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Identification and Characterization of Amelogenin Genes in Monotremes, Reptiles, and Amphibians (Tooth Formation͞evolutionary Innovations)

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Identification and Characterization of Amelogenin Genes in Monotremes, Reptiles, and Amphibians (Tooth Formation͞evolutionary Innovations) Proc. Natl. Acad. Sci. USA Vol. 95, pp. 13056–13061, October 1998 Evolution Identification and characterization of amelogenin genes in monotremes, reptiles, and amphibians (tooth formationyevolutionary innovations) SATORU TOYOSAWA*†,COLM O’HUIGIN*, FELIPE FIGUEROA*, HERBERT TICHY*, AND JAN KLEIN*‡ *Max-Planck-Institut fu¨r Biologie, Abteilung Immungenetik, Corrensstrasse 42, D-72076 Tu¨bingen, Germany Edited by Susumu Ohno, Beckman Research Institute of the City of Hope, Duarte, CA, and approved August 25, 1998 (received for review June 8, 1998) ABSTRACT Two features make the tooth an excellent tified, one on the X and the other on the Y chromosome model in the study of evolutionary innovations: the relative (10–15); in rodents, only the X linked gene has been found (14, simplicity of its structure and the fact that the major tooth- 15). Immunohistochemical analysis has indicated the possible forming genes have been identified in eutherian mammals. To existence of amelogenin-like compounds in reptiles and am- understand the nature of the innovation at the molecular level, phibians (16, 17). The nature of an enamel-like material, it is necessary to identify the homologs of tooth-forming genes sometimes referred to as enameloid, remains unresolved and in other vertebrates. As a first step toward this goal, homologs controversial. As part of a systematic effort to elucidate the of the eutherian amelogenin gene have been cloned and emergence of teeth in evolution, we have initiated a study characterized in selected species of monotremes (platypus and aimed at cloning the major tooth-forming genes of species echidna), reptiles (caiman), and amphibians (African clawed representing gnathostome classes. Here we describe ameloge- toad). Comparisons of the homologs reveal that the ameloge- nin-encoding cDNA clones isolated from reptiles and amphib- nin gene evolves quickly in the repeat region, in which ians as well as single-exon PCR products from monotremes. numerous insertions and deletions have obliterated any sim- ilarity among the genes, and slowly in other regions. The gene organization, the distribution of hydrophobic and hydrophilic MATERIALS AND METHODS segments in the encoded protein, and several other features Source and Isolation of DNA. DNA samples from platypus have been conserved throughout the evolution of the tetrapod (Ornithorhynchus anatinus) and the short-nosed echidna amelogenin gene. Clones corresponding to one locus only were (Tachyglossus aculeatus) were provided by Robert W. Slade found in caiman, whereas the clawed toad possesses at least (Queensland Institute for Medical Research, Royal Brisbane two amelogenin-encoding loci. Hospital, Australia). Tissue samples from 3-day-old smooth- fronted caimans (Paleosuchus palpebrosus) and adult African One of the major innovations accompanying the emergence of clawed toads (Xenopus laevis) were kept frozen at 270°C until jawed vertebrates was the development of teeth, presumably their use. Genomic DNA was isolated from the tissues by from dermal scales (1). The concurrent development of jaws phenol-chloroform extraction (18). and teeth generated a new type of feeding device—biting cDNA Library Construction and Screening. The caimans and structures that enabled gnathostomes to colonize new envi- the African clawed toads were killed under anesthesia, and their ronmental niches and thus allow their adaptive radiation. To jaws were removed and immediately frozen in liquid nitrogen. understand how this innovation occurred, it is necessary to The frozen tissues were homogenized to a fine powder, and total delineate the evolutionary history of the genes involved in RNA was extracted (19). Poly(A)1 RNA isolation and cDNA tooth development, in particular those responsible for the synthesis were performed with the mRNA purification kit (Phar- formation of the two characteristic tooth components, the macia) and the TimeSaver cDNA synthesis kit (Pharmacia), enamel and the dentin. Tooth enamel is one of the most highly respectively. The cDNA was inserted into the EcoRI-digested l mineralized tissues known (2). It is formed in the early stages gt10 vector (Stratagene), and the cDNA library was in vitro- of odontogenesis by ameloblasts, which synthesize and secrete packaged with the help of the Gigapack cloning kit (Stratagene) several matrix proteins. Later, during the stage of enamel and used to transform competent Escherichia coli NM514 bac- maturation, the production of matrix proteins wanes, and the teria. The initial titers of the libraries were 1.2 3 106 plaque- proteins already produced are gradually replaced by hydroxy- forming units (pfu) for the caiman, and 6 3 105 pfu for the apatite crystals. The main matrix protein, amelogenin, is African clawed toad. The caiman and African clawed toad thought to be involved in the regulation of enamel crystallite libraries were amplified once to a titer of 1.5 3 1011 pfuyml and formation, presumably by providing the hydrophobic environ- 1.8 3 1011 pfuyml, respectively. ment necessary for the initiation and growth of calcium PCR Amplification. hydroxyapatite crystals (2). The amelogenin genes of the Amelogenin-encoding cDNA clones have been isolated monotremes (platypus, echidna) and the caiman were ampli- from several representatives of mammals, including humans fied by using primers based on a comparison of human, cattle, (3), cattle (4), pig (5), rat (6), mouse (7), and opossum (8). The presence of amelogenin-encoding genes in wallaby (9) and This paper was submitted directly (Track II) to the Proceedings office. platypus (9) has been indicated by Southern blot hybridization, Abbreviations: Myr, million years; pfu, plaque-forming units; UTR, untranslated region. but no sequence has been presented to date. In the bovids and Data deposition: The sequences reported in this paper have been anthropoid primates, two amelogenin genes have been iden- deposited in the GenBank database [accession nos. AF095566 (platy- pus), AF095567 (echidna), AF095568 (caiman), AF095569 (toad-1), The publication costs of this article were defrayed in part by page charge and AF095570 (toad-2)]. †Permanent address: Department of Oral Pathology, Osaka Univer- payment. This article must therefore be hereby marked ‘‘advertisement’’ in sity Faculty of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, accordance with 18 U.S.C. §1734 solely to indicate this fact. Japan. © 1998 by The National Academy of Sciences 0027-8424y98y9513056-6$2.00y0 ‡To whom reprint requests should be addressed. e-mail: jan.klein@ PNAS is available online at www.pnas.org. tuebingen.mpg.de. 13056 Downloaded by guest on October 2, 2021 Evolution: Toyosawa et al. Proc. Natl. Acad. Sci. USA 95 (1998) 13057 pig, rat, mouse, and marsupial sequences. In the case of the broth, and minipreps were prepared according to the standard monotremes, primers AM1 (sense; 59-TATGGTTACGAA- protocol (20). Two to five micrograms of DNA were used in the CCCATGGGTGGATGG-39) and AM2 (antisense; 59- dideoxy sequencing reactions with the AutoRead sequencing ATCCACTTCTTCCCGCTTGGTCTTGTC-39) were used to kit (Pharmacia). The reactions were then processed by the amplify 420-bp fragments of the amelogenin exon 6 sequence. Automated Laser Fluorescent (ALF) sequencer (Pharmacia). In the caiman, primers AM6 (sense; 59-GAA-CCCATGGGT- To determine the exon-intron organization of the caiman and GGATGGCTGCACCA-39) and AM2 were used for the orig- toad amelogenin genes, the genomic sequence data were inal amplification, and primers AM6 and Tu1360 (antisense; compared with the cDNA sequences and exon–intron bound- 59-GGCAGCAGTGGGGGCAGAGGCTG-39) were then aries were identified by comparison with the published con- used for half-nested PCR to amplify a 370-bp fragment of sensus sequences (21). amelogenin exon 6 sequence from genomic DNA. The primers For Southern blots, 10 mg of genomic DNA was digested with AM8 (sense; 59-CAGACTCTCACACCTCACCACCA-39) the appropriate restriction enzyme, and the resulting fragments and AM7 (antisense; 59-TTGTTGCTGTGGTATAGGCAT- were separated in 0.8% agarose gels (GibcoyBRL) and trans- CAT-39), designed within the 370-bp product amplified by the ferred to a hybridization membrane (Hybond-N1, Amersham) by primers above, were used to recover inserts by anchored PCR. using the VacuGene blotting system (Pharmacia). The filters In the case of the African clawed toad, primer AM10 (sense; were incubated for5hinaprehybridization solution containing 59-CCTGGTTATGTCAACTTCAGTTATGA-39), based on 13 Denhardt’s solution (0.02% polyvinylpyrrolidoney0.02% Fi- the caiman amelogenin sequence, was used to amplify a colly0.02% BSA)y53 standard saline citrate (SSC, 13 SSC 5 fragment of about 600 bp by anchored PCR. The primer AM22 0.15 M sodium chloridey0.015 M sodium citrate, pH 7)y0.2% (antisense; 59-CATCATAGATTGGTA-CCATTT-39), de- SDSy5 3 106 cpm of the probe, which was labeled by using the signed within the fragment of the toad amelogenin product random-priming method with 32P to a specific activity of 1 3 109 amplified by the above primers, was used to recover the 59 cpmymg with the Ready-To-Go DNA labeling kit (Pharmacia). region of the inserts by anchored PCR. To determine the Filters were washed in 23 SSCy0.1% SDS for 20 min at room organization of the caiman and toad amelogenin genes, prim- temperature and then used to expose XAR5 film (Kodak) with ers were designed to amplify regions of the individual exon– intensifying screens for 3–5 days. intron boundaries (Table 1). The primer AM57 (antisense; Data Analysis. The nucleotide sequences and inferred pro- 59-ATTCTGGCTCTCGTGGTCAGGTTT-39) was used to tein sequences were aligned with the aid of the GCG package confirm the identities of the second toad amelogenin clone. (Genetic Computer Group, Madison, WI) and the SEQPUP (ref. Genomic DNA (100 ngyml) or lysate of the cDNA libraries (1 22; available at http:yyiubio.bio.indiana.eduysoftymolbio) ml) were amplified by PCR in 50 ml of PCR buffer (1.5 mM computer program. The evolutionary relationships were then y m y MgCl2 200 M dNTP 10 mM Tris, pH 8.5) in the presence evaluated by the neighbor-joining algorithm (23). of the sense and antisense primers and 2.5 units of Taq polymerase (Pharmacia).
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