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Volume 14 Number 22 1986 Nucleic Acids Research Linkage arrangement in the vitellogenin gene family of Xenopus laevis as revealed by gene segregation analysis Jean-Luc Schubiger* and Walter Wahli Institut de Biologie Animale, University de Lausanne, Batiment de Biologie, CH-1015 Lausanne, Switzerland Received 25 September 1986; Accepted 21 October 1986 ABSTRACT Using restriction fragment length polymorphism (RFLP) we have analyzed the segregation of alleles of the different vitellogenin genes of Xenopus laevis. The results demonstrate that the four genes whose expression 1s controlled by oestrogen, form two linkage groups. The genes Al, A2 and Bl are linked genetically whereas the fourth gene, the gene B2, segregates Independently. The possible origin of this unexpected arrangement 1s discussed. INTRODUCTION Vitellogenin, the precursor of the yolk proteins, 1s encoded 1n a small family of four related genes 1n Xenopus laevis (1). These genes are strictly regulated by oestrogen 1n the liver of mature females where they are coordinately expressed (2,3). The four genes can be divided Into two main groups (A and B) showing a sequence divergence of about 20% within the coding regions. Each group can then be subdivided into two closely-related subgroups (Al and A2, Bl and B2) that show approximately 5% sequence divergence 1n the exon sequences. The 201 divergence between the A and B genes suggest that an A and a B gene arose by a first duplication of an ancestral gene about 150 million years ago (4). Later, about 30 million years ago, a whole genome duplication seems to have occurred 1n Xenopus laevis (5,6). If this were the case, it would have resulted in an A-B gene linkage on one chromosome and a second A-B gene linkage on a second chromosome that arose during the genome duplication event (7). The latter event would have generally produced pairs of genes 1n Xenopus laevis, with each member of a given pair on a separate chromosome. In agreement with this proposal, pairs of related genes have been observed that code for © IRL Press Limited, Oxford, England. 8723 Nucleic Acids Research albumins (8), giobins (9), ribosomal proteins (10,11) and actins (12). Structural studies of the four vitellogenin genes have revealed some features which are consistent with the proposed model of their evolution, while some others contradict 1t. The strongest support comes from the linkage between the genes Al and Bl (7), and from the similar degree of divergence within the homologous exons of the A1/A2 and B1/B2 gene pairs (1). In contrast, the corresponding introns and flanking regions of the A genes are much more different than the corresponding introns and flanking regions of the B genes (13,14). This is inconsistent with a simultaneous appearance of the two A genes and the two B genes as a result of a genome duplication. Clearly, the origin of this gene family could be better understood once Its genomic organization has been elucidated. Furthermore, knowledge of the organization of the vitellogenin genes might also be beneficial for analyses of their oestrogen dependent transcriptional induction, as well as their acquisition of inducibility during development (15,16). Using RFLPs as allelic markers of different members of the gene family, we have determined the linkage groups of these genes as a first step towards this goal. MATERIAL AND METHODS Animals The adult animals that were analyzed were obtained from the African Xenopus Facility (Clareinch, South Africa 7740). Analysis of genomic DNA DNA was prepared from either erythrocyte or hepatocyte nuclei, which were isolated from adult Xenopus, tadpoles at stage 60-64 (17) or nine month-old froglets. The DNA was digested with a 5 to 10-fold excess of restriction endonuclease, electrophoresed on a 0.7S or 0.8S agarose gel and transferred to a nitrocellulose filter (18). Preparation of radioactive probes H13 clones containing the fragments Indicated 1n the text were used to prepare probes. Single strand "+" DNA was Isolated, annealed with a primer complementary to the 5' region of the M13 multiple cloning sites and the primer was elongated with the Klenow fragment of £. coli DNA polymerase in 8724 Nucleic Acids Research the presence of a - [ 32 P] - dATP (3,000 C1/mmol), unlabelled dCTP, dGTP and dTTP for 60 m1n at 15°C as described (19). The labelled probes had a Q specific activity of 1-2 x 10 cpm per gg of input DNA. Preparation of nick Q translated DNA probes (specific activity 1-3 x 10 cpm/ug) was performed as described (20). Hybridization with radioactive probes Prehybr1dizat1on, hybridization and washing were performed as described (21). Alternatively, the filters hybridized with dextran sulfate were prehybridized at 40°C for 4-16 hours 1n Stark's buffer (5x SSC, [1 x SCC = 150 mM NaCl, 15 mM sodium citrate], 25 mM sodium phosphate pH 6,5, 1 x Denhardt's solution [ref. 22], 250 ug/ml denaturated salmon sperm DNA, 50S formamide and 2% glycine; ref. 23). Hybridization was performed at 40°C in 4 volumes of Stark's buffer and 1 volume of 50* dextran sulfate for 20-60 hours in the presence of 1-3 x 10 cpm of probe per ml of hybridization hours in the presence of 1-3 x 10 cpm of probe p< solution. Washing was carried out as described (21). RESULTS Restriction fragment length polymorphisms (RFLP) as ailelic markers for the genes Al, A2 and B2 Based on our analysis of genomic clones, three RFLPs were chosen as genetic markers for the vitellogenin genes Al, A2 and B2. A polymorphism 1n Intron 12 of the gene Al gives rise to either a 1.1 kb or a 1.5 kb EcoRI fragment (Figure la; ref. 24). Similarly, a RFLP 1n the 5' flanking region of the gene A2 gives either a 6.2 kb or a 6.6 kb Hindlll fragment (Figure lb; ref. 24). Finally, the gene B2 EcoRI RFLP 1s also 1n the 5' end region of the gene and 1t shows more variation, yielding fragments of 2.25, 2.5, and 2.7 kb (Figure lc, ref. 21). We Interpret the fact that none of the several animals tested shows more than two bands to mean that these fragments represent allelic forms of their respective genes. Linkage groups within the viteilogenin gene family A breeding pair of frogs that were Imported from Africa was chosen to illustrate the segregation of the viteliogenin alleles (Figure 2 ) . As shown below analysis of their offspring allows determination of the linkage groups within the vitellogenin gene family. The male was heterozygous and had the 8725 Nucleic Acids Research a UO»Ckn»i> 10*. H». 110 10S.1W 5 A1 O«n« y * si a*«• Animal T 1 z/ -J H-f- \ i.i m 01 5 2 3 4 10 U> — 1.5 m ^^^ — — 1.1 — 5- u Oaiu T • . . 1 •• , 1 . . . 01 12S.127 e*ttr 124.125 62 hb 5 5 6 10 U> _6.6 — 6.2 •V I \ / H-4-. 01 5 — 2.7 — 2.5 — 2.25 iai.za.za.m z.7u> Figure 1 Restriction fragment length polymorphisms (RFLP) within the Al, A2 and B2 gene loci. Each scheme gives a map of the cloned region of the different genes with the EcoRI sites (panel a and c) or Hindi 11 sites (panel b ) . The polymorphic region is enlarged. The M l v genomic clones containing an allele corresponding to one of the polymorphic restriction fragment are given on the left (24,14). The EcoRI restriction sites are indicated by an open circle, the Hindi 11 sites by a square. The hatched boxes show the regions used as probes in the hybridizations. On the right, autoradiograms are shown presenting the different alleles observed. In each lane, 10 ug (5 ug for panel c) of Xenopus laevis genomic DNA prepared from erythrocytes of different animals was digested either by EcoRI (panel a and c) or Hindi 11 (panel b ) , electrophoresed on a agarose gel and transferred to a nitrocellulose membrane. For a and b the fragments used as probe were inserted in a M13 cloning vector and the radioactive probe was synthesized by elongation of a primer hybridized to the "+" strand. For c the fragment was nick-translated. Prehybridization, hybridization and washing were performed in the absence of dextran sulfate. 8726 Nucleic Acids Research Parantal animals •* — ^^^^B G»n« B2 ^ *^ " —*2 *3 -- % * 271 27 Offspring .221 — 26225— — 27 H « M I ^T ^ «MW -«^ _2« —22S Figure 2 Genotype, with respect to the gene Al, A2 and B2 polymorphisms, of the male and female parental animals, as well as of three of their offspring. 10 ug of genomic DNA prepared from erythrocytes (parental animals) or from whole tadpoles at stage 60-64 (offspring) were digested by EcoRI (genes Al and B2) or HindiII (gene A2). The DNA was electrophoresed on a agarose gel along with 3 equivalents per haploid genome of the clones XXlv 107, 110, 221 or 228 or 6 equivalents of the clones XXIv 124 or 126 as where indicated. The DNA fragments were then transferred to a nitrocellulose membrane. The probes used are indicated in Figure 1, with exception for the gene B2 analysis, where the probe was the 2.5 kb EcoRI fragment of XXIv 228 (Fig. lc). Hybridization was performed in the presence of dextran sulfate. following genotype : gene Al-1.1 and 1.5 alleles, gene A2-6.2 and 6.6 alleles and gene B2-2.25 and 2.7 alleles. The female was similarly heterozygous with the gene Al-1.1 and 1.5 alleles and the gene A2-6.2 and 6.6 alleles, while being homozygous for the gene B2-2.5 allele. From 29 to 52 progeny of this mating were analyzed, depending on the gene pair tested. As an example, Figure 2b shows the genotype of 3 offspring of this cross. The qualitative and quantitative analyses are given in Table I to III. Table I shows the segregation of the gene A2 versus the gene B2 alleles. Given the alleles involved, four different genotypes are expected if the two genes are linked, while six different 8727 Nucleic Acids Research Table I: Segregation of the A2 and B2 alleles A2a6;6.2 p A2:6.6; 6.2 B2: 2.7; 2.25 B2: 2.5; 2.5 Possible gametes 6.6/2.5 6.6/2.7 6.2/2.7 6.6/2.25 6.2/2.25 62125 F1 29 individuals analyzed Dnked FREQUENC:IES observed expect ed unlinked GENOTYPEES expect;id linked unBnked possible A2: 6.6; 6.6 / B2: 2.7; 2.5 A2: 6.6; 6.2 / B2: 2.7; 2.5 • • 7.2 (0) 3,6 4 ' C) • 7,2 (7.2) 7,2 8 (*) (*) • 0 (7.2) 3,6 8 • 0 (7,2) 3,6 2 (*) • 7,2 (7,2) 7,2 4 • 7,2 (0) 3,6 5 A2: 62; 62 1 B2: 2.7; 2.5 A2:6.6; 6.6 / B2: 2.5; 2^5 A2: 6.6; 6.2 / B2: 2.5; £25 ' A2:6.2; 62 1 B2: 2.5; 2^5 • TOTAL 29 (29) 29 29 Testing 29 Individuals gives a probabftty of 0.98 that all six genotypes would be found S the genes are not linked. In case of linkage, there are two possibilities (the second is given In brakets) for the genotypes expected depending on which of the aDelea form a linkage group in the male. Statistical analysis of the expected unlinked frequencies versus the observed ones gives: %2 - 7 ; 0.2 < p < 0.3. genotypes would result 1f the two genes segregate independently. In the twenty-nine animals tested, all six genotypes were found with frequencies 1n agreement with the expected values. From this result it appears that the genes A2 and B2 are not linked, which is Inconsistent with the simple gene-genome double-duplication model. Thus, we wondered if either the gene A2 or B2 belongs to the already established Al-Bl linkage group (7). To answer this question the inheritance of the alleles of the gene Al relative to those of the gene A2 was analyzed (Table II). In the 36 animals checked only three out of nine possible genotypes were observed, demonstrating that the genes Al and A2 are linked. The gene A2-6.6 allele was found linked to the gene Al-1.1 allele and the gene A2-6.2 allele is linked to the gene Al-1.5 allele in both parental animals. Thus, the gene 8728 Nucleic Acids Research Table II: Segregation of the A1 and A2 alleles A1: 15; 1.1 A1: 15; 1.1 A2: 6.6; 6.2 A2: 6.6; 6.2 P Possible gametes 1.5/6.6 1.1 /6.6 1.5/6.6 1.1 /6.6 1.5/6.2 1.1 /6.2 1.5/6.2 1.1 /6.2 F1 36 individuals analyzed FREQUENC IES GENOTY PES possible expected linked unlinked expec•ted unlinked linked observed • 0 (9) 2,2 0 A1: 1.5; 1.5 / A2: 6.6; 6.2 • 0 (0) 6,6 0 A1:1.5; 1.1/A2: 6.6; 6.6 • 0 (0) 4,4 0 A1: 1.5; 1.1 / A 2 : 6.6; 6.2 ' O • 18 (18) 6,6 17 A1: 1.5; 1.5/A2: 6.2; 6.2 * • 9 (0) 2,2 10 • 0 (0) 4,4 0 9 (0) 4,4 9 A1: 1.5; 1.5/A2: 6.6; 6.6 (') A1: 1.5; 1.1/A2: 6.2; 6.2 AV 1 1-1 1 / A 2 6 6-6 6 A l : 1.1; 1.1/A2: 6.6; 6.2 • 0 (0) 4,4 0 A1:1.1; 1.1 / A 2 : 6.2; 6.2 * 0 (9) 2,2 0 36 (36) C) TOTAL 36 36 Testing 36 individuals give a probability of 0.95 that all nine genotypes would be found if the genes are unDnked. In case of linkage, there are two possibilities (the second is given In brakets) for the genotypes expected depending on which of the alleles form a linkage group. Statistical analysis of the expected finked frequencies versus the observed ones gives: x 2 - 0.834; 0.5 < p < 0.9. In contrast statistical analysis of the expected unlinked frequencies versus the observed ones gives: X2 - 60.6; p < 0.001. A2 belongs to the Al-Bl linkage group; however, Its position and orientation relative to the Al-Bl complex remain to be elucidated. Further confirmation that the gene B2 1s at a different genetic locus 1s confirmed by Its behavior relative to the gene Al (Table III). In the fifty-two offspring tested all six genotypes anticipated from independent segregation of the gene Al versus the gene B2 alleles were observed at frequencies close to the expected values. 8729 Nucleic Acids Research Table III: Segregation of Possible gametes the A1 and B2 alleles A1: 15; 1.1 A1: 15; 1.1 B2: 25; 2.5 B2: 2.7; Z25 1AI23. 1.1/2.5 1.5/2.7 1.1 /2.7 1.5/2.25 1.1 Z2.25 F1 52 individuals analyzed GENOTYF>ES possible A1:15;1.5/B2:Z7;2.5 exp€icted Inked unlinked • A1:15;15/B2:2.7;25 A1: 1.5; 1.1 /B2:Z7;Z5 A1: 15; 1.1 /B2:2.5;Z25 13 (0) 6.5 9 • 0 (13) B.5 5 • 13 (13) 13 16 C) • 13 (13) 13 11 (') • 0 (13) 6,5 5 * 13 (0) 6,5 6 TOTAL 52 (52) (') ' * A1: 1.1; 1.1 /B2:2.5; 2.25 A1:1.1; 1.1 /B2:2.5; 2^5 • • F R E Q U E N DIES expe<3ed observed unlinked Qrtked (') 52 52 Tasting 52 individuals gives a probability of 0.99 that the six genotypes would be found if the genes are unlinked. In case of linkage, there are two possibilities (the iscond Is given In brakets) for the genotypes expected depending on which of the alleles form a Dnkage group in the male. Statistical analysis of the expected unlinked frequencies versus the observed ones gives: x 2 - 4.7; 0.3 < p < 0.5. DISCUSSION RFLP within the vitellogenin loci Previous analysis of numerous vitellogenin genomic clones revealed several polymorphic regions, some of which we have used here to determine the linkage groups within the gene family (13,21,24). Frequently, the RFLPs used are the result of Insertions or deletions 1n Introns or gene flanking regions. For the gene Al alleles, for example, the longer 1.5 kb EcoRI fragment from Intron 12 contains a repeated sequence that 1s absent in the corresponding allelic 1.1 kb fragment (25). Similarly, the polymorphism observed 1n the 5' end region of the gene B2 1s also due to repeated sequences (21). The polymorphism 1n the 5' flanking region of the gene A2 1s again the result of an Insertion or deletion event (24) but 1t has yet to be characterized at the sequence level. 8730 Nucleic Acids Research Definition of the linkage groups within the viteliogenin gene family of Xenopus laevis Linkage between the Al and Bl vitellogenin genes has previously been found by molecular cloning, and the two genes are about 15 kb apart (7). The general organization and linkage of the whole gene family can formally be studied in two ways. The first is to demonstrate the linked or independent segregation of members of the gene family, and the second 1s to "walk" along the chromosome (chromosomal walking) from the Isolated genes towards the next linked member of the family. Here, we have performed an analysis by exploiting the RFLPs discussed above as allelic markers of the genes Al, A2 and B2. The results demonstrate that 1n Xenopus laevis the genes Al, A2 and Bl are linked, while the gene B2 is located elsewhere, most likely on a different chromosome. Clearly, the simple gene-genome double-duplication model (7) only partially explains the present day organization of the gene family. It has been suggested that Xenopus laevis, with 36 chromosomes, 1s "tetraploid" after a genome duplication that occurred about 30 million years ago (5,6). This could have arisen by alloploidization or alternatively, by autoploidization. The fact that only bivalents, rather than multivalents, are observed during meiosis could indicate that the first of these observation plants, two does genes possible not have exclude been events took place autopolyploidization described that control (26). However, since, at bivalent this least 1n pairing in autopolyploids (27,28). Xenopus tropical is with twenty chromosomes would represent a close relative to the now extinct diploid ancestral Interestingly, vitellogenin 1s encoded 1n three genes in form. Xenopus tropicalis, two closely related type-A genes with about 6% divergence and a single type-B gene (29). It will be Interesting to see 1f these genes correspond to the A2, Al-Bl linkage group 1n Xenopus laevis. As shown in Figure 3, our results suggest that 1n the genus Xenopus an A2-A1-B linkage group arose before the genome duplication event 1n Xenopus laevis. First was a duplication to give the A-B gene pair about 150 million years ago (4), followed much later by an A2-A1 duplication shortly before the polyploidization event 1n Xenopus laevis. If the genome duplication 1s the 8731 Nucleic Acids Research AHopolyplo<dy AutopolyploMy A-0 g.n» dupOcaflon . | A1-AI Bam dupflcation di^flcadon *r-AT Ban* aUninatlan Friainl day orgentzaflen Figure 3 Scheme representing the possible origin of the present day arrangement of the viteTlogenin genes in Xenopus laevis. The star Indicates that the position and orientation of the gene A2 relative to the Al-Bl complex are not known. result of autopolyploidy, then two A genes must have been lost on one of the duplicated chromosomes. Alternatively, 1f alloploidy took place, one of the two related species which mated might have only contributed with a B gene that was still very closely related to the B gene of the other species. Alternatively both species had an A2-A1-B type linkage group. In that case, two A genes must again have been lost after the tetraploidization event. This revised gene-genome duplication hypothesis 1s compatible with the observation that the two B genes are more closely related than the two A genes 1n Xenopus laevis. It has been observed that silent mutations accumulate much more rapidly in exon sequences than replacement mutations (30). The accumulation of both has been calculated for the first three exons of the A and the B genes (4). In both pairs of genes (Al and A2, Bl and B2) replacement mutations have been Introduced to nearly the same level (3.4 and 3.3%, respectively), 8732 Nucleic Acids Research but more silent mutations are found between the two A genes (28.8%) compared to the two B genes (22.21). This again suggests that the two A genes formed shortly before the two B genes. If the latter two genes arose as result of the genome duplication event 30 million years ago, this difference suggests that the two A genes might have formed about 10 million years earlier, that is about 40 million years ago. Additional variants of the general hypothesis presented here could be discussed. The chromosomal distribution of the duplicated genes has not necessarily to follow the polyploidization pattern. Indeed, there is a variety of possible genomic rearrangement mechanisms by which duplicated genes might become located on different non-homologous chromosomes. Thus, only further detailed molecular and comparative analysis of the two vitellogenin lod in Xenopus laevis, as well as of the corresponding l o d 1n related species, will help to refine our understanding of the phylogeny of the vitellogenin gene family. ACKNOWLEDGEMENTS We thank Drs. Bob H1psk1nd, Anne Seiler, Philippe Walker, and Riccardo Wittek for comments on the manuscript as well as Hannelore Pagel for secretarial help. This work was supported by the Etat de Vaud and the Swiss National Science Foundation. * Present address : F. Hoffmann-La Roche and Co AG, Grenzacherstrasse 124, CH-4002 Basel, Switzerland. REFERENCES 1. 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