCCTATAATTCCAAAAAA...... . . . . . .ATTCATTCTCATAATTCCTACAGC......
Genomic
. ..AACTCTTCCCCCCTTTGTTCTTGG
GRTH
...AACTCTTCCCCCCTTTGTTCTPGG....
Normal
. ..AACTCl-TCCCCCCTTTGTTCTTGG
... ...ATTCATTCTCATAATTCCTACAGC......
A4,A12
. ..AACTC!I’TCCCCCCTTTGTTCTPGG * *
. . . . . . ATTCATTCTCATAATTCCAAAAAA...... * **
I3 position
[ DNA 1 of
nucleotide
\ 11
A/-Y/
A\ 288
T3/T4
I
291
325
1013
F (Ah A\\
1020
1254
1295
1353
1636
1651
1685
1689
1800
reported nucleotide correct nucleotide reported amino acid
Pro (238)
II0 (332)
Leu (446)
correct emino acid
1 Ar9
I Thr
1 Phe
Fig. 4. Correction of nucleotides in hTR/31 cDNA. (A) Sequence of hTRP clones and comparison to the reported sequence (top). Sequences of genomic clones shown in Fig. 1A (Genomic), amplified cDNA clones of patients with GRTH (GRTH) and normal individuals (Normal), and original cDNA clones (A4, A12) are aligned. Nucleotide sequence numbers are indicated above. Asterisks indicate the position of nucleotide changes found in all clones sequenced. Lowercase in Genomic clone indicates the inttonic sequence. In some patients with GRTH, the hTRB gene is heterozygous and one of two alleles has a G to C substitution at nucleotide 1318 (Sakurai et al., 1989b). (B) hTR/31 cDNA is shown schematically as described in the legend for Fig. 2A. The positions of nucleotides to be corrected are shown with both reported and correct nucleotides. If these changes alter the reported amino acid sequence, these codons and the correct amino acids are also noted.
of hTR/3 in tumorigenesis await further investigation. Finally we note a correct cDNA sequence for hTRj31. These corrections must be especially im-
portant for researchers studying the structure or functions of hTRP using PCR technique, since mismatch of nucleotides greatly reduces the effectiveness of PCR.
91
Note added in proof We recently noted that correction of the originally reported (Weinberger et al., 1986) A at nucleotide 288 to G produces a new initiation codon. The deduced amino acid sequence of the N terminus of hTRj31 is therefore as follows: 286 ATG ACT CCC AAC AGT ATG --MET
Thr
Pro
Asn
Ser
Met
---
This amino acid sequence is identical to that of the N terminus of rTRj31, and the total amino acid length of hTRP1 is 461 instead of 456. This length is also the same as for rTRj31. References Bale, A.E., Usala, S.J., Weinberger, C., Weintraub, B.D. and McBride, O.W. (1988) Nucleic Acids Res. 16, 7756. Drabkin, H., Kao, F.-T., Hartz, J., Hart, I., Gazdar, A., Weinberger, C., Evans, R.M. and Gerber, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9258-9262. Evans, R.M. (1988) Science 240, 889-895. Gareau, J.-L.P., Houle, B., Leduc, F., Bradley, W.E.C. and Dovrovic, A. (1988) Nucleic Acids Res. 16, 1223. Green, S. and Chambon, P. (1987) Nature 325, 75-77. Green, S., Kumar, V., Theulaz, I., Wahh, W. and Chambon, P. (1988) EMBO J. 7, 3037-3044. Hodin, R.A., Lazar, M.A., Wintman, B.I., Darling, D.S., Koenig, R.J., Larsen, P.R., Moore, D.D. and Chin, W.W. (1989) Science 244, 76-79. Huckaby, C.S., Conneely, O.M., Beattie, W.G., Dobson, A.D.W., Tsai, M.-J. and O’Malley, B.W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8380-8384. Koenig, R.J., Wame, R.L., Brent, G.A., Hamey, J.W., Larsen, P.R. and Moore, D.D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5031-5035. Koenig, R.J., Lazar, M.A., Hodin, R.A., Brent, G.A., Larsen, P.R., Chin, W.W. and Moore, D.D. (1989) Nature 337, 659-661. Kuiper, G.G.J.M., Faber, P.W., van Rooij, H.C.J., van der Korput, J.A.G.M., Ris-Stalpers, C., Klaassen, P., Trapman, J. and Brinkmann, A.O. (1989) J. Mol. Endocrinol. 2, Rl-R4. Lawn, R.M., Fritsch, E.F., Parker, R.C., Blake, G. and Maniatis, T. (1978) Cell 15, 1157-1174. Lazar, M.A., Hodin, R.A., Darling, D.S. and Chin, W.W. (1989a) Mol. Cell. Biol. 9, 1128-1136. Lazar, M.A., Hodin, R.A. and Chin, W.W. (1989b) Proc. Natl. Acad. Sci. U.S.A. 86, 7771-7774. Leduc, F., Brauch, H., Hajj, C., Dobrovic, A., Kaye, F., Gazdar, A., Harbour, J.W., Pettengill, O.S., Sorensen, G.D., van den Berg, A., Kok, K., Campling, B., Paquin, F.,
Bradley, W.E.C., Zbar, B., Minna, J., Buys, C. and Ayoub, J. (1989) Am. J. Hum. Genet. 44, 282-287. Markowitz, S., Haut, M., Stellato, T., Gerbic, C. and Molkentin, K. (1989) J. Clin. Invest. 84, 1683-1687. Maxam, A. and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 560-564. Mitsuhashi, N., Tennyson, G.E. and Nikodem, V.M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 58045808. Miyajima, N., Horiuchi, R., Shibuya, Y., Fukushige, S., Matsubara, K., Toyoshima, K. and Yamamoto, T. (1989) Cell 57, 31-39. Murray, M.B., Zilz, N.D., McCreary, N.L., MacDonald, M. and Towle, H.C. (1988) J. Biol. Chem. 263, 12770-12777. Nakai, A., Seino, S., Sakurai, A., Szilak, I., Bell, G.I. and DeGroot, L.J. (1988a) Proc. Natl. Acad. Sci. U.S.A. 85, 2781-2785. Nakai, A., Sakurai, A., Bell, G.I. and DeGroot, L.J. (1988b) Mol. Endocrinol. 2, 1087-1092. Oppenheimer, J.H., Dillman, W.H. and Schwartz, H.L. (1979) Fed. Proc. 38, 2154-2161. Ponglikitmongkol, M., Green, S. and Chambon, P. (1988) EMBO J. 7, 3385-3388. Refetoff, S. (1982) Am. J. Physiol. 243, E88-E98. Refetoff, S., DeWind, L.T. and DeGroot, L.J. (1967) J. Clin. Endocrinol. Metab. 27, 279-294. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Science 239, 487-494. Sakurai, A., Nakai, A. and DeGroot, L.J. (1989a) Mol. Endocrinol. 3, 392-399. Sakurai, A., Takeda, K., Ain, K., Ceccarelli, P., Nakai, A., Seino, S., Bell, G.I., Refetoff, S. and DeGroot, L.J. (1989b) Proc. Natl. Acad. Sci. U.S.A. 86, 8977-8981. Sanger, F., Coulson, A.R., Barrell, B.G., Smith, A.J.H. and Roe, B.A. (1980) J. Mol. Biol. 143, 161-178. Sap, J., Munoz, A., Damm, K., Goldberg, Y., Ghysdeal, J., Leutz, A., Beug, H. and Vennstrom, B. (1986) Nature 324, 635-640. Sheer, D., Sheppard, D.M., LeBeau, M., Rowley, J.D., San Ramon, C. and Solomon, E. (1985) Ann. Hum. Genet. 49, 167-171. Tso, J.Y., van der Berg, D.J. and Kom, L.J. (1986) Nucleic Acids Res. 14, 2187-2200. Usala, S.J., Bale, A.E., Gesundheit, N., Weinberger, C., Lash, R.W., Wondisford, P.E., McBride, O.W. and Weintraub, B.D. (1988) Mol. Endocrinol. 2, 1217-1220. Usala, S.J., Tennyson, G.E., Bale, A.E., Lash, R.W., Gesundheit, N., Wondisford, F.E., Accili, D., Hauser, P. and Weintraub, B.D. (1990) J. Clin. Invest. 85, 93-100. Weinberger, C., Thompson, C.C., Ong, E.S., Lebo, R., Gruol, D.J. and Evans, R.M. (1986) Nature 324, 641-646. Wyllie, F.S., Lazarou, L.P. and Wynford-Thomas, D. (1988) Nucleic Acid Res. 16, 5224. Zahraoui, A. and Cuny, G. (1987) Eur. J. Biochem. 166,63-69.