Transthyretin (prealbumin) gene expression in choroid plexus is strongly conserved during evolution of vertebrates

Comp. Biochem. Physiol. Vol. 99B, No. 1, pp. 239-249, 1991

0305-0491/91 $3.00+ 0.00 © 1991PergamonPress plc

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TRANSTHYRETIN (PREALBUMIN) GENE EXPRESSION IN CHOROID PLEXUS IS STRONGLY CONSERVED D U R I N G EVOLUTION OF VERTEBRATES PAUL J. HARMS,GuO-FEN TU, SAMANTHAJ. RICHARDSON,ANGELAR. ALDRED, ANTHONYJAWOROWSKIand GERHARDSCHREIBER The Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Melbourne 3052, Australia (Tel: 613 344 5914) (Received 29 October 1990)

Abstract--1. The major protein synthesized and secreted by the choroid plexus from mammals, birds, reptiles and probably amphibians is similar in subunit structure to transthyretin. 2. In mammals and birds the proportion of transthyretin mRNA is much higher in choroid plexus RNA than in liver RNA. No transthyretin mRNA is found in brain outside the choroid plexus. 3. Transthyretin-like protein, such as that secreted by the choroid plexus, was not detected in amphibian serum and was present in very low levels in reptile serum. 4. It is proposed that transthyretin synthesis and secretion arose earlier in evolution in the choroid plexus than in the liver.

INTRODUCTION A protein with an electrophoretic mobility greater than that of albumin, a "prealbumin", was initially detected in human cerebrospinal fluid (Kabat et al., 1942a,b) and serum (Seibert and Nelson, 1942). Mammalian prealbumin has been studied extensively with amino acid sequences determined for the protein isolated from human (Kanda et al., 1974) and rabbit (Sundelin et al., 1985) serum, and the structure of human prealbumin determined by o X-ray crystallography with a resolution of 1.8 A (Blake et al., 1978). Mammalian prealbumin is composed of four identical subunits with a molecular weight of 15,000, and, like albumin, does not contain carbohydrate (Putnam, 1975). The name prealbumin was changed to transthyretin to indicate its role in the transport of thyroid hormones and retinol in the bloodstream, and to avoid possible confusion with the precursor form of albumin, proalbumin [International Nomenclature Committee of the International Union of Biochemistry Newsletter (1981) J. biol. Chem. 256, 12-14]. The name transthyretin is used hereafter. The primary structure of human (Mita et al., 1984), mouse (Wakasugi et al., 1985), rat (Dickson et al., 1985b; Duan et aL, 1989) and sheep (Tu et al., 1989) transthyretins have been deduced from nucleotide sequences of corresponding cDNA clones. Transthyretin, thyroxine-binding globulin and albumin are three thyroid hormone-binding proteins found in the serum of higher animals (for a review see Robbins and Edelhoch, 1986). Mutations leading to the absence of the protein from the bloodstream are known for albumin in humans (Bennhold et al., 1954; Boman et al., 1976; Dammacco et al., 1980) and rats (Nagase et al., 1979), and for thyroxine-binding globulin in humans (see Refetoff, 1989; Refetoff and Larsen, 1989). These mutations do not lead to an impairment of health and, in particular, do not result

in difficulties related to an insufficient supply of thyroid hormones to tissues. However, an absence of transthyretin from the bloodstream, due to a genetic mutation, has never been recorded, suggesting a fundamental function of transthyretin early in the developing embryo. Since such an absence can easily be tolerated for the thyroid-hormone carriers of lower affinity (albumin) or higher affinity (thyroxinebinding globulin) for thyroid hormones than transthyretin, the fundamental role in early life for transthyretin is not likely to be the transport of thyroid hormones in the bloodstream. The question arises then as to what other biological functions transthyretin might have. Most proteins secreted into the bloodstream are synthesized by the liver (for a review see Schreiber, 1987) and the synthesis of transthyretin (Dickson et al., 1982) and the presence of transthyretin mRNA (Dickson et aL, 1985a,b, 1986; Soprano et al., 1985, 1986; Dickson and Schreiber, 1986; Herbert et al., 1986; Kato et al., 1986; Mita et al., 1986) have been reported in both adult and fetal rat liver. However, extrahepatic sites of synthesis of transthyretin have been reported such as the visceral yolk sac (Soprano et al., 1986; Schreiber, 1987; Fung et al., 1988) and the choroid plexus (Dickson et al., 1985a,b; Thomas et al., 1988, 1989). In particular, the proportion of transthyretin mRNA in RNA extracted from the choroid plexus of the rat (Dickson et al., 1985a,b, 1986; Dickson and Schreiber, 1986; Stauder et aL, 1986) and human (Dickson and Schreiber, 1986; Herbert et al., 1986; Mita et aL, 1986; Fung et al., 1988) is very much higher than in RNA extracts from liver. The expression of the transthyretin gene in presumptive choroid plexus was observed at a very early stage in the development of rats (Fung et al., 1988; Thomas et al., 1988, 1989). It has been proposed that transthyretin synthesis and secretion by the choroid plexus is involved in the

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t r a n s p o r t of thyroxine from the b l o o d s t r e a m to the b r a i n (Dickson et al., 1987; Schreiber et al., 1990). Since the supply o f thyroid h o r m o n e s is critical for the early d e v e l o p m e n t of the brain, one would expect a strong conservation o f the expression o f the t r a n s t h y r e t i n gene in the c h o r o i d plexus. This p a p e r reports data showing t h a t a protein similar to t r a n s t h y r e t i n is synthesized a n d secreted by the choroid plexus of a wide range of vertebrates from m a m m a l s to a m p h i b i a n s , b u t that this protein appears to be in relatively low a b u n d a n c e in the serum of reptiles a n d absent from a m p h i b i a n serum. MATERIALS AND METHODS

Animals and tissues Only adult animals were used. New Zealand White rabbits (Oryctolagus cuniculus) and Dunkin Hartley guineapigs (Cavia porcellus) were from the Department of Pharmacology, University of Melbourne, inbred C57 Black × CBA mice (Mus musculus) from the Centre for Early Human Development, Monash Medical Centre, Melbourne, and inbred Balbc mice from the Walter and Eliza Hall Institute for Medical Research, Melbourne. Material from Merino Cross sheep (Ovis aries), Large White pigs (Sus scrofa), Hereford cattle (Bos taurus) and chickens (Gallus gallus) was obtained from local abattoirs and tissue from a German Short-Haired Pointer Cross dog (Canis familiaris) from a local veterinary clinic. Stumpy-tailed lizards (Trachydosaurus rugosus) and cane toads (Bufo marinus) were purchased or collected locally. Buffalo and Brown-Norway rats (two strains of Rattus norvegicus) were from inbred colonies in the Department of Biochemistry, University of Melbourne. Human liver, from a cadaver used for kidney transplantation, was obtained through the Royal Melbourne Hospital. A human lateral ventricle choroid plexus from a 6 month old boy was provided by the Clinical Pathology Department of the Royal Children's Hospital (Melbourne), the sample being obtained after death due to circulatory collapse following pneumonia.

Chemicals, chicken transthyretin standard, cDNA probes The Klenow fragment of E. coli DNA Polymerase I and [ct-32P]dATP ( > 3000 Ci/mmol) were purchased from Bresatec, restriction endonucleases from Promega, guanidine hydrochloride and agarose from Sigma Chemical Company, GeneScreen Plus membrane from Dupont NEN Research Products, L-[Y,5'J25I]thyroxine (1.2 Ci/mg), L-[U14C]leucine (350Ci/mol) from Amersham, Ampholines (pH 3.5-10 and pH 5-7) from LKB, sodium phenobarbitone from Bomac Lab., New South Wales, tricaine (MS222) from Sandoz, and acrylamide from Kodak. Chicken transthyretin was purified from pooled chicken serum by preparative polyacrylamide gel electrophoresis using the system described earlier (Urban et al., 1974). A 610bp transthyretin cDNA insert was prepared by digestion of sheep transthyretin cDNA in ~gt 10 from a sheep choroid plexus cDNA library (Tu et al., 1989) and used as a probe. A 670 bp chicken transthyretin cDNA probe was provided by W. Duan (Melbourne).

Incubation of choroid plexus Stumpy-tailed lizards were anaesthetized by intramuscular injection of sodium phenobarbitone, 75 mg/kg body wt, and cane toads by transcutaneous absorption of tricaine from a 0.1% solution in water. Rats and mice were killed under ether anaesthesia and rabbits under carbon dioxide. The lateral and fourth ventricle choroid plexus in the brain of the mammals and chickens and the third and fourth ventricle choroid plexus of lizards and cane toads were dissected out immediately after death and placed in serum-

free medium at 25°C, supplemented with amino acids (minus leucine), for 30 min. The medium (Edwards et al., 1976) and the concentrations of amino acids added (Schreiber and Schreiber, 1973) have been described previously. After initial incubation under shaking at 37°C for mammals and chickens, the medium was replaced with fresh incubation medium containing 12/l M L-[U-14C]leucine. For cane toads and stumpy-tailed lizards, incubations were performed at 25°C and the medium was Hams F-12 (minus leucine) containing 30#M L-[U-14C]leucine. Incubations were for 8 hr. After this time, the medium was decanted and any remaining solid debris centrifuged in microfuge tubes at 15,000g for 5min. The supernatants were then stored at - 20°C. Incorporation of L-[U-~4C]leucine into protein was measured according to Mans and Novelli (1961) in a Rackbeta Model 1217 liquid scintillation spectrometer using toluene/0.3% PPO/0.03% POPOP as a scintillation medium.

Analysis of proteins by polyacrylamide gel electrophoresis For analysis under denaturing conditions, samples of medium from choroid plexus incubations were separated in vertical sodium dodecyl sulphate polyacrylamide slab gels (15% polyacrylamide) using a 4.5% polyacrylamide stacking gel and the discontinuous buffer system of Laemmli and Favre (1973). Following electrophoresis (100 V, room temperature) proteins were fixed in 40% methanol, 12% acetic acid and then stained with Coomassie Brilliant Blue R250. To visualize radioactivity in proteins, gels were impregnated in scintillant (Amplify, Amersham), dried, and then fluorographed at - 7 0 ° C using Kodak XAR-5 film for the indicated times. Alternatively, samples were analysed by 2-dimensional polyacrylamide gel electrophoresis as described by O'Farrell (1975) using isoelectric focusing in tube gels (0.3 x 10 cm) as the first dimension and electrophoresis in I1 or 12.5% polyacrylamide, vertical slab gels (10 × 13 × 0.I cm unless stated otherwise) containing 0.1% sodium dodecyl sulphate, for the second dimension. Gels were fixed, dried and fluorographed as described above. Serum and choroid plexus incubations were also analysed under non-denaturing conditions using 10% polyacrylamide gels and the discontinuous buffer system of Laemmli and Favre (1973) except that sodium dodecyl sulphate was omitted from all buffers. Gels were stained with Coomassie Brilliant Blue R250 followed by the silver-staining procedure of Merril et al. (1981). Individual lanes were also cut out and the proteins subjected to electrophoresis in a second dimension in the presence of 0.1% sodium dodecyl sulphate as described above using 15% polyacrylamide gels. Thyroxine-binding proteins were analysed following incubation of serum at 20°C for 1 hr with 1 nM [t25I]thyroxine by electrophoresis under non-denaturing conditions, using 10% polyacrylamide gels and 0.05 M Tris-glycine pH 8.6 buffer. Gels were subjected to pre-electrophoresis for 90 min at 125 V. Electrophoresis was carried out at 125 V (5 hr, room temperature) with buffer recirculation to maintain constant pH throughout the gel. Gels were dried, then autoradiographed at - 7 0 ° C for the indicated times.

Estimation of protein concentration in serum Protein concentrations of animal sera were estimated by the microbiuret procedure of Itzhaki and Gill (1964) using defatted bovine serum albumin as the standard,

Purification of RNA and Northern analysis Livers were obtained within 5 min after death. Brains were kept at 4°C. Choroid plexus tissue was dissected from lateral, third and fourth ventricles, dissection being completed within 4 hr after death. Tissue samples were stored at -70°C until use. Total cellular RNA was purified from tissue as described (Dickson et al., 1985b). RNA was denatured and separated

Gene expression in choroid plexus by electrophoresis (with buffer circulation) in 1.2% agarose gel containing 6.6% formaldehyde (Dickson et aL, 1985b). After transfer to GeneScreen Plus membranes, RNA was hybridized at 42°C overnight with a eDNA probe which had been labelled with [~-32p]dATPby random hexanucleotideprimed synthesis (Fein,ber-~and Vogelstein, 1983, 1984). All other conditions for hybridization were as described earlier (Tu et al., 1990). RESULTS

Pattern o f proteins synthesized and secreted by choroid plexus The incorporation of [~4C]leucine has been used in previous work to investigate the synthesis and secretion of proteins by the rat choroid plexus (Dickson et al., 1986; Aldred et al., 1987; Thomas et al., 1989). This system was used in the present study to investigate the secretion of newly synthesized protein by the choroid plexus from mammals (cattle, sheep, pigs, rabbits, guinea-pigs, mice, rats), birds (chickens), reptiles (stumpy-tailed lizards) and amphibians (cane toads). Choroid plexus were removed, incubated with [14C]leucine, and proteins secreted into the medium were analysed as described in the Materials and

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Methods section. The data in Fig. 1 show that the choroid plexus from all vertebrates studied incorporate radioactivity at a high rate into a secreted protein which has an apparent subunit tool. wt of about 15,000, except for amphibians where the protein has a slightly larger apparent subunit mol. wt of 20,000. Control incubations containing 5 0 # M cycloheximide confirmed that incorporation of radioactivity was due to de novo protein synthesis. The 15,000 mol. wt polypeptide synthesized by rat choroid plexus has been identified by immunological techniques as the subunit of transthyretin (Dickson et al., 1986). To further characterize the transthyretin-like protein synthesized and secreted by vertebrate choroid plexus, the incubation media analysed on Fig. 1 were analysed by 2-dimensional polyacrylamide gel electrophoresis as described previously (Thomas et al., 1989). Representative results are shown in Fig. 2a--e, In mammals, the predominant protein synthesized and secreted by the choroid plexus was resolved into two components: a major form with an isoelectric point of about 6.2 and a minor form with an isoelectric point of about 5.8. This is typical of transthyretin present in either serum or cerebrospinal fluid

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"9,-FRONT Fig. 1. Comparison of proteins synthesized and secreted by choroid plexus of various animal species by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Choroid plexus, dissected from the species indicated, were incubated with [14C]leucine and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis as described in the Materials and Methods section. Resolving gel dimensions, 14 x 13 x 0.15crn. The gel was dried, then fluorographed for 7 days at -70°C. Samples analysed contained 4000 clam protein-associated radioactivity, except for lizard which contained 3000 cpm proteinassociated radioactivity. Protein standards used and subunit mol. wts assumed were phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100) and ct-lactalbumin (14,400). Control lanes, medium from rat and cane toad choroid plexus incubated with [z4C]leucinein the presence of 50 ~ M cycloheximide: volumes loaded were identical to those for the rat and cane toad experimental samples, respectively. CBPB ~/I--P

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(Thomas et al., 1989). In chicken, the same pattern of spots was observed, except that the isoelectric points were shifted by about 1 pH unit towards the acidic range (Fig. 2c). These two radioactive spots co-migrate with transthyretin purified from chicken serum (data not shown). In the choroid plexus incubations from stumpy-tailed lizards, the 15,000 mol. wt polypeptide was also resolved into two spots, a major spot and a minor, more acidic, form with isoelectric points of 5.7 and 5.4, respectively (Fig. 2d), whereas for cane toads, the 20,000 tool. wt two

polypeptide was resolved into a series of four to five spots with isoelectric points between 4.5 and 7.0 (Fig. 2e). Levels o f transthyretin m R N A in the choroid plexus f r o m different species From a comparison of the nucleotide sequences published previously (Mita et al., 1984; Dickson et al., 1985b; Wakasugi et al., 1985; Sundelin et al,, 1985; Fung et al., 1988; Duan et al., 1989; Tu et al., 1989) strong cross-hybridization among mammalian

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Fig. 2(e) Fig. 2.2-Dimensional polyacrylamide gel electrophoresis of choroid plexus secreted proteins for several animal species. Choroid plexus, dissected from the species indicated, were incubated with [~'C]leucine and analysed by 2-dimensional polyacrylamide gel electrophoresis as described in the Materials and Methods section. (a) Rat, (b) sheep, (c) chicken, (d) lizard and (e) cane toad. Protein-associated radioactivity in samples analysed: rat and chicken (5000 cpm), sheep, cane toad and lizard (10,000 cpm). Gels were dried and ftuorographed at -70°C: (a), (b), (c) and (e) 14 days, (d) 5 days.

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Fig. 3(c,d,e,f) Fig. 3. Northern analysis of transthyretin mRNA in liver (L), choroid plexus (CP) and brain without ehoroid plexus (RB) for various species. (RATI), Buffalo rats; (RAT2), Brown Norway rats. Ten microgrammes of total RNA were loaded into each well and separated by eleetrophoresis in 1.2% agarose gel, transferred to GeneSereen Plus membranes and hybridized to a sheep transthyretin eDNA probe (for human, sheep, dog, bovine, pig, rabbit, guinea-pig, mouse and rat RNA samples) or to a chicken transthyretin eDNA probe (for chicken RNA samples) as described in the Materials and Methods section. For mice, the results obtained with a C57 Black × CBA strain are presented in the figure. Similar results were obtained with inbred Balbc mice (figure not shown). Autoradiography for 3 hr at - 7 0 ° C with an intensifying screen. The positions of 28S and 18S ribosomal RNA bands are indicated at the right margins of the figures.

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PAUL J. HARMSet al.

species can be expected in Northern analysis of transthyretin mRNA. In preliminary experiments, this was found to be the case. Therefore, the proportions of transthyretin m R N A in total RNA from choroid plexus, total brain without choroid plexus ("rest of brain") and liver extracted from various mammalian species were measured using a heterologous sheep transthyretin cDNA probe (Tu et aL, 1989). For the analysis of chicken RNA an homologous chicken cDNA probe was used. The analysis of R N A extracted from human tissues shows that choroid plexus RNA contains a much higher proportion of transthyretin m R N A than R N A from liver (Fig. 3a): a strong band corresponding to transthyretin m R N A was seen in the track containing 10/~g R N A from choroid plexus, but only a weak signal was observed in the track containing 10/~g liver RNA. Longer exposures produced a distinct band for liver RNA, but led to gross over-exposure for the choroid plexus track. Similar results were obtained for all mammalian and bird species examined (Fig. 3b-f). In all species studied the RNA from choroid plexus contained a much greater proportion of transthyretin m R N A than the R N A from liver, but the extent of the difference varied between species, with rabbit (Fig. 3d) and chicken (Fig. 3e) appearing to have the strongest signal for the liver. A transthyretin m R N A of 700 bases was found in all species. Expression of transthyretin in the rest of the brain was examined, and in no case was transthyretin m R N A detected in the brain from which the choroid plexus had been removed. R N A extracted from mouse choroid plexus showed a second, distinct band which hybridized to transthyretin cDNA (Fig. 3f). Hybridization with oligo(dT) followed by treatment with ribonuclease H, which leads to removal of poly(A) tails, showed that the difference in the sizes of the two mouse transthyretin mRNAs was not due to differing lengths of poly(A) tails (data not shown). Neither mammalian nor avian transthyretin cDNA probes cross-hybridized to reptilian or amphibian choroid plexus RNA, under a variety of hybridization and washing conditions. Therefore, investigation of transthyretin synthesis by reptilian or amphibian choroid plexus by this approach was not possible.

plasma. Serum isolated from a reptile (stumpy-tailed lizard) and an amphibian (cane toad) was incubated with [125I]thyroxine, and the proteins were separated by polyacrylamide gel electrophoresis under nondenaturing conditions. Human serum incubated with [125I]thyroxine was analysed by electrophoresis in an adjacent lane for comparison. The thyroid hormone-binding proteins were demonstrated by autoradiography and their electrophoretic mobility compared with the prominent [14C]-labelled protein in the corresponding choroid plexus incubation medium (Fig. 4). In both cane toad and lizard serum, a single thyroxine-binding protein was detected (lanes 4, 5). The electrophoretic mobility of this protein corresponded in both cases to that of serum albumin, i.e. the major Coomassie-stained band in the serum (data not shown), and was similar to the electrophoretic mobility of human serum albumin. Both lizard and toad transthyretin-like proteins migrate faster than albumin under these conditions (i.e. as a "prealbumin") and with an electrophoretic mobility similar to that of human transthyretin. 2-Dimensional

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The above data show that the choroid plexus of mammals and birds contain high levels of transthyretin m R N A and synthesize and secrete a protein with a subunit mol. wt and an isoelectric point similar to that of transthyretin. In the rat this protein has been demonstrated to be serum transthyretin. Therefore, it is concluded that the choroid plexus of homeothermic vertebrates specializes in the synthesis and secretion of transthyretin (see Dickson et al., 1986). In reptiles and amphibians, the choroid plexus synthesizes a protein which is tentatively identified as transthyretin based on its electrophoretic behaviour and the fact that it is synthesized and secreted at a high rate by the choroid plexus. The question arises as to whether the transthyretinlike proteins from cane toad and stumpy-tailed lizard choroid plexus incubations are thyroxine carriers in

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phoresis of serum proteins and proteins synthesised by choroid plexus in ~itro. Serum: 4 #1 serum from indicated species, preincubated with 1 nM [~25I]thyroxine; C.P.: 5 #1 medium of choroid plexus from indicated species incubated with [~4C]leucine (containing the following protein-associated radioactivity: cane toad, 7600 cpm; lizard, 6300 cpm); C.P. (+Ser): 5#1 medium of cane toad choroid plexus incubated with [~4C]leucinemixed with 4/~1 serum from the same species, prior to electrophoresis. Autoradiography was for 10 days, -70°C with intensifying screen. Positions corresponding to human thyroxine-binding globulin (TBG), albumin (ALB) and transthyretin (TTR) are indicated.

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analysed by 2-dimensional electrophoresis as described in the Materials and Methods section, using non-denaturing 10% polyacrylamide gel electrophoresis in the first dimension and 15% polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulphate in the second dimension. Labelled proteins were detected by fiuorography for 7 days at -70°C. Molecular weight standards were as described in Fig. I; o, origin; e, end of gel; f, dye front. polyacrylamide gel electrophoresis was used to verify that this "prealbumin" seen in toad choroid plexus incubations has the same subunit mol. wt as the transthyretin-like protein resolved in Fig. 1 (see Fig. 5). No thyroxine-binding protein with an electrophoretic mobility similar to the transthyretin-like protein was observed in either toad or lizard serum under these conditions (Fig. 4; lane 4 compared with lane 2, and lane 5 compared with lane 6), or in experiments using conditions of electrophoresis described by Larsson et al. (1985) (data not shown). Mixing of serum with the choroid plexus incubation medium did not alter the electrophoretic mobility of the transthyretin-like protein from toad choroid plexus (Fig. 4; lane 2 compared with 3) and lizard choroid plexus (data not shown). These results demonstrate that transthyretin is not the major thyroid hormone-binding protein in serum of either reptiles or amphibians. Experiments were therefore carded out to determine the levels or lower limits of detection of transthyretin-like protein in lizard and toad serum by polyacrylamide gel electrophoresis and silver staining. The data in Fig. 6 show that a small amount of a protein with an electrophoretic mobility greater than that of albumin (and corresponding to the transthyretin-like protein secreted by the choroid plexus) is present in lizard serum, but not in toad serum. The staining intensity of the band corresponding to the transthyretin-like protein was compared with that of transthyretin in various dilutions of human serum analysed on the same gel. From this measurement it was concluded that the concentration of transthyretin-like protein in lizard serum is 2-5%, and in toad serum less than 0.5%, of the concentration of transthyretin in human serum.

DISCUSSION The unambiguous identification of serum proteins for inter-species comparisons, particularly when lower species are to be included in the comparison, has presented problems. The most widely used criterion for identifying and classifying serum proteins has been their electrophoretic mobility (cf. Engle and Woods, 1960) and specific binding of ligands (e.g. the binding of iron to transferrin or thyroid hormones to transthyretin, e.g. Larsson et al., 1985). Unambiguous identification for comparative purposes requires information about the primary structures of the proteins. This rigorous approach has been used for only a restricted number of plasma proteins. Identification by methods relying on primary structure has become more accessible in recent years by the introduction of specific cDNA probes and their use in hybridization to individual mRNAs in Northern analysis. This approach was used in the present work. Because of the strong conservation of the primary structure of the coding sections of plasma protein genes in mammals, a cDNA probe for sheep transthyretin could be used for identification of transthyretin m R N A in choroid plexus and liver R N A from a large number of mammalian species. Although sheep and rat transthyretin cDNA probes were found to cross-hybridize with avian transthyretin m R N A (data not reported), a chicken transthyretin cDNA probe was used in the Northern analysis of R N A from chicken liver, choroid plexus and the rest of the brain for reasons of greater sensitivity. The results show that transthyretin m R N A is present in large amounts in the choroid plexus of all mammals and birds investigated and that

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from cane toads synthesized a protein with slightly larger subunit tool. wt, but similar electrophoretic mobility to transthyretin from higher animals. Since transthyretin synthesis by the choroid plexus is highly conserved in birds and mammals, we have tentatively identified both of these proteins as transthyretins, although an unambiguous identification can only be made when sequence data become available. It remains to be determined whether either protein binds thyroxine. The presumptive transthyretin was not detected in toad serum and was present in only very low amounts in lizard serum. These observations are consistent with the interpretation that expression of the transthyretin gene appeared first in evolution in the choroid plexus, and only later in the liver, perhaps correlated with the role of thyroxine in regulation of the rate of oxidation and heat production (homeothermy).

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Fig. 6. Non-denaturing 10% polyacrylamide gel electrophoresis of lizard and cane toad serum. Twenty microlitres of serum were analysed by polyacrylamide gel electrophoresis under non-denaturing conditions as described in the Materials and Methods section. Gels were stained with Coomassie Brilliant Blue R250, followed by siNer-staining, then individual lanes scanned with a Molecular Dynamics Model 300A computing densitometer. (A) Lizard serum (0.88 mg protein), (B) cane toad serum (0.44 mg protein). The peak corresponding to serum albumin in each species is indicated. the choroid plexus synthesizes and secretes a protein which, in rats, has been shown to be transthyretin. In addition, in all cases, expression of the transthyretin gene in the brain was located exclusively in the choroid plexus and the proportion of m R N A which codes for transthyretin was much higher in R N A preparations from choroid plexus than in R N A extracts from livers. It is concluded that the predominant protein synthesized and secreted by the choroid plexus of homeotherms is transthyretin which is presumably secreted into the cerebrospinal fluid, as has been demonstrated in sheep (Schreiber et al., 1990). Failure to obtain hybridization between reptile and amphibian transthyretin m R N A and mammalian and avian c D N A probes, meant that the expression of the transthyretin gene in choroid plexus of stumpytailed lizards and cane toads could not be analysed by this approach in the present study. However, choroid plexus from stumpy-tailed lizards synthesized and secreted large quantities of a protein with a similar subunit size, isoelectric points and electrophoretic mobility under non-denaturing conditions as transthyretin of higher animals, and the choroid plexus

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