Polymorphism of the vitamin D binding protein (DBP) among primates An evolutionary analysis.код для вставкиСкачать
AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 73:365-377 (1987) Polymorphism of the Vitamin D Binding Protein (DBP) Among Primates: An Evolutionary Analysis J. CONSTANS, C. GOUAILLARD, C. BOUISSOU, AND J.M. DUGOUJON Centre d’Hhtotypologie, CNRS, CHU Purpan, 31300 Toulouse, France KEY WORDS evolution Vitamin D binding protein, Polymorphism, Primate ABSTRACT The distribution of the DBP (vitamin D binding protein) polymorphism is now well characterized among human populations but for primates only limited results are known. The aim of this paper is to describe the electrophoretic polymorphism of this protein among various species. Using three different electrophoretic methods, we are able to detect a n unknown polymorphism and to classify the different alleles observed. These results may be used to set a n international nomenclature for further comparisons. The different electrophoretic mobilities between Old and New World Monkeys show that: 1)the Cercopithecoidea are presenting the largest genetic heterogeneity; 2) the DBP among the Galago corresponds to the lowest isoelectric points observed among Primates; 3) during the evolution from nonhuman Primates to Man, the DBP is able to keep its affinity for vitamin D derivatives despite the occurrence of significant molecular modifications; 4) among Anthropo’idea, the electrophoretic patterns of DBP are very close to the human Gcl proteins. These results show that evolution at the DBP level can be considered as a continuous mechanism of structural modifications. A significant transition occurs during the differentiation between Cercopithecoldea and Anthropoi’dea. It is not too speculative to consider that some electrophoretic forms detected among Gorilla, Pongo, or Pan may be identical to rare variants observed among humans. The human vitamin D binding protein (DBP) is a n a2 globulin, also called groupspecific component (Gc) (Daiger et al., 1975). It is the main carrier protein of the vitamin D derivatives in the serum. The amino acid seq-uence has now been fully determined by different teams (Schoentgen et al., 1985; Yang et al., 1985; Schoentgen et al., 1986). The most original observation obtained is its striking similarity with albumin and alpha fetoprotein whose loci have been syntenic on the same chromosome for over 400,000 years (Constans, 1984). Biochemical investigations on the DBP revealed that the protein evolved after triplication of the ancestral gene (Cooke and David, 1985), but very little is known about the different steps of the DBP modifications in the course of evolution. For these reasons, we undertook the determination of the DBP polymorphism among most of the primate species living today. The aim was to 0 1987 ALAN R. LISS, INC. present a reliable pattern of the electrophoretic mobilities of the DBP in order to develop a nomenclature for further studies and comparisons of results between the different laboratories. Additionally, this study attempted to identify some characteristic traits of the DBP structure during primate evolution using human DBP as a reference. MATERIALS AND METHODS Serums stored a t -20°C were analysed. These samples were provided by several laboratories as well as directly from field capture of animals in Africa and Bolivia. The detailed list of the species investigated, the origin of the samples, and their numbers are included in Table 1. More than 380 animals were studied, these representing more than Received July 30, 1986;revision accepted January 27,1987 366 J. CONSTANS ET AL. TABLE 1. Sera examined in this study: Animal identifications and origins Animals Pan P. paniscus P. troglodytes Gorilla G.g. gorilla Pongo P.p. abelei P.p. pygmaeus Papio P. ursinus P. hamadryas Theropithecus T. gelada Macaca M.f. fuscata M. tonkeana M. sylvana Cercocebus C. albigena C. torquatus C. torq x C. alb Cercopithecus C. nictitans C. ascanius C. petaurista C. cephus C. erythrotis C. pogonias c. wolfi C. l’h. l’hoesti C. I’h. solatus C. l’h. preussi C. neglectus C. hamlyni C. aethiops C. mona C. pog. x C. asc. C. pog. x C. mona Allenopithecus A. nigroviridis Miopithecus M. talapoin Erythrocebus E. patas Cebus C. apella Saimiri S. sciureus Galago G.c. crassicaudatus G.c. argentatus Lemur L.f. albifrons L.f. mavotensis L. catti Coming from - N 23 5 Yerkes RLL Yerkes RPC 14 Yerkes RPC (13) Zoo, France (1) 23 4 Yerkes RPC Yerkes RPC 3 14 South Africa Montpellier (Clin Midy), France 39 Ethiopia 30 9 Japan Univ. Louis Pasteur (Strasbourg), France Zoo, France 42 18 4 7 2 1 10 1 3 1 5 1 1 7 2 23 1 4 1 St. Biol. Paimpont + CIRMF, Gabon Station Biologique Paimpont, France CIRMF (Gabon) + Station Biol. Paimpont CIRMF Station Biologique Paimpont Station Biologique Paimpont Station Biol. Paimpont + CIRMF Station Biol. Paimpont CIRMF Station Biol. Paimpont CIRMF Station Biol. PaimDont + CIRMF Zoo Mulhouse M. Hist. Nat. Paris CIRMF, Gabon Zoo Mulhouse, France Station Biologique Paimpont Zoo de Mulhouse Delta RPC + Barbados Islands Station Biologique Paimpont Station Biologique Paimpont Station Biologique Paimpont + + + + Zoo Mulhouse 5 M. Hist. Nat. Paris 3 Station Biologique Paimpont 8 Delta RPC 10 Bolivia 31 Bolivia 12 2 Oregon RPC Oregon RPC 6 3 6 Museum #Histoire Naturelle Paris Malaeazv MalagaG DBP POLYMORPHISM AMONG PRIMATES 35 taxonomic groups, and for most of these animals, pedigree information was provided by their breeding centers or the zoo. The electrophoretic procedures used in this investigation are similar to those used for human populations: isoelectric focusing (IEF) in a pH range 4-6 and in the presence of 3M urea as well as polyacrylamide gel electrophoresis (PAGE)(Constans et al., 1983). The pattern obtained after PAGE was detected by staining with a Coomassie blue solution. When standard IEF and IEF in 3M urea were run, the DBP band was located by print immunofixation in cellulose acetate strips imbedded with a n IgG antihuman DBP solution. Some PAGE patterns were also confirmed by the immunological procedure. Additional identity of the primate protein with DBP protein was obtained by following the electrophoretic shift of the protein induced by addition of a saturating dose of 25 OHDs (ethanol solution) to the serum protein (Constans et al., 1980). Neuraminidase treatment Sera showing two or more DBP bands were incubated with neuraminidase (clostridium perfringens) at 37°C after equilibration with a n acetate buffer pH 5.5 according to the standardized procedure (Cleve and Patutschnick, 197913). The object of neuraminidase treatment was to ascertain whether the additional bands were due to the presence of sialic acid. Time sequences were used in order to determine the number of sialic acids released by the enzyme, and, for some experiments, the samples were incubated overnight. Treated samples were examined on IEF gels. Results The use of three electrophoretic methods resulted in a large number of bands with different mobilities. When the bands were classified and compared with a human Gc21F phenotype, several distinctive patterns of bands were found and each was characteristic of a taxonomic group (Figs. 1,2). The effect of neuraminidase treatment on some samples is shown in two figures (Figs. 3, 4). The isoelectric point of each band was determined. Results are reported in Table 2. We verified that the various DBP phenotypes may be controlled by the existence of autosoma1 and codominant alleles at a single locus. Allele frequencies were calculated according 367 to the Hardy-Weinberg formula, and the results are shown in Tables 3 and 4. The nomenclature used is the same as that adopted by previous workers (Cleve and Patutschnick, 197913; Constans et al., 1983; Dykes et al., 1985). A single-band pattern is called Gc2 while the double-band pattern corresponds to Gcl proteins. Only among Galago serum was a three-band pattern observed. It was also called Gc’.This denomination is further documented in the text. We called Gc2 the most frequent single band within a species, and the less frequent additional bands were called GcZAif the electrophoretic mobility was more anodal than the Gc2 protein and GcZc when the mobility was cathodal. This nomenclature gives the electrophoretic mobility of the protein and indicates which allele is most frequent or rare in one species. Any new protein detected in the future can be compared with those included in our figures and named according to this nomenclature. Discussion The DBP typing among nonhuman Primates requires the use of at least the three electrophoretic procedures if hidden differences in the protein are to be detected between animals belonging to the same or to different species. For this reason, it is difficult to compare our electrophoretic patterns with those already published for DBP (Kitchin et al., 1965; Barnicot et al., 1971; Barnicot et al., 1972; Kitchin et al., 1967). In some studies DBP was also confused with “post albumin” which include some other proteins such as alphal-antitrypsin (Lucotte et al., 1979). The most discriminating technique is the IEF procedure with or without urea. The significant result obtained in this study is the observation of different electrophoretic forms of the DBP between any species, which prevents any phylogenic comparison based on electrophoretic criteria only (Lucotte and Ruffie, 1982a). Here IEF migrations are based on the difference between apparent isoelectric points (PI) of the proteins. The isoelectric point of a protein is considered as a characteristic of the molecule. It represents a balance between electric charges located at the surface of the protein and charges due to its amino acid composition. Somehow the comparison of PI may be relevant for the 368 J. CONSTANS ET AL. l?A.G.E. 0 - - ?IF W21*lIt2II 2 2 A 3 2 A 2 2nr 2 2 ( l 212 2*, - - I - 2 2 zrz 1 xi 2('3 2 2('l 2r2 2Cl ZC2 2 (('1 l('3 1.E.E 3M-UREA 0 1- I -----_ I40 5 50 a- i 0 2A1 Zr\2 2 A I 2 HOMO ' Pan Macaca Gorilla 2g 1 2( 2 ZA! 2 Theropirhecus 2 21, 2c2 z('3 Papio 2 2('1 zt2 2 2('( Z('2 Cercocebus Eryrhrocebus Ponao HOMlNOlDEA >< CERCOPITHECOIDEA Fig. 1. DBP polymorphism among Primates. IEF, IEF 3M urea, and PAGE patterns obtained with the samples belonging to Pan, Gorilla, Pongo, Macaca, Theropithecus, Papio, Cercocebus, and Erythrocebus species. DBP POLYMORPHISM AMONG PRIMATES 369 F?A.G.E. 2 ZCI 2A1 2 2CS 2C1 2 C l 2A2 2Cl 2C6 2C8 ZC3 2CZ 2CI 2 2UI 2 2 2 2 2 2 2 1P 1AI 1s lC1 lC3 ICZ 2 21'2 2.41 2C1 2 21 2 2l.I 2.41 1.E.E 3M-UREA 2 ZCI 2A2 2 2CZ 2n 2C3 ZCS 2C8 2C6 2C9 2A1 7x4 2C7 1 8 IF W3 1s K2 1Ci ZCI 1.E.E 2 ZCI Miopithecus 2 A 2 2 ~ 1 2 2cI 2C2 ZC3 2Ck 2CS ZC6 ZCl 2C9 2C8 Cercopithecus 2 2c1 1Al IF IS Y'1 1C2 U S 2 I('( Zf'Z 2 A1 Allenopilhecus Cebus Saimri Galago Lemui CERCOPITHECOIDEA Fig. 2. DBP polymorphism among Primates. IEF, IEF 3M urea, and PAGE patterns obtained with the samples belonging to Miopithecus, Cercopithecus, Allenopithecus, Cebus, Saymiri, Galago, and Lemur species. ' 370 J. CONSTANS ET AL. 1 2 3 4 5 6 7 8 Fig. 3. IEF pattern obtained after neuraminidase treatment of the Pongo GclC2 protein. References, human samples: 1,5, human Gc2-IS; 2, human Gc2; 3, Pongo GclC2; 4, 3 + neuraminidase overnight; 6, Pongo Gc2; 7,6 neuraminidase overnight; 8, human Gc IF-1s. + evolution of the protein structure. From these data (Table 2), Primates may be distributed into three groups. The first group consists of Cebus, Galago, and Lemur genera. These animals are characterized by the lowest PI which corresponds to the most primitive form of the DBP. The second group comprises the Cercopithecoydea only. In these animals, DBP is already more basic than that found in the first group. Higher isoelectric points are obtained and they appear to correlate with a n increase in positive charges due to a n accumulation of basic or neutral amino acid substitutions. The third group is composed of the Hominoidea whose DBP has a large PI range (ApI 0.6), which is also observed with Cercopithecus. In these animals, the extent of genetic variation for DBP is the highest obtained. Among Pan, five protein forms are observed. Only one allele GclF is common to both Pan paniscus and P troglodytes. In the P troglodytes the DBP polymorphism is more variable with the occurrence of 4 alleles. We confirm that the anodal band of the two-band pattern is affected by the neuraminidase treatment (Cleve and Patutschnick, 1979b). As is the case with human Gcl protein, one sialic acid molecule is present on the anodal band. The addition of the hydroxylated vitamin D derivative (25-OH-D3)to the DBP of Pan induces a n anodal shift of the holoprotein form as observed in man (Hay and Watson, 1977). Among Gorilla and Pongo sera, DBP shows the same electrophoretic pattern as the one discussed for Pan. Three alleles are present, GclA2 allele being common and most frequent in both the Gorilla and Pongo. No genetic variation is detected among Gorilla while both Pongo p. abelei and Pongo p. pygmaeus have GclM and Gclc2 alleles. Only a single Gc2band pattern was observed among Pp, pygmaeus (Fig. 1). These differences are in agreement with those obtained by Bruce and Ayala (1979) on the mobility of adenosine desaminase (ADA), by Lucotte and Smith (1982b) on post-albumin, by Dugoujon et al. (1981, 198413) on the Gm phenotypes of immunoglobulins, and by Seaunez et al. (1979) on chromosome analysis. These data clearly demonstrate genetic differences between the two orang utan subspecies. The anodal band of the two GclC2 isoproteins is only affected by the neuraminidase degradation and shows a mobility similar to DBP POLYMORPHISM AMONG PRIMATES 371 1 2 3 4 5 6 7 8 9 1 0 1 1 Fig. 4. IEF patterns obtained with Galago samples with and without neuraminidase treatment. References, human samples. A) 1, human Gc2-1F; 2, Galago GclF1C2; 3, Galago GclS-1Cl; 4, Galago GclF-1S 5, Galago GclF; 6, Human GclF-1s. B) 1,3,11, human Gc2-1s 2, Human Gc2; 4, Human GclF-1s; 5, Galago GclF; 6 , Galago GclA1; 7, 6 + neuraminidase during 4 h; 8, 5 + neuraminidase during 4 h; 9, 6 + neuraminidase overnight; 10,5 + neuraminidase overnight. 372 J. CONSTANS ET AL. TABLE 2. Isoelectric points of the differentforms of the DBP obtained after IEF migration for all the species included in this studv PI Range variations Human Pan Gorilla Pongo Papio Theropithecus Macaca Cercopithecus Cercocebus Allenopithecus Miopithecus Erythrocebus Cebus-Salmiri Galago Lemur I 5.30 - 4.70 5.30 - 4.75 or 4.90 5.25 5.20 5.30 5.50 5.35 5.20 5.40 5.50 4.90 4.65 4.75 5.20 5.15 5.00 4.90 5.20 5.10 5.10 5.40 4.75 4.40 4.60 that of the second cathodal band. Only one sialic acid is released by the anodal protein during the treatment P i g . 3). A similar structure is present among human Gcl proteins (Constans et al., 1985). According to the IEF mobility, the GclA2 protein seems to be very close to the human GclF protein. Additional similarity to the human DBP polymorphism is the simultaneous presence of the two Gcl and Gc2 protein forms in Pongo. Orang utan could be considered as the primate closest to man. Brown et al. (1982) gives the same hypothesis based on molecular phylogeny while Diamond (19841, using also mDNA data, suggests a different classification. Considering the mobilities of human DBP variants, it is possible to find some similarities with one of the proteins observed in Pan, Pongo, or Gorilla but it would be hazardous to go any further in the absence of sequence data. From the DBP variation among Hominoidea, we can conclude that Pongo and Gorilla but not Pan may originate from a common ancestor (King and Wilson, 1975; Schwartz, 1984). The emergence of the human species is associated with the presence of three different alleles, GcIF, Gc", and Gc2, which are not detected in any sample belonging to the Primate group (Constans et al., 1985). Among Papio, Theropithecus, and Macaca, the DBP polymorphism is significantly less extensive than among Hominoidea (Scheffrahn and Ziggiotti, 1981). Similar results were observed with the immunoglobulin markers studied by Dugoujon (1985). A single-band pattern is the usual form of DBP in these species (Moore and Lalley, 19841, but between them no common form of DBP is present. The absence of polymorphism in €? hamadryas may be due to the social structure of their groups but also to the selection of the animals in the breeding centers. Dykes et al. (1985) previously noted the presence of two alleles in the same species. The Gc2 protein thus described by Dykes et al. (1985) is probably identical with Gc2C3 in our investigation. The DBP polymorphism in €? ursinus is represented by three alleles, despite the fact that only three animals were examined. No allele common to the two Papio genera was found. Two alleles, Gc2*l and Gc2 are observed among Theropithecus gelada. The homologous proteins are different from the ones present in Papio or Macaca species. In Macaca syluana, DBP shows no variation. Considerable polymorphism is observed in M. fuscata with three alleles, and M. tonkeana differs from M. fuscata by having GcZc2and GcZA2alleles. The unexpected observation is that the three Macaca species did not present any common allele. The reason of these genetic differences are not clearly understood but it can be speculated that these results may reflect different ancestral origins for the three species or a severe genetic drift among isolate groups during speciation. Two Cercocebus species and their hybrids were examined, the DBP polymorphism corresponds to the presence of three alleles. Gc2 is common to C. albigena and C. torquatus and to the hybrid C. torquatus x C. albigena. Gc2C1 protein seems to be limited to C. albigena while Gc2C2 protein is found in C. torquatus and the hybrid. These data agree with a common ancestor between C. torquatus and C. albigena. These species show a molecular phenotype similar to that of the Papioninae confirming their evolutionary closeness (Dutrillaux et al., 1982). These results have already been observed by various studies of immunoglobulin polymorphism (Dugoujon et al., 1981)or cytogenetic analysis (Dutrillaux, 1979; De Grouchy et al., 1978). As we have discussed previously, genetic divergence may have occurred and may be responsible for the differences observed within the Cercocebus species. In species of the African forest such as Allenopithecus, Miopithecus, and Erythrocebus, the DBP polymorphism is represented by a singleband pattern as we already described for Papio, Macaca, and Cercocebus. Each band has DBP POLYMORPHISM AMONG PRIMATES a distinctive electrophoretic mobility. Further the PI of DBP in Erythrocebus is very high and approaches the values observed in some Cercopithecus samples. The Gc2C1 of Miopithecus talapoin is also a basic protein. It is interesting to note that DBP is quite polymorphic in these three genera with the presence of two or three alleles. Ruvolo (1982) described four alleles among Erythrocebus patas, while only three were detected in this series. The small difference reveals the difficulty in obtaining a precise information on protein polymorphism in animal populations because of the selection of the samples (breeding centers, zoo, geographical area, and social structure of the troops) and of the limited size of the samples available for such investigations. Cercopithecus is probably one of the most important and most puzzling genera in the Primates when their history is read through the DBP. Three points are worth noting: 1) the DBP variability in Cercopithecus is one of the largest among Primates (Table 2); 2) 12 alleles are detected for a total of 14 species or subspecies or about one specific allele per subspecies (Fig. 2); and 3) the confirmation of a common origin for all Cercopithecus animals and probably a genetic speciation which appeared very early; the Gc2 protein can be considered as the oldest form as it was present in all animals examined. Similar conclusions could be drawn using other genetic systems such as the red blood cells enzymes (Ruvolo, 1982; Dugoujon, 19851, or the karyotype, (Dutrillaux, 1979). These data confirm the complex nature of the evolution and speciation of Cercopithecus. The protein Gc2C7 is present in C. neglec tus, C. I'h. l'hoesti, and C. cephus and its presence may be explained by the occurrence of interbreeding between these species after the emergence of the Cercopithecus ancestor. It also implies that the three species were living in the same geographic zone a long time ago. On the contrary, all other species have evolved in isolated situations with specific speciation. C. aethiops sabens is a significant example of the genetic differentiation (Barbados isolate) occurring within a species (Dracopoli et al., 1983). C. nictitans, C. neglectus, and C. ascanius can also illustrate a similar analysis. Among Cercopithecus, the determination of the DBP polymorphism is a useful tool together with other proteins for paternity test- 373 ing, identification of the subspecies, and identification of the hybrids. However, the situation is different with the genera Cebus and Sairniri. Despite the examination of 40 samples, DBP is monomorphic. The electrophoretic pattern is represented by a single protein band associated with PI values lower than the ones obtained for Cercopithecus, Miopithecus, Allenopithecus, and Cercocebus. The changes in the structure of DBP among Cebus and Sairniri seem to correspond to a n intermediate step between Anthropoidea and Prosirni in the evolution of DBP. In contrast, among Prosimians, Galago presents a n original pattern of the DBP polymorphism. This protein has the lowest isoelectric point in Primates (Fig. 4A). The reason for this appears to be a n accumulation of acid residues. This observation was confirmed by determining the number of sialic acids in each protein band (Fig. 4-B). GclF and GclAl proteins were treated by the neuraminidase solution from 5 min to 24 h in order to detect successive steps of desialylation and to obtain the final protein form. From the beginning to the end, the PI of the DBP increased from 4.4 to 4.80. The progressive removal of sialic acids shows that the most cathodal of the three bands (original form) bears at least three sialic acid residues. Each additional band possesses probably one more sialic acid radical, giving a total of five for the most anodal protein (PI 4.40). Since the removal of one sialic acid residue is followed by a 0.1 pH unit shift of the PI, the total PI variation for the anodal band (PI0.5) confirms the presence of five sialic acid residues on this protein. The experiment also shows that the electrophoretic heterogeneity of the DBP among Galago is only due to posttranscriptional addition of sialic acids because, in the end, a single band is obtained. The well-equilibrated pattern of the three native proteins indicates that the glycan chains are progressively saturated by sialic acids during posttranscriptional steps and that sialylation is equally affected by transferases for addition of three, four, and five sialic acids. This analysis also reveals the existence of a t least two kinds of glycan chains on this DBP. All those posttranscriptional modifications show that the metabolism of the DBP and of the vitamin D derivatives is also probably exceptional in the Galago species. 374 J. CONSTANS ET AL. TABLE 3. Allele distribution and frequencies observed in each species and subspecies GclAl GclA2 - Pan P. paniscus P. troglodytes 0.013 Gorilla C.g. gorilla Pongo P.p. abelei P.p. pygmaeus Papio P. ursinus P. hamadryas Theropithecw T. gelada Macaca M.f. fuscata M. tonkeana M. sylvana Cercoceb us C. albigena C. torquatus C . torq x C. alb Cercopithecus C. nictitans C . ascanius C. petaurista C. cephus C . ervthrotis C. pogonias c . wolfi C . l’h. l’hoesti C. l’h. solatus C. l’h. preussi C . neglectus C. hamlyni C. aethiops C. mona C. pog. x C. asc. C. pog. x C. mona A lenopithecus A. nigroviridis Miopithecus M. talapoin Erythrocebus E. patas Cebus C . apella Saimiri S. sciurea Galago G.c. crassicaudatus G.c. argentatus Lemur L.f. albifrons L.f. mayotensis L. catta GclF 0.9 0.895 GclS GclCl GclC2 GclC3 Gc2A1 Gc2A2 Gc2A3 0.10 0.019 0.072 1.00 0.260 0.250 0.740 0.500 0.397 0.125 0.889 1.00 0.065 0.130 1.00 0.084 0.375 0.50 0.50 0.208 0.042 0.167 0.125 1.00 375 DBP POLYMORPHISM AMONG PRIMATES TABLE 3. Allele distribution and frequencies observed in each species and subspecies (continued) Gc2 x2 Gc2C1 Gc2C2 Gc2C3 Gc2C4 Gc2C5 Gc2C6 Gc2C7 Gc2C8 Gc2C9 0 0.148 0 0.100 0 0.250 0.167 0.333 0.500 0 1.00 0.603 0.315 0.833 0.042 0.875 0.125 0.875 0.750 0.857 0.071 0.750 1.00 0.95 1.00 1.00 1.00 0.700 1.00 0.50 0.113 0 1.736 0.125 0.250 0.003 0 0 0.004 0.071 0.250 0.05 0.100 0.006 0.50 0.857 0.50 0.804 1.00 1.00 1.00 0.60 0 0.100 0.100 0 0.143 0.143 0.50 0.654 0.40 0.167 0.834 0.687 0.250 0.063 0 1.00 0 0.667 0.50 0.333 0.50 0 376 J. CONSTANS ET AL. An additional and interesting observation among Galago is the large polymorphism, especially, the presence of 6 alleles in G.c. crassicaudatus. The polymorphism is represented by 4 frequent alleles; in addition, GclAl and GclF are both G.c. crassicaudatus and G.c. argentatus. Lemurs show the presence of four alleles responsible for the synthesis of proteins with low PI. These values are in the range obtained for Galago but the main difference is the occurrence of single-band pattern. The DBP in L.f: albifi-ons and L.f mayotensis is polymorphic, and of the two alleles present, Gc2C2 is common to the two subspecies. Among L. catta, DBP is not polymorphic in our sample and the protein detected is not present in the other Lemur subspecies examined. CONCLUSIONS metabolism is probably a key to the understanding of the molecular mechanisms responsible for the differentiation of the species during evolution (Fontaine, 1984).This study shows that for some species, the DBP polymorphism is limited and small whereas among Cercopithecus as well as in the great apes this polymorphism is more extensive. Keeping in mind some limitations which we analysed in this paper, such as animal sampling, size of the group, and data on the population structures of wild animals, only general hypotheses can be discussed: 1)It can be considered that a limited polymorphism may be associated with geographical isolation and a n important genetic drift (Jones, 1986). The influence of selective forces would be important in such a situation. 2) On the contrary, when the DBP distribution is more polymorphic, it can be explained by a higher mutation rate in the course of adaptative process. Speciation of the involved animals would probably be more recent. Founder effect due to male behaviors and to female reproduction are also to be taken in account when considering the DBP polymorphism amont Primates. These data suggest that evolution at the DBP level during Primate differentiation is very likely a directional process associated with large discontinuities during speciation. Different patterns of protein evolution are to be expected according to the structure of the molecule and to the biological activity of the protein during the fetal period and the development of the organism. This study on the DBP among Primates is only the first step in the investigaton of larger samples for more detailed information at the molecular level, in order to test the reliability of the hypotheses developed in this analysis. DBP is present in all primate species, though the modifications of the protein sequence are probably limited. In other words, from Prosimi to Human, the ancestral structure of the DBP is largely maintained because of small antigenic differences between the various DBP forms present among Primates. Polyclonal IgG antibody produced against human DBP is able to detect the same protein among Primates as shown in Figures 3 and 4. This was recently confirmed by using monoclonal antibodies (Pierce et al., 1985). The binding site for the vitamin D derivatives is also preserved (Hay and Watson, 1977; Adams et al., 1985) throughout Primate evolution, despite differences in the protein structures (PI and polymorphism variations). If minute PI variations were underlined in this paper (the largest range variation is from 4.4 to 5.5 for the total samACKNOWLEDGMENTS ple examined), this value is no more than one pH unit. Some variations are accepted We are grateful to Dr. L.C. Lay for his by the protein as long as the PI remains interest in this work and for his fruitful parabout 5, which probably represents a signifi- ticipation in the preparation of the manucant PI for the biological activity of the pro- script. tein. In addition Hay and Watson (1977) LITERATURE CITED described significant differences in the DBP level between Old World Monkeys and the Adams, JS, Gacad, MA, Baker, AJ, Gonzales, B, and Rude, RK (1985) Serum concentration of 1.25-dihyNew World Monkeys. A similar observation droxyvitamin D3 in Platyrrhini and Cararrhini: a phywas made by Pugeat et al. (1984)when inveslogenetic appraisal. Am. J. Primatol. 9:219-224. tigating another serum carrier protein: the Barnicot, NA, and Hewet-Emmet, D (1971) Red cell and cortisol binding globulin. serum proteins of talapoin patas, and vervet monkeys. 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