Gene expression of thyroid hormone receptors TR alpha and TR beta during early development in tilapia, oreochromis mossambicus

C LONING AND SEQUENCING OF THYROID HORMONE RECEPTORS TR α AND TRβ IN TILAPIA, OREOCHROMIS MOSSAMBICUS 6 2.1 I NTRODUCTION.... L OCALIZATION OF THYROID HORMONE RECEPTORS TR α1 AND TRβ1 I

AND TR β DURING EARLY DEVELOPMENT IN TILAPIA,

OREOCHROMIS MOSSAMBICUS

A SHARMILA

NATIONAL UNIVERSITY OF SINGAPORE

2003

DURING EARLY DEVELOPMENT IN TILAPIA, OREOCHROMIS

MOSSAMBICUS

By

A Sharmila M.Sc., M.Phil

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

NATIONAL UNIVERSITY OF SINGAPORE

2003

I would like to express my deep gratitude to Prof T J Lam, for his guidance, enthusiasm and support throughout the course of this project Most of all, his unfailing encouragement is very much appreciated

I am thankful to A/Prof W K Chan and A/Prof A D Munro for their advice and help in carrying out the studies

I am extremely grateful to Dr J Walford, Dr Konda P Reddy, Dr P Sukumar and Dr B Sivaloganathan for their helpful discussions, constant support and practical help and most importantly for their invaluable suggestions during the preparation of this thesis

I would like to take this opportunity to express my gratitude to Dr Y W Liu,

for her help in guiding me in whole mount in situ hybridization work

I would also like to thank my friends, Sudha and Shanti for their timely help during the course of this study

Finally, the award of a Research scholarship by the National University of Singapore is gratefully acknowledged This study was supported by a NUS Research Grant RP3972374 to Prof T J Lam

A CKNOWLEDGEMENTS i

T ABLE OF CONTENTS ii

L IST OF ILLUSTRATIONS v

L IST OF TABLES viii

S UMMARY ix

Chapter 1 G ENERAL INTRODUCTION 1 Chapter 2 C LONING AND SEQUENCING OF THYROID HORMONE RECEPTORS TR α AND TRβ IN TILAPIA, OREOCHROMIS MOSSAMBICUS 6 2.1 I NTRODUCTION 6

2.2 M ATERIALS AND METHODS 10

2.2.1 Cloning of TRα1 10

2.2.2 Partial cloning of TRβ1 11

2.2.3 Cloning of TRβ2 11

2.2.3.1 5' and 3' -Rapid amplification of cDNA ends 11

2.2.3.2 Long distance PCR (LD PCR) 12

2.2.4 Analysis of TRα and TRβ PCR Products 12

2.2.4.1 Agarose gel electrophoresis 12

2.2.4.2 Extraction of PCR products from agarose gel 13

2.2.4.3 Quantification of DNA by spectrophotometry 13

2.2.4.4 DNA ligation 13

2.2.4.5 Preparation of competent cells 13

2.2.4.6 Transformation 14

2.2.4.7 Colony screening PCR 15

2.2.4.8 Isolation and purification of plasmid DNA 15

2.2.4.9 Automatic sequencing 16

2.2.4.10 Database search and sequence analysis of TRα and TRβ 17

2.3 R ESULTS 17

2.3.1 Cloning and sequencing of TRα1 17

2.3.2 Cloning and sequencing of TRβ 18

2.3.2.1 Partial cloning and sequencing of TRβ1 18

2.3.2.2 Cloning and sequencing of TRβ2 18

2.4 D ISCUSSION 29

3.1 I NTRODUCTION 32

3.2 M ATERIALS AND METHODS 33

3.2.1 Source of tilapia eggs/larvae 33

3.2.2 Total RNA isolation 34

3.2.3 RNA gel electrophoresis 34

3.2.4 Synthesis of first strand cDNA 34

3.2.5 Semi-quantitative RT-PCR analysis 35

3.2.6 Statistical analyses 35

3.3 R ESULTS 36

3.3.1 Changes in the levels of TRα1 and TRβ1 expression during early development 36

3.4 D ISCUSSION 40

Chapter 4 L OCALIZATION OF THYROID HORMONE RECEPTORS TR α1 AND TRβ1 IN EMBRYOS AND THEIR TISSUE-SPECIFIC EXPRESSION IN TILAPIA, OREOCHROMIS MOSSAMBICUS 44 4.1 I NTRODUCTION 44

4.2 M ATERIALS AND METHODS 46

4.2.1 Whole mount in situ hybridization 46

4.2.1.1 Synthesis of labeled RNA probe 46

4.2.1.1.1 Linearization of plasmid DNA 46

4.2.1.1.2 Probe incubation and precipitation 47

4.2.1.2 Preparation of tilapia embryos for in situ hybridization 48

4.2.1.3 Proteinase K treatment of embryos 48

4.2.1.4 Prehybridization 48

4.2.1.5 Hybridization 49

4.2.1.6 Post-hybridization washes 49

4.2.1.7 Antibody incubation 49

4.2.1.7.1 Preparation of preabsorbed DIG and fluorescein antibody 49

4.2.1.7.2 Incubation with preabsorbed antibodies 49

4.2.1.8 Color development 50

4.2.1.9 Mounting and photography 50

4.2.2 Extraction of total RNA from adult tissues 51

4.2.3 Statistical analyses 51

4.3.2 Expression of TRα1 and TRβ1 in various adult tissues 52

4.4 D ISCUSSION 56

Chapter 5 A UTOINDUCTION OF THYROID HORMONE RECEPTORS TR α1 AND TRβ1 DURING EARLY DEVELOPMENT IN TILAPIA, OREOCHROMIS MOSSAMBICUS 59 5.1 I NTRODUCTION 59

5.2 M ATERIALS AND METHODS 61

5.2.1 Source of tilapia larvae 61

5.2.2 Hormone treatment 62

5.2.2.1 Preparation of treatment media 62

5.2.2.2 Experiment 1 62

5.2.2.3 Experiment 2 62

5.2.2.4 Experiment 3 62

5.2.2.5 Preparation of samples 63

5.2.3 Quantification of mRNA using real-time RT-PCR 63

5.2.3.1 In vitro transcription 63

5.2.3.2 The Standard curve 64

5.2.3.3 Real-time RT-PCR analysis 64

5.2.4 Statistical analyses 65

5.3 R ESULTS 65

5.3.1 Quantification of mRNA using real-time RT-PCR 65

5.3.2 Experiment 1 66

5.3.3 Experiment 2 66

5.3.4 Experiment 3 66

5.4 D ISCUSSION 72

Chapter 6 G ENERAL DISCUSSION AND C ONCLUSIONS 77 L ITERATURE CITED ……… 81

cDNA The open reading frame encodes a polypeptide of 409 amino

acids Putative zinc fingers are underlined The presumptive

translation start is indicated by ATG codon and TGA codon is

marked by asterisk……… 21 2.2 Comparison of tilapia TRα1 with those of salmon, flounder, halibut

and zebrafish A dot represents a gap insertion to maximize matches

in the multiple alignment Amino acids identical to tilapia TRα1 are

shown as dashes Numbers represent amino acid number starting

with methionine……… 22

2.3 Partial nucleotide sequence (669 bp) of tilapia TRβ1 with the

predicted amino acid sequence The nine amino acid insertion is

represented as broken lines Degenerate primer sequences are in

bold letters Gene specific primers and nested primers are also

2.4 Nucleotide and deduced amino acid sequence of a cDNA encoding

tilapia TRβ2 The sequence contains an open reading frame of 1185

nucleotides which encodes a polypeptide of 395 amino acids An

insertion sequence of nine amino acids in the ligand binding domain

is represented as broken lines Putative zinc fingers are underlined… 24 2.5 Comparison of tilapia TRβ2 with those of seabream, flounder,

zebrafish and salmon Amino acids identical to tilapia TRβ1 are

represented as dashes A dot represents a gap insertion to maximize

the number of matches The 9 amino acid insertion is indicated in

bold letters Numbers represent amino acid number starting with

25

2.6 Phylogenetic tree of vertebrate TRα and TRβ receptors Phylogram

of Bootstrap analysis (1000) replicates are identical to this tree

Bootstrap percentages are given above the branches Species and

their Genbank accession numbers for sequences included for

analysis: Atlantic salmon TRα1 (AF146775), flounder TRαA

(D16461), flounder TRαB (D16462), halibut TRα1 (AF143296),

zebrafish TRα1 (U54796), Xenopus TRαA (M35343), chicken

TRα1 (Y00987), human TRα1 (X55005), Atlantic salmon TRβ1

(AF302251), flounder TRβ1 (D45245); sea bream TRβ1 (Nowell et

al., 2001); zebrafish TRβ1 (AF109732), conger eel TRβ1 and TRβ2

(Kawakami et al., 2003); Xenopus TRβ1 (M45245), chicken TRβ1

(X17504) and human TRβ1 (X04707)……… 27

3.1 Developmental changes in TRα1 and TRβ1 mRNA levels during

early life stages of tilapia, Oreochromis mossambicus Data

presented as mean ± standard error from three batches of 10 pooled

3.2 Gel photographs showing changes in the expression of (A) TRα1

(B) TRβ1 genes during early development of tilapia and (C) β actin

is used as an internal control……… 39

4.1 Localization of TRα1 and TRβ1 in 6 dpf embryos by whole mount

in-situ hybridization in O mossambicus (A) No expression with

sense TRα1 probe (B) antisense-TRα1 expressed in optic tectum,

cerebellum and medulla of brain and tail regions (C) No expression

with sense TRβ1 probe (D) antisense-TRβ1 mRNA expressed in

brain and tail regions Abbreviations: b- brain; ot- optic tectum; ce-

cerebellum; md- medulla; t- tail……… 53

4.2 Semi-quantitative RT-PCR analysis of tilapia TRα1 and TRβ1

expression in various adult tissues Data presented as mean ±

standard error from triplicate experiments……… 54

4.3 Gel photographs showing the expression of (A) TRα1 (B) TRβ1 in

various adult tissues of tilapia and (C) β actin used as an internal

5.1 Real time PCR of TRα1 standard curve using in-vitro transcribed

RNA as template Ten fold serial dilutions of the templates, ranging

from 2.38-7.38 log molecules (log molecules= log N, where N is the

copy number of that molecule)……… 67 5.2 Real time PCR of TRβ1 standard curve using in-vitro transcribed

RNA as template Ten fold serial dilutions of the templates, ranging

from 2.4 -7.4 log molecules (log molecules= log N, where N is the

copy number of that molecule)……… 68 5.3 Quantification of TRα1 and TRβ1 expression on day 7 post

treatment of newly hatched larvae with different concentrations of

T3 Data presented as mean ± standard deviation from triplicate

experiments Means with the same alphabet are not significantly

different (p>0.05, One Way ANOVA followed by SNKM)

69

5.4 Quantification of TRα1 and TRβ1 expression on day 7 post

treatment of newly hatched larvae with different concentrations of

T4 Data presented as mean ± standard deviation from triplicate

experiments Means with the same alphabet are not significantly

different (p>0.05, One Way ANOVA followed by SNKM).………

70

5.5 Real time PCR analysis of expression of TRα1 and TRβ1 in

different adult tissues 16 hrs after injection of tilapia with a dose of

LIST OF TABLES

TABLE PAGE

NO NO 2.1 List of primers used in obtaining full length cDNA of TR β2 ……….20

3.1 Sequences of primers used in RT-PCR analysis……… 37

Complementary DNA (cDNA) of thyroid hormone receptor specific for the alpha subunit (TRα1) was cloned from tilapia adult liver tissue by library screening

using a cDNA probe derived from the Xenopus TRα1 cDNA The tilapia cDNA

sequence was found to contain an open reading frame of 1227 nucleotides encoding

a polypeptide of 409 amino acids Two TRβ variants have been identified in O

mossambicus The TRβ1 cDNA of 669 bp was cloned from total RNA of tilapia adult brain by reverse transcription polymerase chain reaction using degenerate primers designed based on consensus sequence of zebrafish and flounder TRβ1 The deduced amino acid sequence possessed a teleost specific nine amino acid insertion within the ligand binding domain The full length sequence of thyroid hormone receptor TRβ2, obtained after 5’ and 3’- RACE, indicated an open reading frame of

1185 nucleotides which encodes a polypeptide of 395 amino acids also with a teleost specific insertion sequence of nine amino acids in the ligand binding domain

To understand the effect of thyroid hormones on tilapia development, gene expression of TRα1 and TRβ1 was studied during early development of tilapia from

2 dpf (days post fertilization) until 20 dph (days post hatching) by semi-quantitative RT-PCR analysis Both TR gene transcripts were low at 2 dpf but increased during larval development TRα1 mRNA levels reached a peak on 13 dph and declined thereafter TRβ1 mRNA levels reached a peak on 15 dph, followed by a significant decline on 17 dph; however, TRβ1 mRNA levels remained higher than those of TRα1 on 20 dph Detection of TRα1 and TRβ1 gene expression before the

early development

Localization of tilapia TRα1 and TRβ1 mRNA was carried out using 3 dpf

(days post fertilization) to 6 dpf embryos (just prior to hatching) by in situ

hybridization (ISH) with digoxigenin labeled antisense probe TRα1 and TRβ1 transcripts were seen in the brain and the tail bud region in developing embryos from

3 dpf onwards Semi-quantitative RT-PCR analyses of expression of TRα1 and TRβ1 in adult tissues indicated that their levels of expression varied substantially in different tissues

Treatment with exogenous T3 and T4 resulted an increase in the expression of TRα1 and TRβ1 in newly hatched larvae and adult tissues as analyzed using real-time RT-PCR analysis The quantified transcripts of TRβ1 appeared to be higher than the TRα1 transcripts for all the treatments in this study and also in the controls The observed upregulation of TRs by thyroid hormones suggests induction of receptors by their ligands and such a response occurs even before the production of endogenous thyroid hormones This is an important observation as TRs are expressed constitutively early in development concomitant with the presence of maternal thyroid hormones It is concluded that both TRα1 and TRβ1 play a role during early development of tilapia and that TRβ1 may play a more important role in the later developmental stages

CHAPTER 1 GENERAL INTRODUCTION

The physiological roles of thyroid hormones, thyroxine (T4) and triiodothyronine (T3), in vertebrates have been well established The major functions

of thyroid hormones in metabolism, development and differentiation have been

reviewed by Schwartz (1983) and Oppenheimer et al (1995) In addition, thyroid

hormones also play important roles during embryogenesis and organogenesis (Sachs

et al., 2000) Thyroid hormones exert their effect by binding to multiple thyroid

hormone receptors (TRs) that act as DNA-binding transcription factors TRs bind to DNA sequences known as thyroid hormone response elements (TREs) found in the regulatory regions of target genes, and depending on the nature of TREs, gene expression may be enhanced or inhibited (Wu and Koenig, 2000) Multiple isoforms

of TRs are reported to be derived from two distinct genes, TRα and TRβ TRs belong to a nuclear receptor superfamily that also includes the receptors for retinoids, steroid hormones, vitamin D, fatty acids and prostaglandins, as well as “orphan receptors” with no identified ligands

Studies on the molecular basis of thyroid hormone action in amphibians are extensive (Shi, 1996) The timing and specific changes during metamorphosis are

highly tissue dependent, and are controlled by thyroid hormones (Kikuyama et al., 1993) and in addition they trigger metamorphosis (Shi et al., 1996) Addition of

exogenous thyroid hormones to the rearing water of premetamorphic tadpoles can induce precocious metamorphosis, whereas inhibiting the synthesis of endogenous thyroid hormones blocks this transition Furthermore, the responses of different organs to thyroid hormones are autonomous since organs such as limb, tail, and

intestine can be induced to undergo metamorphosis with thyroid hormones even

when cultured in vitro (Tata et al., 1991) Thyroid hormones have been shown to

affect gene expression in metamorphosing tissues The rising concentration of endogenous thyroid hormones following embryonic development and subsequent tadpole growth period, induces the gene expression in different tissues that leads to the removal or remodeling of larval organs and differentiation of adult tissues (White

and Nicoll, 1981; Kikuyama et al., 1993) The transcriptional regulatory properties

of thyroid hormones through its receptors suggest that thyroid hormones induce a gene regulation cascade in each tissue that undergoes metamorphosis (Shi, 1996) In amphibians, detailed studies from the laboratory of D D Brown have characterized both TRα and TRβ genes of Xenopus (Yaoita et al., 1990; Shi et al., 1992; Shi, 1994) Many of the thyroid hormone response genes have been isolated and characterized Both the TR genes are developmentally and hormonally regulated, which has led to the concept that the autoregulation of TR genes by thyroid hormones is crucial to the hormonal regulation of metamorphosis itself (Tata, 1996)

In mammals, studies on understanding the mechanism underlying thyroid hormone action have grown exponentially over the past decade with TR cloning, identification and cloning of the TR heterodimer partner RXR (retinoid X receptor) and the identification of transcriptional coregulators An understanding of the specific functions of each TR isoform, TR homo- and heterodimers and TR coactivator and corepressor is required in order to understand the expanded view of

TR target genes in each target tissue and cell type under different physiological conditions (Zhang and Lazar, 2000)

Studies in teleosts have demonstrated the presence of thyroid hormones in

unfertilized eggs and developing embryos (Brown et al., 1987; Kobuke et al., 1987; Tagawa and Hirano, 1987; Reddy et al., 1992) The presence of thyroid hormones in

eggs is probably of maternal contribution This has been confirmed in the study in which injection of thyroid hormones into gravid females, resulted in increased levels

in unfertilized eggs (Brown et al., 1988; Brown and Bern, 1989; Ayson and Lam,

1993) Furthermore, thyroid hormones induced metamorphosis in flounder,

Paralichthys olivaceus (Inui and Miwa, 1985; Miwa and Inui, 1987) Also, early

stages of development in a variety of teleost species are stimulated by treatment with

thyroid hormones (Lam, 1980; Lam and Sharma, 1985; Lam et al., 1985; Reddy and Lam, 1987; Brown et al., 1988; Brown and Bern, 1989; Reddy and Lam, 1992a, b; Brown and Kim, 1995; Tachihara et al., 1997) but retarded when treated with inhibitors of thyroid hormone synthesis (Okimoto et al., 1993) High doses of

exogenous thyroid hormones have a negative effect on growth, survival and development, although the hormone concentrations at which this occurs, appear to be species specific (Woodhead, 1966; Nacario, 1983; Lam, 1985; Lam and Sharma, 1985; Brown and Kim, 1995)

Studies on the developmental expression patterns of TRs in teleosts is largely restricted to a few species In flounder, the temporal and regional distribution of the

TR subtypes suggests that thyroid hormone actions are controlled at the receptor level by the differential expression of TRs (Yamano and Miwa, 1998) Expression of TRα and TRβ during embryogenesis in zebrafish (Essner et al., 1997; Liu et al.,

2000) and seabream (Nowell et al., 2001) indicates that TRs have developmental

roles during organogenesis Elevated levels of TR mRNAs during Atlantic halibut

metamorphosis show the TR transcripts to be present in virtually every remodeled

organ (Llewellyn et al., 1999) In salmon, low levels of TRα expression were

detected during organogenesis, followed by a gradual increase during larval

development (Jones et al., 2002)

However, in teleosts, not much information is available on the receptor mediated thyroid hormone action although physiological effects of thyroid hormones

on the growth and development have been reported in many species including

Oreochromis mossambicus There are also a few reportss on the cloning and sequencing of TRs and expression during development of teleosts (Yamano et al., 1994; Yamano and Inui, 1995; Essner et al., 1997, 1999; Yamano and Miwa, 1998; Llewellyn et al., 1999; Liu et al., 2000; Nowell et al., 2001; Marchand et al., 2001; Jones et al., 2002; Kawakami et al., 2003) However, until now there has been no report on TRs during the development of O mossambicus, although the

physiological effects of thyroid hormones during ontogeny have been very well studied in this species (Lam, 1994)

Thus the present study explores the presence of isoforms of TR, their tissue specific distribution, their expression during early development and the

autoinduction of these receptors in the tilapia, O mossambicus

In order to understand gene expression of TRs during development in O mossambicus, cloning and sequencing of thyroid hormone receptors were carried out

(Chapter 2) This enabled studies on the expression profiles of TRs during development and delineation of the ontogeny of TRα and TRβ (Chapter 3) Localization during early development and differential expression of TRα and TRβ

in adult tissues were also studied (Chapter 4) and the possible role of thyroid hormones on TRs was investigated (Chapter 5)

CHAPTER 2 CLONING AND SEQUENCING OF THYROID HORMONE RECEPTORS

TRα AND TRβ IN TILAPIA, OREOCHROMIS MOSSAMBICUS

2.1 INTRODUCTION

The thyroid hormone receptors (TRs) belong to a family of activated nuclear receptors which includes the receptors for retinoids, vitamin D3 and steroid hormones Thyroid hormones regulate the physiological function of many

hormone-organs by gene transcription through thyroid hormone receptors (Oppenheimer et al.,

1995) TRs are encoded by two different genes TRα and TRβ Alternative splicing from each gene generates multiple TR isoforms, including TRα1, TRα2 and TRα3 from the TRα gene and TRβ1 and TRβ2 from the TRβ gene (Lazar, 1993) The TRα isoforms differ in their carboxyl-termini The TRα1 isoform binds to T3 and analogues and serves as a ligand-regulated transcriptional regulator By contrast, the TRα2 isoform does not bind the hormone and functions dominantly as a transcriptional repressor Both the TRβ1 and TRβ2 isoforms are generated through alternative usage of promoters in the 5’ portion of the gene for initiation of transcription TRβ1 and TRβ2 isoforms have divergent amino-termini and both

function as ligand-regulated transcriptional regulators (Ribeiro et al., 1998)

TRs have a similar domain organization as that found in all nuclear receptors TRs have modular structures with six regions (A-F) and three functional domains: an amino-terminal A/B domain, a central DNA-binding domain containing two “zinc fingers” (DBD), a hinge region containing the nuclear localization signal, and a

carboxy-terminal ligand-binding domain (LBD) (Yen, 2001)

The amino-terminal regions have variable lengths and divergent sequences among the TR isoforms Among different species, this region is less well conserved The rat and human TRs are 97 and 99% identical in their DBDs and LBDs,

respectively, against 85% identity in their amino-terminal domains (Koenig et al.,

1988) The role of this domain in transcriptional activation is still controversial Deletion of the amino-terminal domain of TRβ1 had no effect on T3 –dependent transcriptional activation by TRβ1, which suggests that it does not contain a major activation function domain like the glucocorticoid receptor (Thompson and Evans,

1989; Yen et al., 1995) On the other hand, studies on TRα1 and TRβ1 from several

species have shown that the amino-terminal domain may be important for

transcriptional activation and interactions (Baniahmad et al., 1993; Hadzic et al., 1995; Tomura et al., 1995)

The DBD is located in the central portion of TR and has two zinc fingers, each composed of four cysteines coordinated with a zinc ion The integrity of each zinc finger is critical, as deletion of zinc fingers or amino acid substitution of these cysteine residues abolishes DNA-binding and transcriptional activity of steroid and

thyroid hormone receptors (Green et al., 1988; Severne et al., 1988; Yen et al.,

1995)

Within the first zinc finger, there is a “P box”, comprised of amino acids

located between and just distal to the third and fourth cysteines (Danielson et al.,

1989; Umesono and Evans, 1989) This critical region has been shown to be important in sequence-specific recognition of hormone response elements (TREs) by

different members of the nuclear hormone superfamily (Nelson et al., 1995;

Rastinejad et al., 1995) There are other important contact points within the minor

groove of the TRE just downstream from the second zinc finger called “A box region” Within the DBD of TR, there are dimerization interfaces in the upstream of the first zinc finger The retinoid X receptor (RXR) dimerization surfaces are located

in the second zinc finger including an arginine located in the D box, a region which

is important for distinguishing spacing between half sites of hormone response

elements (Luisi et al., 1994; Nan et al., 1998)

Critical roles of LBD include transactivation, dimerization and basal repression by unliganded TR Ligand is buried deep within a hydrophobic pocket in the LBD formed by discontinuous stretches that span almost the entire LBD, and the

carboxy-terminal region contributes its hydrophobic surface (Brzozowski et al.,

1997)

The hinge region between the DBD and T3-binding domain contains an amino acid sequence that is associated with nuclear localization TRs are likely imported into the nucleus shortly after synthesis and can bind DNA even in the absence of hormone (Evans, 1988) Recent studies using green fluorescent fusion proteins of wild-type TRβ and TRβ hinge region mutants demonstrated that this region is important for T3-mediated translocation of TR into the nucleus (Zhu et al.,

1998)

TRs and other nuclear hormone receptors can modulate transcriptional activities of each other This binding can occur via several mechanisms: formation of heterodimers, and competition for cofactors (Schule and Evans, 1991; Yen and Chin, 1994)

In vitro studies indicated that TRs can bind to TREs as monomers,

homodimers and heterodimers (Bigler and Eisenman, 1995) TRs can form heterodimers with RXRs which also are members of the nuclear hormone receptor

superfamily (Ikeda et al., 1995) TR/RXR heterodimers bind with a specific polarity

which in turn can modulate transcriptional activity The shape of the TR complex formed with TREs may be important in protein-protein interactions with coactivators and corepressors that link the liganded TR/RXR heterodimer with the transcriptional machinery (Yen, 2001) TRs can also heterodimerize with TR auxillary proteins (TRAPs) and bind better to TREs than TR homodimers (Murray and Towle, 1989;

Burnside et al., 1990)

TRα and TRβ1 isoforms can bind T3 and various TH analogs with subtle differences in affinity TRα binds T3 with slightly higher affinity than TRβ1

(Schueler et al., 1990) Triac (3,5,3’-triiodothyroacetic acid) binds to TRα1 with

similar affinity as that of T3 and binds to TRβ1 with two- to threefold higher affinity than T3 (Chiellini et al., 1998) Scattered throughout the LBD are discontinuous

heptad repeats that have been proposed to form hydrophobic interfaces for TR homo and heterodimerization (Forman and Samuels, 1990)

In contrast to steroid hormone receptors that are transcriptionally inactive in the absence of ligand, TRs can also activate transcription in the absence of ligand

(Helmer et al., 1996) Unliganded TRs bind to TREs and may modulate transcription

of target genes Unliganded TRs have been found to repress basal transcription of

positively regulated TREs (Brent et al., 1989; Baniahmad et al., 1992) Additionally,

several groups have reported ligand-independent gene transcription in neuroblastoma

and Xenopus cells (Pastor et al., 1994; Nagl et al., 1995) Recent studies have

suggested that ligand-independent activation may be mediated by TR recruitment of corepressors and this may be mediated by protein-protein interactions of DNA

(Tagami et al., 1997, 1999) Besides, co-repressors are also known to regulate the repression of TRs (Chen and Evans, 1995; Horlien et al., 1995; Sande and Privalsky,

1996) TRs are also regulated by coactivators such as steroid receptor coactivator-I

(SRC-1) (Onate et al., 1995) which enhance their ligand-dependent transcription

In teleosts, not much is known about the characterization of the TRs Information on either the partial or full length sequence of TRs (TRα/TRβ) and

developmental expression patterns is available only for a few species (Yamano et al., 1994; Yamano and Inui, 1995; Essner et al., 1997; Yamano and Miwa, 1998; Llewellyn et al., 1999; Liu et al., 2000; Nowell et al., 2001; Marchand et al., 2001; Jones et al., 2002; Kawakami et al., 2003) Although the developmental role of thyroid hormones in O mossambicus has been established (Reddy and Lam, 1992a; Reddy et al., 1992), nothing is known about the TRs in this species with respect to

gene expression In order to understand the gene expression, information on the gene

sequence of TRs is required Hence the cloning and sequencing of TRs in O mossambicus were done in order to characterize the temporal and spatial expression

of these receptors in this species (chapters 3 & 4)

2.2 MATERIALS AND METHODS 2.2.1 Cloning of TR α1

Poly A+ mRNA was extracted from adult tilapia liver using OligotexTMmRNA mini kit (Qiagen) A cDNA library in λGEM-2 (Promega) was constructed

using Poly A+ mRNA The library was screened with a cDNA encoding Xenopus

TRαA (Genbank accession number, M35343) A positive clone was selected for sequencing The insert sequence was subcloned into pGEM-T Easy Vector (Promega)

2.2.2 Partial Cloning of TR β1

Total RNA was extracted from adult tilapia brain using TRIZolTM reagent (Life Technologies) Total RNA (5 µg) was reverse transcribed into cDNA using SuperscriptTM RT-PCR kit (Life Technologies) and then the cDNA was amplified using the degenerate primers (Forward primer 5’- CAGAAGACNGTNTGGGAYCG -3’; Reverse primer 5’- CCARAARTGNGCNACYTTGTG-3’) designed from highly conserved amino acid sequences of zebrafish and flounder TRβ1

2.2.3 Cloning of TR β2

2.2.3.1 5’- and 3’ – Rapid amplification of cDNA ends

The full length cDNA of TRβ2 was obtained using SMARTTM RACE cDNA Amplification Kit (Clontech, USA) The advantage 2 Polymerase Mix used in RACE had a Taq DNA polymerase with a nuclease-deficient N-terminal deletion, a minor amount of a proofreading polymerase and Taq antibody for hot start PCR Gene-specific primers were designed for the 5’ and 3’- RACE reactions (GSP1 and GSP2, respectively) from the partial tilapia TRβ1 cDNA (669bp) Nested gene specific primers (NGSP1 and NGSP2) were also designed to facilitate analysis of the RACE products (see Fig 2.3, Table 2.1)

First strand cDNA for 5’-RACE was synthesized using a modified docking oligo (dT) primer (5’-CDS- 5’-RACE cDNA Synthesis Primer) and the

lock-SMART II oligo (5’ AAGCAGTGGTAACAACGCAGAGTACGCGGG) The RACE cDNA was synthesized using a traditional reverse transcription reaction, but with a special oligo (dT) primer This 3’-RACE cDNA Synthesis Primer (3’-CDS) primer includes the lock docking nucleotide positions as in 5’-CDS primer and also has a portion of the SMART sequence at its 5’ end By incorporating the SMART sequence into both the 5’ and 3’ RACE Ready cDNA populations, both RACE PCR reactions can be primed using the Universal Primer Mix (UPM), that recognizes the

3’-SMART sequence, in conjunction with distinct gene-specific primers

2.2.3.2 Long distance PCR (LD PCR)

After completing 5’ and 3’- RACE analysis, LD PCR was carried out using 5’ RACE Ready cDNA as template to construct the full length sequence of TRβ2 cDNA The primers were designed in the 5’ and 3’ UTR regions (Table 2.1)

2.2.4 Analysis of TR α and TRβ PCR products

The standard molecular techniques followed for cloning the PCR products are described in this section

2.2.4.1 Agarose Gel Electrophoresis

Typical DNA electrophoresis was performed in 1% agarose gel unless particular requirements present The agarose powder was dissolved in 1XTAE (0.04

M Tris-acetate; 0.001 M EDTA) by heating After the solution was cooled to 60°C, ethidium bromide was added to a final concentration of 0.5 µg/ml and mixed thoroughly A voltage of 1-5 v/cm was applied during the electrophoresis

2.2.4.2 Extraction of PCR products from agarose gel

QIAquick Gel Extraction Kit (QIAGEN, USA) was used to recover the PCR products of interest from agarose gel according to manufacturer’s instructions

Briefly, the gel slice containing DNA band of interest was cut and melted at 50°C in Buffer QX1 for 10 minutes and then loaded into a QIAquick spin column The volume of the Buffer QX1 was approximately three times that of the gel slice The column was centrifuged at 14,000 rpm for 1 minute, washed by adding 0.75 ml

of Buffer PE, and spun again After removing residual Buffer PE by spinning at 14,000 rpm for 1 minute, 15-20 µl of H2O was added to the top of the column The column was incubated at room temperature for 1 minute and DNA fragment was eluted into a 1.5-ml centrifuge tube by centrifugation at 14,000 rpm for 2 minutes

2.2.4.3 Quantification of DNA by Spectrophotometry

DNA was quantified by optical density reading at 260nm using UV-1601 spectrophotometer (SHIMADZU, Japan)

µl The ligation reaction was carried out overnight at 4°C

2.2.4.5 Preparation of competent cells

Competent cells were prepared from Escherichia coli (E.coli) strain DH5α

for the transformation of ligated mixture

For the preparation of competent bacteria cells, 2 ml of LB broth was

incubated with a single fresh colony of E.coli strain DH5α at 37°C with 250 rpm

shaking overnight Five hundred µl of the culture was re-inoculated into a 250 ml flask containing 50 ml of LB broth and shaken at 250 rpm at 37°C until OD600

reached around 0.5 The culture was chilled on ice for 15 minutes before centrifugation Cells were pelleted by centrifugation at 1,000 g at 4°C for 15 minutes and the pellet was re-suspended in RF1 (100 mM RbCl; 50 mM MnCl2; 30 mM Potassium acetate; 10 mM CaCl2 and 15% glycerol) with 1/3 volume of the original culture After incubation on ice for 15 minutes, the cells were spun down and resuspended in 1/12.5 of the original volume of RF2 (10 mM MOPS; 10 mM RbCl;

75 mM CaCl2; 15% glycerol) This followed a 15 minute-incubation on ice, before the competent cells were aliquoted into microcentrifuge tubes The cells were then frozen in liquid nitrogen and stored at -80°C

2.2.4.6 Transformation

Ligation reaction (5 µl) was added into prechilled 100 µl of E.coli DH5α competent cells and left on ice for 20 minutes The mixture was then incubated at 37°C for 5 min followed by an immediate cooling on ice for 2 minutes 400 µl of

LB medium was added to the mixture and incubated with shaking (225rpm) at 37ºC for 1 hr After the incubation, 1/10 and 9/10 of the transformation reaction mixture was spread onto two separate LB plates containing 100 µg/ml ampicillin, 0.5 mM IPTG and 50 mg/ml X-Gal and incubated at 37°C overnight The plates with colonies grown were stored at 4°C

2.2.4.7 Colony Screening PCR

The colony screening by PCR is more sensitive and less time-consuming than restriction enzyme digestion analysis of extracted and purified plasmids

For PCR screening, colonies to be examined were marked in numerical order

A toothpick was used to touch the colony and the attached bacteria were spread to the bottom of a PCR tube, which was preloaded with 20 µl pf PCR mixture, containing 0.6 U of Taq DNA polymerase, 2 µl of 10X PCR buffer, 1 µl of 2mM dNTP mix and 0.2 µg of each sense and antisense primers For pGEM-T Easy vector, SP6 and T7 primers were used as primer pair PCR program includes initial denaturation at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 45 seconds and elongation at 72°C for 1.5 minutes PCR product was examined in 1~1.5% agarose gel Colonies that yielded PCR products with expected size were inoculated for plasmid DNA preparation

2.2.4.8 Isolation and Purification of Plasmid DNA

Small-scale preparation of plasmid DNA was carried out using Wizard Miniprep kit (Promega, USA) or Qiagen Plasmid Purification kit (Qiagen, USA) The protocol involved alkaline lysis followed by binding of plasmid DNA to a silica-based resin DNA was eluted in buffer EB (10 mM Tris.Cl, pH 8.5)

Firstly, the bacteria in LB (Luria-Bertani) liquid medium with appropriate antibiotics were harvested by centrifugation at 12,000 rpm for 1 minute using the 5417C centrifuge (Eppendorf, Germany) The bacterial pellet was resuspended in

200 µl of Cell Resuspension Solution (100 mg/ml RNAse A; 10mM EDTA; 25 mM Tris-HCl, pH 7.5) 200 µl of Cell Lysis Solution (0.2 M NaOH; 1% SDS) was added

and mixed by gently inverting the tube for several times This mixture was neutralized by adding 200 µl of Neutralization Buffer (1.32 M KOAc, pH 4.8) which was followed by centrifugation in microcentrifuge at 14,000 rpm for 5 minutes The supernatant was transferred into a fresh 2-ml tube to which 1 ml purification resin was added The Resin/DNA mix was transferred into a minicolumn and washed with

2 ml of Column Wash Solution (200 mM NaCl; 5 mM EDTA; 20 mM Tris-HCl, pH

7.5; 75% EtOH) The resin was drained by spinning the minicolumn at 14,000 rpm

for 2 minutes The minicolumn was then transferred to a new microcentrifuge tube and 30 µl of water was added After 2 minutes incubation at room temperature, plasmid DNA was eluted from the column by centrifugation at 14,000 rpm for 2 minutes

2.2.4.9 Automatic Sequencing

Automated sequencing reactions were carried out using the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer) The kit contains a sequencing enzyme AmpliTaq® DNA Polymerase called FS and a set of dye labeled terminators for fluorescent cycle sequencing larger fragments with more accuracy

Each sequencing reaction (20 µl) contains 8 µl of Terminator Ready Reaction Mix, 200-500 ng of double strand DNA, and 1 µl of primer (0.2 µg/µl) PCR was performed on the GeneAmp PCR System 9600 (Perkin Elmer) or Peltier Thermal cycler PTC200 (MJ Research, USA) with 25 cycles of 96°C/10 seconds, 50°C/5 seconds and 60°C/4 minutes, and finally held at 4°C Ethanol precipitation was carried out to purify the extension products Two µl of 3 M NaOAc (pH 4.6) and 50

µl of 95% ethanol was mixed with the 20-µl reaction mix, and incubated at room temperature for 15 minutes The tube was spun for 20 minutes at 14,000 rpm, 4°C The pellet was rinsed with 250 µl of 70% ethanol and air-dried

The DNA pellet was dissolved in 6 µl of loading dye [50 ml contains EDTA (25 mM, pH 8.0) 1 ml; 10 ml deionised formamide; 50 mg dextran blue and 39 ml

H2O] and heated to 92°C for 3 minutes Samples then were chilled on ice for 2 minutes before being loaded into the sequencing gel (18g urea; 5 ml 10X TBE; 5 ml

long range gel solution and 26 ml H2O; 250 µl 10%APS and 35 µl TEMED) The electrophoresis was carried out at 1,690 volts for 5-9 hours The cycle sequencing products were analyzed automatically with the ABIPrismTM377 (Perkin Elmer, USA)

and software

2.2.4.10 Database search and sequence analysis of TR α and TRβ

The known amino acid and nucleic acid sequences of TRα and TRβ genes were searched from Genbank [National Center for Biotechnology Information (NCBI)] Homology search was performed using BLAST (Basic Local Alignment Search Tool) The homology and phylogenetic analysis were established using DNAMAN software

2.3 RESULTS 2.3.1 Cloning and Sequencing of TR α1

The tilapia TRα1 cDNA sequence was found to consist of 409 amino acids in the coding region of 1227 bp The cDNA nucleotide sequence of tilapia TRα1 is shown in Fig 2.1 and the protein contained two putative cystein-rich zinc fingers which are characteristic of nuclear hormone receptors, including TRs

Homology alignment of tilapia TRα1 with its counterparts from Atlantic salmon TRα1 (AF146775), flounder TRα1 (D16461), halibut TRα1 (AF143296) and zebrafish TRα1 (U54796) is shown in Fig 2.2 Homology analysis of tilapia TRα1 in the DNA and ligand binding domain with those of other teleosts showed that tilapia TRα1 is close to flounder TRα2 (Fig 2.2)

2.3.2 Cloning and Sequencing of TR β

2.3.2.1 Partial Cloning and Sequencing of TR β1

The TRβ1 cDNA (669 bp) fragment obtained had a teleost specific insertion sequence composed of nine amino acids in the ligand binding domain as shown in Fig 2.3 The partial fragment obtained lies in the hinge region and ligand-binding domain

2.3.2.2 Cloning and Sequencing of TR β2

The full length sequence of thyroid hormone receptor TRβ obtained after 5’ and 3’- RACE, LD PCR and sequencing indicated an open reading of 1185 nucleotides which encodes a polypeptide of 395 amino acids also with a teleost specific insertion sequence of nine amino acids in the ligand binding domain (Fig 2.4) The protein contained two putative zinc fingers

The region of TRβ2 corresponding to the TRβ1 fragment (partial sequence) seem to be different in its nucleotide and amino acid sequence These when compared for their nucleotide and amino acid identity indicated 97.65% and 98.65% identity respectively

Homology alignment of amino acid sequences of TRβ2 for the tilapia with sea bream TRβ1 (Nowell et al., 2001), flounder TRβ1 (D45245), zebrafish TRβ1

(AF109732), zebrafish TRβ2 (AF322218), salmon TRβ1 (AF302251) and salmon TRβ2 (AF302252) is represented in Fig 2.5 Homology analysis of tilapia TRβ2 in the DNA and ligand binding domain with those of other teleosts showed that tilapia TRβ2 is close to flounder TRβ1 (Fig 2.5)

A phylogenetic tree was constructed for TR genes to analyse the relationships among the vertebrates Highly conserved domains (DBD and LBD) were considered for analysis The analysis indicated fish and other vertebrate isoforms to cluster separately (Fig 2.6)

Table 2.1 List of primers used in obtaining full length cDNA of TRβ2

AATCCCGTTGTTGTCGCCAGCCTTGGCTTCAAGGCCAGCTGATTTTACCCGTGGAGCCTCTCATCTTTTCATATT 75 TCCCAGCATCACCGTGAGCAACGACAACACCGGGTTGCACTCCTTTTGGAGAAACGCCCAAAACCCACTTTCCAA 150 GGACCCCAGGGACCGGCCCTTAGGGACCGGCCCTTGTTATGTTTTGTTGACCGACTCGTCCCAACCGGCGAATGA 225

A C H A S R F L H M K V E C P N E L F P P L F L E

GTCTTCGAGGACCAGGAGGTGTGAGACACACTTGCCGGCACGCAAAGGGAAGAGAAAGTTGGAGCGAAACAGAAG 1575

V F E D Q E V *

ATGGGTTAGTCCATCGTGTTTGTGGAGGAAGAATGGAGGGAGAATCCTCTCTGCAGGAATCTGCATTTCTCTGAC 1650 ATCACATTCACAGTATGTGGTTTTAAAACTGGAAACAACAATATCAGAAACAACACACCAGCAACAGGAGAGTGA 1725 AGCAAACAGAGGATTTTGTTTTGTGAACACCCTCTTCTCCACCTCTTTCTCCTCCCTATACCAACCCCTGTTTTA 1800 AAATCTGTTTGGGGGCCCTTCTGTCTGAGCATGATGTCTTATCTAGTAAGAGTTGTACGACAGCAACGGAATTCG 1875

Fig 2.1 Nucleotide and amino acid sequence of the O mossambicus TRα1 cDNA

The open reading frame encodes a polypeptide of 409 amino acids Putative zinc fingers are underlined The presumptive translation start is indicated by ATG codon and TGA codon is marked by asterisk

tilapia α1 MEHMP.KEQDSNPSEGEEKQWLNGPKRKRKNSQCSVKSMT GYIPSYLEKDEPC 52 atlantic salmon α1 -EPIS.NVEDPNSSE-DEKRWPDGPKRKRKNSTCSVKSMSALSLSVQ I - 59 flounder α1 -EPMS.NKQDSNSSE-DEKGWPDVPKRKRKNSQCSMKSMSALSVSVP I - 59 flounder α2 -AQWPEKEEEEQPMF-EEY T I - 33 halibut α1 -EPMS.NKQDSNSSE-DEKGWPDVPKRKRKNSQCSMKSMSALSVSVP I - 59 zebrafish α1 .MENTEQEHNLPE-DETQWPNGVKRKRKNSQCSMNSTSDKSISVP V - 57 tilapia α1 VVCGDKATGYHYRCITCEGCKGFFRRTIQKNLHPTYSCKYDGCCIIDKITRNQCQLCRFK 112 atlantic salmon α1 -AY DG -R 119 flounder α1 -SY EG -K 119 flounder α2 -SY DC -K 93 halibut α1 -AY EG -K 119 zebrafish α1 -SY DS -R 117 tilapia α1 KCIAVGMAMDLVLDDSKRVAKRRLIEENRERRKKEEMVKSLQSRPEPTGAEWELIRLVTE 172 atlantic salmon α1 -A-C -K KD-I-KT-QA DSS -HV-E 179 flounder α1 -S-G -K RE-M-RT-QI DTA -MA-D 179 flounder α2 -A-G -R KE-I-KT-QN TGA -MV-E 153 halibut α1 -S-G -K RE-M-RT-QV DTA -MA-D 179 zebrafish α1 -S-G -K KE-I-KT-HN TVS -MV-E 177 tilapia α1 AHRHTNAQGSQWKQKRKFLPEKIGQSPVAPTSDGDKVDLEAFSEFTKIITPAITRVVDFA 232 atlantic salmon α1 -SH -PED -S-RA P-G -I - 239 flounder α1 -SS -SDD -S-MV S-G -M - 239 flounder α2 -AQ -PDK -S-VA S-G -I - 213 halibut α1 -SS -SDD -G-MV S-G -M - 239 zebrafish α1 -PH -PED -S-.A S-N -I - 236 tilapia α1 KKLPMFSE.LPCEDQIILLKGCCMEIMSLRAAMRYDPDSETLTLSGEMAVKREQLKNGGL 291 atlantic salmon α1 -. -V E -SG K - 298 flounder α1 -. -V D -NS K - 298 flounder α2 -Q -M E -SG K - 273 halibut α1 -. -V E -NG K - 298 zebrafish α1 -. -V E -SG S - 295 tilapia α1 GVVSDAIFDLGKSLAQFNLDDTEVALLQAVLLMSSDRSGLTCTDKIEKCQETYLLAFEHY 351 atlantic salmon α1 -S-A -S L -LTLVD -K -T - 358 flounder α1 -E-G -T M -HQCME -Q -A - 358 flounder α2 -S-A -T L -LTCMD -K -T - 333 halibut α1 -S-A -T M -LTSLE -Q -A - 358 zebrafish α1 -S-S -S L -LTCVE -K -M - 355 tilapia α1 INYRKHNIPHFWPKLLMKVTDLRMIGACHASRFLHMKVECPNELFPPLFLEVFEDQEV 409 atlantic salmon α1 H -P -D -PNELFPPLFLEVFEDQEV 416 flounder α1 Y -P -D -PSELFPPLFLEVFEDQEV 416 flounder α2 Y -P -D -PNELFPPLFLEVFEDQEV 391 halibut α1 Y -P -D -SSELFPPLFLEVFEDQEV 416 zebrafish α1 H -S -N -PTELFPPLFLEVFEDQEGST 415 tilapia α1 409 atlantic salmon α1 416 flounder α1 416 flounder α 391 halibut α1 416 zebrafish α1 GVAAQEDGSCLR 427

Fig 2.2 Comparison of tilapia TRα1 with those of salmon, flounder, halibut and zebrafish A dot represents a gap insertion to maximize matches in the multiple alignment Amino acids identical to tilapia TRα1 are shown as dashes Numbers represent amino acid number starting with methionine

TCCATAGCAAACACTGACACAGTCGCTCACACCGTTAGCTCACCCTGCACTCAGTCCACTCCGCGCGCATAGACC 75 TCGCCTGATTTGCACTGGCGCGGGTCATTTCAGGTTAAACCCTGGCGCTGTGTCCCGTTCACTTTAACCGACGGA 150 GATCAACGCTGGTTCCAGTTGAGGCGGAATGTACACCCACTGGGACTCCAAGTGATATTTGGACACGGGCTTCGT 225 CTTGCATATGCGGTAGGAACCCAGATGTCTGATGAGGCTCTGAGGATGACGTCTTTGCCTTGAAGCCCCACCAGT 300 ATGTCAGAGCCAGCAGAAAAATGCTCCCCCCGCTGGAAAGATGAGGCCATTCAAAATGGGTACATACCAAGTTAC 375

Fig 2.4 Nucleotide and deduced amino acid sequence of a cDNA encoding tilapia TRβ2 The sequence contains an open reading frame of 1185 nucleotides which encodes a polypeptide of 395 amino acids An insertion sequence of nine amino acids in the ligand binding domain is represented as broken lines Putative zinc fingers are underlined

Atlantic salmon TRβ1 conger eel TRβ2 zebrafish TRβ1

63

flounder TRβ1 sea bream TRβ1 88

tilapia TRβ2 67

31

conger eel TRβ1 91

chicken TRβ1 human TRβ1 82

Xenopus TRβ1 85

71

Atlantic salmon TRα1 flounder TRαB tilapia TRα1 96

55

zebrafish TRα1 32

flounder TRαA halibut TRα1 99

29

chicken TRα1 Xenopus TRαA 59

human TRα1 50

100

Fig 2.6 Phylogenetic tree of vertebrate TRα and TRβ receptors Phylogram of Bootstrap analysis (1000) replicates are identical to this tree Bootstrap percentages are given above the branches Species and their Genbank accession numbers for sequences included for analysis: Atlantic salmon TRα1 (AF146775), flounder TRαA (D16461), flounder TRαB (D16462), halibut TRα1 (AF143296), zebrafish TRα1

(U54796), Xenopus TRαA (M35343), chicken TRα1 (Y00987), human TRα1

(X55005), Atlantic salmon TRβ1 (AF302251), flounder TRβ1 (D45245); sea bream TRβ1 (Nowell et al., 2001); zebrafish TRβ1 (AF109732), conger eel TRβ1 and TRβ2 (Kawakami et al., 2003); Xenopus TRβ1 (M45245), chicken TRβ1 (X17504) and human TRβ1 (X04707)