Proc. Nadl. Acad. Sci. USA Vol. 82, pp. 3712-3715, June 1985 Evolution A truncated immunoglobulin e pseudogene is found in gorilla and man but not in chimpanzee (qualitative molecular evolution/human evolution) SHINTAROH UEDA*, OSAMU TAKENAKAt, AND TASUKU HONJO0§ *Department of Anthropology, Faculty of Science, The University of Tokyo, Tokyo 113, Japan; tDepartment of Biochemistry, Primate Research Institute, Kyoto University, Aichi 484, Japan; and tDepartment of Genetics, Osaka University Medical School, Osaka 530, Japan Communicated by Motoo Kimura, January 22, 1985 ABSTRACT Molecular genetic analyses ofthe young pseu- able to provide a qualitative answer to determine the branch- dogenes of the immunoglobulin CE genes were carried out to ing sequence. We studied the organization of the CE genes of obtain qualitative evidence for the phylogenetic branching nonhuman primates, including 13 species of Old World pattern of hominoid primates. We found that Old World monkeys and 5 species of hominoids, to analyze their monkeys had two C. genes, one ofwhich was processed. Among evolutionary relationships to man. the hominoids examined only the gorilla and human genomes contained three C, genes: an active, a truncated, and a MATERIALS AND METHODS processed gene. Other hominoids so far examined, including chimpanzee, contained two C, genes: one active and the other The species of nonhuman primates examined include 13 processed. These results suggest that the processed C, pseudo- species of Cercopithecoidea (Old World monkeys): Macaca gene was generated before the divergence between Old World fuscata (Japanese monkey), Macaca mulatta (rhesus mon- monkeys and hominoids and that the gorilla is more closely key), Macaca fascicularis (crab-eating monkey), Macaca related to man than the chimpanzee is, unless the chimpanzee arctoides (red-faced macaque), Macaca nemestrina (pig- has lost the CE2 gene after the divergence of this species. tailed macaque), Macaca cyclopis (Formosan monkey), Macaca radiata (bonnet monkey), Macaca assamensis (As- The immunoglobulin genes in the heavy-chain constant (CH) samese monkey), Theropithecus gelada (gelada), Papio region cluster are divided into five classes, C,,, C8 C,, C., and anubis (anubis baboon), Papio hamadryas (hamadryas ba- Ca. There are three CE genes in the human genome (1-3). We boon), Erythrocebus patas (patas monkey), and Cercopith- have called the CE genes in the 2.7-, 5.9-, and 8.0-kilobase (kb) ecus aethiops (green monkey); and 5 species of Hominoidea BamHI fragments the CE1, CE2, and CQ3 genes, respectively (hominoids): Pan troglodytes (chimpanzee), Gorilla gorilla (2, 4, 5). One of them (C71) is active, whereas the remaining (gorilla), Pongopygmaeus (orangutan), Hylobates lar (white- two are pseudogenes. One pseudogene (CE2) is truncated by handed gibbon), and Hylobates agilis (agile gibbon). recombination (3, 4) and the other (CE3) is processed (5, 6). DNA was prepared from lymphocytes of peripheral blood The latter lacks introns entirely and is translocated from (8), except for DNAs of orangutan and gorilla. DNA of chromosome 14 to 9, suggesting that this gene was created by orangutan was prepared not only from lymphocytes but also reverse transcription of an aberrantly transcribed Cf se- from an Epstein-Barr-virus-transformed cell line ofthe same quence. Since the CH gene family of mouse contains only one individual. DNA of gorilla was obtained from lymph nodes CE gene (7), the creation of two CE pseudogenes in the human and further purified by using equilibrium sedimentation in a genome seems to have taken place after mammalian radia- cesium chloride density gradient. Two micrograms of DNA tion. Comparison of the nucleotide sequences (4, 5) allowed was digested with appropriate amounts of restriction en- us to estimate that the CE2 and CE3 genes diverged from the zymes, electrophoresed in 0.5% agarose gels, and transferred CE1 gene 6.6-8.9 x 106 and 39 x 106 years ago, respectively. to nitrocellulose filters (9). DIAA fragments used as probes The primate superfamily Hominoidea includes man, the were labeled with [a-32P]dCTP by nick-translation to a chimpanzee, the pygmy chimpanzee, the gorilla, the orangu- specific activity of500-1000 cpm/pg ofDNA (10). Hybridiza- tan, and the gibbons. The branching sequence ofthe lineages tion was carried out in 1 M NaCl at 650C as described and the datings of the divergence nodes are still in dispute. A previously (11) and filters were washed three times (30 min large number of studies on hominoid relationships have been each) in 0.15 M NaCl/0.015 M sodium citrate/0.1% sodium based on morphology, fossils, behavior, and molecular com- dodecyl sulfate at 650C. parison. Usually the most powerful evidence for the study of phylogeny is derived from fossil records and molecular RESULTS comparisons, both of which have, unfortunately, limitations for studies on hominoid evolution. The fossil records of The organization of CE genes in DNAs from various species hominoids, especially nonhuman hominoids, are too scarce of primates was studied by the Southern hybridization to draw a definitive conclusion. Comparison of amino acid method, using human CE probes. The number of C, genes in and nucleotide sequences ofprimates is not convincing either the genome of each species was estimated by using a 1.2-kb because the divergence of the sequences is too small to BamHI/Hpa I fragment of the human CE1 gene as probe quantitate accurately a small difference in the branching time. (probe A shown in Fig. 1), while the processed CE gene was In this report we present another strategy to determine the analyzed by using a 1.0-kb Acc I/Acc I fragment ofthe human branching sequence of the lineages: using human CE pseudo- CE3 gene as probe (probe B shown in Fig. 1). Probe A genes, which evolved very recently. The presence or the cross-hybridized with the other CE genes, while probe B was absence of young pseudogenes in DNA of various species is specific for the processed CE gene under the stringent washing The publication costs of this article were defrayed in part by page charge Abbreviation: kb, kilobase. payment. This article must therefore be hereby marked "advertisement" §Present address: Department of Medical Chemistry, Faculty of in accordance with 18 U.S.C. §1734 solely to indicate this fact. Medicine, Kyoto University, Kyoto 606, Japan. 3712 Downloaded by guest on September 30, 2021 Evolution: Ueda et al. Proc. Natl. Acad. Sci. USA 82 (1985) 3713 BamHI BglU Hpal A Ce1 -A r_. "-"-F-} probe A probe B A 1 2 3 4 5 1 2 3 4 5 C- Accl AccI i. CM 2 2 1 3 1 31 * 7.6 W B i 7.14 ig 7 l 200 bp lp 7. FIG. 1. Structure of the human CGj, CE2, and CE3 genes. The CH exons are shown by open boxes with domain numbers. The 5' flanking region homologous in the CGl and CQ3 genes and the 3' untranslated sequence are shown by hatched and open boxes, respectively. The regions homologous to the CQ1 gene in the C,2 and Cf3 genes are shown by rectangles, while nonhomologous regions are shown by lines. Fragments (A, B, and C) used as probes are indicated B by horizontal bars. bp, Base pairs. probe A probe C 1 2 3 4 5 4 5 conditions, although another, very weak, band correspond- ing to the Cj1 gene sometimes appeared under the mild washing conditions. Probe A detected two hybridizing bands in BamHI digests 15- , of all the Old World monkey DNAs examined (Fig. 2A): 2.7- 8. and 7.1-kb bands in all the macaques, baboons, and gelada; -7:0 6.9-;-.28.2 2 n -8.0 7.1- and 7.4-kb bands in the patas monkey; and 7.1- and -5.6 -5.9 7.6-kb bands in the green monkey. Probe B detected only 7.1-kbBamHI bands in all the Old World monkey DNAs (Fig. _ 272 7- -2 7 2.7-__ -2.7 2A). As shown in Fig. 2B, BamHI digests ofgibbon, orangu- tan, and chimpanzee DNAs contained two bands, each hybridizing with probe A. BamHI bands other than 2.7 kb were also detectable with probe B (data not shown). We examined 11 individual chimpanzee DNAs, but there was no C variation in hybridization patterns. probe A probe C In contrast to the hominoid DNAs mentioned above, there were three bands (2.7, 6.9, and 15 kb) hybridizing with probe 1 2 3 4 5 6 7 6 7 A in BamHI digests ofthe gorilla DNA (Fig. 2B). When probe B was used, the 15-kb BamHI band was shown to contain the -30 2:'# 2;SV processed C. gene (data not shown). So we examined -30 30 302=25 whether gorilla DNA contained a truncated CE (CE2) gene as 22-7 in the human genome. To identify the CE2 gene, which lacks -9.2 -7 7 9.2- 992 92-4*O- 9.2 7.7 the CH1 and CH2 exons in addition to their flanking sequences 'O (3, 4), we have used as probe the 0.65-kb BamHI/Bgl II -6.8 human Cjl gene fragment (probe C shown in Fig. 1), which 6.8 4- is deleted in the human CE2 gene. Since probe C cross-hybrid- izes with the CE3 gene, which contains the pseudo-CH1 and Mi..* pseudo-CH2 exons, the Cf gene fragments that do not hybridize with probe C are the truncated Cf2 gene fragments. These are the 5.9-kb BamHI band ofthe human DNA and the FIG. 2. Southern hybridization of primate DNAs with human C, 6.9-kb BamHI band of the gorilla DNA (Fig. 2B). In DNAs probes. Numbers indicate sizes of the hybridizing fragments in kb.