Proc. Natl. Acad. Sci. USA Vol. 96, pp. 10267–10271, August 1999 Evolution Late changes in spliceosomal introns define clades in vertebrate evolution BYRAPPA VENKATESH*†,YANA NING‡, AND SYDNEY BRENNER*‡ *Marine Molecular Genetics Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609; and ‡Molecular Sciences Institute, 2168 Shattuck Avenue, Berkeley, CA 94704 Contributed by Sydney Brenner, July 2, 1999 ABSTRACT The evolutionary origin of spliceosomal in- in the rhodopsin gene, which has been shown to contain introns trons has been the subject of much controversy. Introns are in mammals (22) and in primitive chordates such as lampreys proposed to have been both lost and gained during evolution. (23) and skates (24), but not in some teleosts (25). If the gain or loss of introns are unique events in evolution, they can serve as markers for phylogenetic analysis. We have METHODS made an extensive survey of the phylogenetic distribution of seven spliceosomal introns that are present in Fugu genes, but PCR. Genomic DNA was extracted from fresh or frozen not in their mammalian homologues; we show that these tissues by using standard protocols. Gene fragments were introns were acquired by actinopterygian (ray-finned) fishes amplified by PCR with AmpliTaq DNA polymerase (Perkin– at various stages of evolution. We have also investigated the Elmer) or the Expand Long Template PCR system (Boehr- intron pattern of the rhodopsin gene in fishes, and show that inger Mannheim). A typical PCR cycle consisted of an initial the four introns found in the ancestral chordate rhodopsin denaturing step of 92°C for 2 min, 35 cycles of 92°C for 30 sec, gene were simultaneously lost in a common ancestor of 55°C for 1 min, and 72°C for 2 min, followed by a final ray-finned fishes. These changes in introns serve as excellent elongation step at 72°C for 6 min. For some combinations of markers for phylogenetic analysis because they reliably define primers and templates, higher (60°C) or lower (50°C) anneal- clades. Our intron-based cladogram establishes the difficult- ing temperatures were used to optimize the amplification to-ascertain phylogenetic relationships of some ray-finned conditions. The following sense and antisense primers com- fishes. For example, it shows that bichirs (Polypterus) are the plementary to the exons flanking the intron were used in PCR. sister group of all other extant ray-finned fishes. Growth hormone intron 4a (Gh4a), TGYTTYAARAAR- GAYATG and AGRTANGTYTCNACCT flanking the Fugu Two competing theories have been proposed to explain the growth hormone gene from codon 175 to 177 (15) (the antisense primer includes two bases complementary to AG, origin of spliceosomal introns, which are widespread in eu- Ј karyote genomes but absent from prokaryotes. The ‘‘introns which are the invariant 3 end bases of introns, and thus no early theory’’ states that the introns are ancient and have been PCR product was obtained when the gene lacked the intron). lost in different lineages (1, 2). On the other hand, the ‘‘introns Mhc class II B-chain intron 2a (Mhc2a), TGCWGYGYR- late theory’’ maintains that the spliceosomal introns were TAYGRSTTCTACCC and AGGCTKGKRTGCTCCACC- inserted into the eukaryote genes later in evolution (3–6). WRRCA (extended primers Tu97 and Tu40) (26). Although the distribution of intron phases and the correlation Mixed lineage leukemia intron 25a (Mll25a), GCNCGNTC- between intron positions and protein module boundaries have NAAYATGTTYTTYGG and ATRTTNCCRCARTCRT- been proposed as evidence for the ancient origin of introns (2), CRCTRTT flanking the Fugu Mll gene from codon 3124 to the restricted phylogenetic distribution of some introns sug- 3376 (16). gests that they arose late in evolution (7–10). Dystrophin intron 6a (Dyst6a), ATGGCNGGNYTNCAR- We and others have identified extra spliceosomal introns in CARAC and GCNARNCCRTCRTTCCARCT flanking the the pufferfish (Fugu) and some other teleosts that are absent human dystrophin gene from codon 134 to 166 (27). from mammals (refs. 11–18; B. Peixoto and S.B., unpublished Dystrophin intron 10a (Dyst10a), TAYCARACNGCNYT- work). Likewise, extra introns were also found in some mam- NGARGA and TGNGTRTGRAAYTGNTCYTT flanking malian genes (16, 19, 20). These discordant introns could be the human dystrophin gene from codon 350 to 376 (27). the result of either loss of ancestral introns or gain of novel RAG1 intron a (RAG1a), AARTTYTCNGANTG- introns in different lineages. Because the spliceosomal introns GAARTT and ACRTCNACYTTRAANACYTT flanking are not self-splicing and are not known to be mobile, the loss the zebrafish RAG1 gene from codon 27 to 157 (21). or the gain of spliceosomal introns in a lineage is likely to be RAG1 intron b (RAG1b), TTYGCNGARAARGAR- a unique event, occurring at a specific point in its evolution; GARGG and TACATYTTRTGRTAYTGRCT flanking the hence it might serve as a decisive marker for evolutionary zebrafish RAG1 gene from codon 456 to 511 (21). studies. To evaluate and confirm whether the extra vertebrate Rhodopsin, CCNTAYGAYTAYCCNCARTAYTA and introns are the result of a loss or gain of introns, we made an TTNCCRCARCAYAANGTNGT flanking the teleost rho- extensive survey of the phylogenetic distribution of seven of dopsin gene from codon 30 to 319 (25). these introns, including one each from the growth hormone gene (15), the major histocompatibility class II B-chain (Mh- Abbreviations: Dyst6a and Dyst10a, novel introns found in the Fugu cII) gene (13) and the mixed lineage leukemia-like (Mll) gene dystrophin gene; Gh4a, fifth intron in the growth hormone gene; Mhc2a, intron 2a in the Mhc class II ␤-chain gene; Mll25a, intron 25a (16), and two each from the dystrophin gene (14) and the in the Fugu mixed lineage leukemia gene; RAG1a and RAG1b, novel RAG1 gene (21). We also analyzed the distribution of introns introns in the RAG1 gene. Data deposition: The sequences reported in this paper have been The publication costs of this article were defrayed in part by page charge deposited in the GenBank database (accession nos. AF134595– AF134630; AF134919–AF134976; AF137083–AF137130; AF137132– payment. This article must therefore be hereby marked ‘‘advertisement’’ in AF137262; AF142553–AF142564; and AF148142–AF148144). accordance with 18 U.S.C. §1734 solely to indicate this fact. †To whom reprint requests should be addressed. E-mail: mcbbv@ PNAS is available online at www.pnas.org. imcb.nus.edu.sg. 10267 Downloaded by guest on October 1, 2021 10268 Evolution: Venkatesh et al. Proc. Natl. Acad. Sci. USA 96 (1999) FIG. 1. Phylogenetic distribution of spliceosomal introns. The phylogenetic tree is based on Nelson’s classification of fishes (28). A plus sign indicates that no PCR fragment was obtained, presumably (ء) ϩ) indicates presence and a minus sign (Ϫ) indicates absence of intron. An asterisk) because of the large size of the intron. The letter ‘‘a’’ indicates a failure to clone by PCR because of the highly variable coding sequence in this region of RAG1. Data from previous studies (Gh4a, refs. 11 and 15; Mll25a, ref. 16; Dyst6a and Dyst10a, ref. 14; Mhc2a, refs. 12, 13, and 29; RAG1a and RAG1b, B. Peixoto and S.B., unpublished work and refs. 21 and 30; Rhod, refs. 22, 24, and 25) are enclosed in parentheses. Gh4a, (15); Mll25a, Downloaded by guest on October 1, 2021 Evolution: Venkatesh et al. Proc. Natl. Acad. Sci. USA 96 (1999) 10269 FIG. 2. Cladogram showing the phylogenetic relationships of fishes, inferred by using the presence or absence of introns as character states. The numbers in the boxes represent introns cloned by us (1, rhodopsin; 2, RAG1b; 3, RAG1a; Gha4a; 5, Mll25a; 6, Dyst10a; 7, Dyst6a; and 8, Mhc2a). B is an extension of the Division Teleostei from A. The rhodopsin primers amplified the gene when there was the major groups of ray-finned fishes; we confirmed the no intron present but failed to amplify the gene when introns presence or absence of an intron by cloning and sequencing the were present (e.g., Polypterus), presumably because of the large PCR products. We also cloned the introns from shark͞torpedo size of the gene that contained introns. The presence of intron (Chondrichthyes) which are the common ancestors of ray- in the Polypterus (bichir) rhodopsin gene was subsequently finned fishes and the tetrapods. Fig. 1 summarizes our results confirmed by amplifying and sequencing the second intron and shows that most of the intron gain or loss events occur at together with its flanking exons by using the primers comple- unique points in the lineage. mentary to the flanking exons (GTNGTNTTYACNTG- The MhcII locus is an apparent exception to this general GATHATGGC and CCRCANGARCAYTGCATNCCYTC). rule. Sequences without introns and sequences with small The absence of Gh4a intron from the Torpedo (electric ray), introns can always be amplified by PCR. However, if a large Lepisosteus (gar), and Amia (bowfin) was confirmed by cloning intron is present, we often fail to obtain a PCR product. Thus and sequencing the growth hormone gene fragments spanning we find MhcII fragments (more than one in each species) with exon 3, intron 3, and exon 4 by PCR with primers specific for no intron until we encounter paracanthopterygians, for which the genes. Because of the sequence similarity between the no product was obtained. We conclude that the paracan- dystrophin and its related protein utrophin, the dystrophin thopterygian MhcII has an intron, but it is too large to be primers that we used also amplified the corresponding utro- amplified. In Mugil (mullet) and some other fishes including phin fragments. Utrophin fragments from all of the fishes were the tetraodontoid boxfish (Ostracion), two PCR products were cloned and sequenced, and none contained introns at positions found, one without and one with an intron. corresponding to Dyst6a and Dyst10a (data not shown). In other recent fishes only the intron-containing fragments Cloning and Sequencing.