EVOLUTIONARY CONSERVATION OF EQUINE Gc ALLELES AND OF MAMMALIAN Gc/ALBUMIN LINKAGE

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1 EVOLUTIONARY CONSERVATION OF EQUINE Gc ALLELES AND OF MAMMALIAN Gc/ALBUMIN LINKAGE LOWELL R. WEITKAMP Depariment of Psychiatry, Division of Genetics and Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York AND PETER Z. ALLEN Department of Microbiology, Universiiy of Rochester School of Medicine and Dentistry, Rochester, New York Manuscript received January 14, 1979 Revised copy received May 14, 1979 ABSTRACT Ancient origin of the equine vitainin D binding protein (Gc) polymorphism is suggested by the finding of two alleles, GcF and G@, in each of three equine subgenera, Equus, Asinus and Hippotigris. The equine Gc and albumin loci are closely linked (lod score = 6). Although no recombinants were observed, the data are not inconsistent with a map distance similar to the 2 centimorgans reported for the human albumin/gc linkage relationship. Gametic association between the Gc" and A2b" alleles appears probable in the American Standardbred horse, perhaps as a result of population structure. Since Gc and albumin are both polymorphic in rodents and possibly other orders, this linkage group will be useful for studies of the evolution of mammalian linkage groups, as well as for a comparison of meiotic recombination frequencies and linkage disequilibria in different species. OLYMORPHISM for a human e2 globulin, group-specific component (Gc Pprotein) was first reported by HIRSCHFELD (1959). More recently, DAIGER, SCHANFIELD and CAVALLI-SFORZA (1975) demonstrated that antiserum against Gc protein co-precipitates Gc and vitamin D, and that Gc and vitamin D binding protein electrophoretic variants are superimposable. These workers also reported the occurrence of vitamin D binding proteins in sera of the mouse, rat, guinea pig, rabbit horse, cow, dog, rhesus monkey and chimpanzee. Cross-reactivity with anti-human Gc reagents has been previously used to demonstrate Gc protein in the serum of a number of mammalian species (REINSKOU 1968). Using immunofixation electrophoresis with an anti-human Gc, we have reported polymorphism of Gc in the horse and mouse (WEITKAMP 1978a,b; WEITKAMP, ALLEN and SELANDER 1978). Gc polymorphism has also been demonstrated in the horse and cattle using l4c-vitamin D, (GAHNE and JUNEJA 1978). The Gc locus is linked to the albumin locus in man (WEITKAMP, RUCKNAGEL and GERSHOWITZ 1966). Here we report the homologous linkage between the equine albumin and Genetics 92: August, 1979.

2 1348 L. R. WEITKAMP AND P. Z. ALLEN Gc loci, as well as a comparison of Gc proteins in the sera of four wild Equus species, several domestic breeds and some other mammals. MATERIALS AND METHODS Serum or plasma samples were obtained from four wild species of the Genus Equus (Order Perissodactyla) and from domestic horses, donkeys and mules. These include four Mongolian wild horses (Equus ferus przewalski) from the Netherlands, five Asiatic wild asses or onagers (Equus hemionus onager), two Grant zebras (Equus burchelli boehmi), and two Hartmann s mountain zebras (Equus zebra hartmannae) from the Philadelphia Zoological Gardens, and an additional zebra of unknown species from an American traveling circus. The domestic equines include registered American Standardbreds (312), Thoroughbreds (61), Arabians (four), and Quarter Horses (four), and Fresian horses (16) from the Netherlands. Samples were also obtained from unregistered Shetland ponies (32), Welsh ponies (two), Donkeys (26), and mules (three). The samples from the registered horse breeds include parents and offspring. The degree of relatedness of the other equines is unknown. Blood samples from representatives of the mammalian orders Carnivora (four dogs and one cat), Lagomorpha (eight rabbits), Artiodactyla (single samples of pig, cattle, goat and deer), Rodentia (one rat, one guinea pig and 27 inbred mouse strains), as well as from one amphibian (Xenopus Zaevus) and one bird (chicken) were obtained locally. Samples were also obtained from two specimens each of Mus musculus castaneus, Mus corolli, Mus dunni, Mus cervicolor cervicolor and Mus cervicolor popaeus captured in Southeast Asia. GC immunofixation electrophoresis in agarose gel was performed as described previously ( WEITKAMP 1978b). Polyacrylamide gel electrophoresis (PAGE) in a discontinuous Tris-HC1- glycine buffer system and autoradiography with 14C labeled vitamin D, was done according to the method of DAIGER and CAVALLI-SFORZA (1977). Immunodiffusion was carried out in agar containing 1% polyethylene glycol (ALLEN et al. 1977), using a goat anti-human Gc protein obtained from Atlantic Antibodies (Westbrook, Maine). Albumin phenotyping was performed according to the method of GAIINE (1966). RESULTS The relative mobilities of the 14C-vitamin D binding serum proteins in equids on PAGE autoradiography are shown in Figure 1 (samples 6-1 1). The three phenotypes consist of a single fast band (F). a double band (FS) and a single slow band (S) corresponding to the three phenotypes previously reported for domestic horses on agarose immunofixation electrophoresis using goat antihuman Gc antibody (WEITKAMP 1978a,b). The faint band anodal to the major band (F) in the samples from an onager (sample 9) was not consistently observed in other samples of the F type. In addition to the 12 horses previously typed (WEITKAMP 1978b), Gc phenotypic frequencies of equids have now been extended to include four wild species of the genus, as well as domestic donkeys, mules and 329 additional domestic horses (Table 1). All of the wild equine species and a portion of the domestic species were typed by both PAGE autoradiography and agarose immuno ixation electrophoresis; the majority of domestic horses were typed by the latter method only. Note that F and S phenotypes were observed in each of the subgenera Equus, Asinus and Hippotigris. No species-specific or species-associated antigenic difference among the Gc proteins of various equids could be shown by immunodiffusion. Zebra, onager, domestic

3 EQUINE GC PROTEIN 1349 donkey, Przewalski horse, domestic horse and mule antigens each gave a single band of precipitation showing complete fusion. All other mammals tested showed one or two major bands on PAGE autoradiography. The sample from Mus ceruicolor popaeus (sample 3, Figure 1) demonstrates two of six different electromorphs observed in the ten wild mice from Southeast Asia ( WEITKAMP, ALLEN and SELANDER 1978). The faint band cathodal to the major band in the rabbit (sample 4, Figure 1) was more apparent on a repeat test of this sample. The major anodal and minor cathodal bands were also observed in seven other rabbits, both by autoradiography and by immunofixation electrophoresis. In a single chicken, a relatively weak but distinct band was seen in PAGE autoradiography migrating more slowly than any of the mammalian bands; no band was observed in a single amphibian, Xenopus Zaeuis. On immunofixation electrophoresis in agarose gel, no bands were seen in the deer, cattle and chicken samples or in Xenopus; relatively weak bands were seen in the rodent samples. However, on immunodiffusion all mammals tested (except the goat) were cross-reactive, producing a band of precipitation. No precipitin band was observed in the samples from the chicken or Xenopus. Differences in the cross-reactive behavior of the Gc antigens in various mammals was evident by spurring of the human antigen over rodent antigen (rat, mouse and guinea TABLE 1 Gc phenotypes in the genus Equus Subgenus Equus (1) Domestic horse, Equus caballus Standardbred Thoroughbred Fresian Arabian Quarter Horse Shetland Welsh (2) Mongolian wild horse, Equus ferus przewalski Subgenus Asinus (1) Domestic donkey, Equus minus (2) Asiatic wild ass, Equus hemionus onager Subgenus Hippotigris (1) Hartmann s mountain zebra, Equus zebra hartmannae (2) Grant s zebra, Equus burchelli boehmi (3) Zebra, cpecies unknown Hybrid Mule Phenotype F FS S

4 135 L. R. WEITKAMP AND P. 2. ALLEN + ORIGIN FIGURE 1.-PAGE autoradiography of vitamin D binding proteins in a discontinuous tris- HC1-glycine buffer system. Sample 1, human (2-2); 2, dog; 3, Mus cervicolor popaeus; 4, rabbit; 5, guinea pig; 6, donkey (S); 7, Fresian horse (F); 8, Przewalski horse (F); 9, onager (F); 1, zebra (S); 11, Standardbred horse (FS); and 12, human (2-1). pig), of lagomorph antigen (rabhit) over horse Gc, and of the horse band over the dog, deer and cattle bands. Close linkage between the Gc and albumin loci in the horse has been reported in preliminary form (WEITKAMP 1978c) and is demonstrated by the data in Table 2. The results are presented in the form of lod scores since, with the exception of three nonrecombinants, the data consist of 26 offspring from nine phase-unknown parents. The peak lod score is 6.2 at zero recombination frequency. A score of three is usually considered significant evidence of linkage (MORTON 1955). In order to test for linkage disequilibrium, the Gc and albumin phenotypes were tabulated for those horses for which neither parent was typed. Among 125 TABLE 2 JW scores for the linkage relationships of the equine loci for albumin and Gc Recombination frequency Sex Male Female Total I.26 I

5 EQUINE GC PROTEIN 1351 horses in the parental generation, 24 were doubly heterozygous. The phase of linkage could be determined from the offspring of 23 of the double heterozygotes, assuming no recombination between the albumin and Gc loci. For the 124 parental generation horses in which haplotypes could be determined, there is an excess of GCF-aZbF and G8-aZbs haplotypes (Table 3, x: = 7.4, p <.1). DISCUSSION Horse F, FS and S phenotypes have previously been shown to be determined by two alleles, GcF and GcS (WEITKAMP 1978b). From data presented in Table 1, the frequency of the F allele is.82 in 431 domestic horses and contrasts to a frequency of.6 in 26 donkeys. The difference is consistent with the observation that all three mules tested are heterozygotes. The finding of Gc F and S electromarphs in sera from each of the three equine subgenera, Equus, Asinus and Hippotigris, suggests that polymorphism for Gcr and GcS antedates speciation within the genus Equus. Recurrent mutation and selection or hidden allelic differences are possibilities that cannot of course be excluded. The fossil record indicates that the genus Equus is about 5 million years old (GROVE 1974). In man, the common alleles. Gc and Gc2, have been found in all 19 populations studied ( CLEVE 1973). Among several hominoideae, all of which diverged more than 5 million years ago, only Pongo pygmcreus shows variations similar to the human Gc phenotypes (reviewed in CLEVE 197). Although detailed pedigree records are available on many breeds of horse, little is yet known about the autosomal gene map of this species. Loose linkage between the 6-PGD and K blood-group loci ( SANDBERG 1974) and close linkage between the albumin and tobiano coat-spotting pattern loci (TROMMERHAUSEN- SMITH 1978) have been reported. Very closely linked groups of genes, uiz., hemoglobin, immunoglobulin, histocompatibility and amylase genes, are known to have been conserved in man, mouse and a few other mammals. Conservation of mammalian X-linked genes has been observed in many species (OHNO 1973), but data on the comparative linkage relationships of autosomal genes that are not related in structure or function are available for only mouse and man. Most of the comparative map TABLE 3 Albumin and Gc haplotypes in 124 parental generation americrrn standardbred Horses Albumin Gc F S Total F (19.81) (74.19) S (38.19) (25.81) Total The expected numbers in parentheses were derived from the marginal totals.

6 1352 L. R. WEITKAMP AND P. Z. ALLEN has been constructed from interspecific cell hybrids, using the formation of heteropolymers in mouse/human hybrid cells to establish homology between the genes in each species. Nine autosomes carry homologous pairs or groups of genes (summarized in LALLEY, MINNA and FRANCKE 1978). Comparative recombinational data are available for two loci: PGD and PGM, (Pgm-2 in the mouse), which have a male map distance of 5 centimorgans in man and 24 centimorgans in the mouse (HAMERTON and COOK 1974; CHAPMAN 1975), and glyoxalase-1 and HLA (H-2), which have a 3 to 5 % recombination frequency in human and mouse males (MEo, DOUGLAS and RIJNBECK 1977; WEITKAMP and FRANCKE 1978). The demonstration of a vitamin D binding protein that has immunologic cross-reactivity in all mammals studied provides strong evidence for the evolutionary homology of the protein. Although the genus Equus is only about 5 million years old, there is considerable variation in chromosome number within the genus (2n = 66 for Equus przewalski, 64 for Equus caballus, 62 for Equus asinus, 56 for Equus heminous onager, 44 for Equus burchelli boehmi and 32 for Equus zebra hrtmannae; BENIRSCHKE 1967). Presumably, there has been substantial opportunity for the development of chromosomal rearrangements between horse and man. Nevertheless, the recombination frequency between the albumin and Gc loci in the horse (equivalent to in 2 recombinants for the peak lod score at % recombination frequency) is probably similar to the 2% frequency observed for man (WEITKAMP et al. 197). Determination of the recombination frequency between the albumin and Gc loci should be possible in rodents and, perhaps, mammals from other orders. Such data will contribute to the question of taxonomic differences in meiotic recombination frequency between homologous loci. They may also provide evidence regarding the constraints on disruption of linkage groups during evolution. Of particular interest would be a comparison of the conservation of the major histocompatibility region in relation to the conservation of linkage between albumin and Gc, two loci with no apparent structural or functional relationship. Tightly linked loci, e.g., immunoglobulin heavy chains, amylases, the MNSs blood groups, hemoglobin /3 and S chains, HLA-B and properdin factor B (Bf) have a high degree of linkage disequilibrium in man. Gametic disequilibrium also exists for the HLA-A and HLA-B loci (recombination frequency -1%) but not for the adjacent and more loosely linked GLO and Bf loci (WEITKAMP 1977; OLAISON, personal communication). Whether disequilibrium in the HLA region results from selection or from human population structure is unknown. However, there is some evidence for gametic disequilibrium between the G-6PD and color-blindness loci in Sardinians (FILIPPI et al. 1977) and between the albumin and Gc loci in Europeans (WEITKAMP 1977), both of which have a recombination frequency of -1-3%. In contrast, disequilibrium between the Rh and a-fucosidase loci, which also have a recombination frequency of -1 to 3%, has not been observed (FISHER, NOADES and ROBSON 1978). Virtually all of the parental generation 1 Standardbreds included in Table 3 share ancestors within the past five or six generations. Thus, the albumin-gc

7 EQUINE GC PROTEIN 1353 linkage disequilibrium can be accounied for most simply by the extent of inbreeding in our sample. The rate o decline of linkage disequilibrium may be taken as (l-s)., where is the recombination frequency and n is the number of generations. At =.2, disequilibrium will decline to.5 or.1 of its original value in 34 or 114 generations, respectively. Since Standardbreds were developed as a separate breed in the last century and since a small number of foundation sires have been particularly influential (WRENSCH 1948), linkage disequilibrium between the albumin and Gc loci may well be characteristic of the breed. If population structure rather than gametic selection does indeed account for the disequilibrium in Standardbreds and possibly in man, one would predict that a natural population with a much shorter generation time, e.g., rodents, might not demonstrate a similar disequilibrium. This research was supported in part by National Foundation grant and by a gift from MRS. JOHN L. WEHLE. Ms. LORRAINE FRANCISCO, E. JOHNSTON and S. GUTTORMSEN provided excellent technical assistance. We also express our appreciation to WILBUR AMAND of the Philadelphia Zoological Gardens for the gift of sera from onagers and zebras, to JAN GOUDS- WAARD, University of Utrecht, for the Przewalski and Fresian horse sera and to ROBERT KENNEY of University of Pennsylvania School of Veterinary Medicine for the sera from donkeys and mules. We thank EARL SPEECE, NELSON WERT, JOHN WITMER, JOSEPH ODEA, ROBERT HILLMAN, ANN TROMMERHAUSEN-SMITH, PETER BOYCE and ROBERT PIERSON for assistance in obtaining blood samples from domestic horses, and VERNE CHAPMAN for supplying the samples from the Asian wild mice. LITERATURE CITED ALLEN, P. Z., E. J. DALTON, S. KHALEEL and R. M. KENNEY, 1977 Studies on Equine Immunoglobulins V. Horse Antibodies to Donkey IgG. Immunochemistry 14: BENIRSCHKE, K., 1967 Sterility and fertility of interspecific mammalian hybrids. pp In: Comparatiue Aspects of Reproductive Failure. Edited by K. BENIRSCHRE. Springer- Verlag, New York. CHAPMAN, V., Phosphogluconate dehydrogenase (PGD) genetics in the mouse: Linkage with metabolically related enzyme loci. Biochem. Genet. 13 : CLEVE, H., 197 The group-specific component (Gc) in chimpanzees. Hum. Hered. 2: , 1973 The variants of the group-specific component. Israel J. Med. Sci. 9: DAIGER, S. P. and L. L. CAVALLI-SFORZA, 1977 Detection of genetic variation with radioactive ligands. 11. Genetic variants of vitamin D-labeled group-specific component (Gc) proteins. Am. J. Hum. Genet. 29: DAIGER, S. P., M. S. SCHANFIELD and L. L. CAVALLI-SFORZA, 1975 Group-specific component (Gc) proteins bind vitamin D and 25-hydroxy-vitamin D. Proc. Natl. Acad. Sci. U.S. 72: FILTPPI, G., A. RINALDI, R. PALMARINO, E. SERAVALLI and M. SINISCALCO, 1977 Linkage disequilibrium for two X-linked genes in Sardinia and its bearing on the statistical mapping of the human X chromosome. Genetics 86: FISHER, R. A., J. E. NOADES and E. B. ROBSON, 1978 group. Cytogenet. Cell Genet. 22: Studies on the Rh:ru-fucosidase linkage GAHNE, B., 1966 Studies on the inheritance of electropholretic forms of transferrins, albumins, prealbumins and plasma esterases of horses. Genetics 53:

8 1354 L. R. WEITKAMP AND P. Z. ALLEN GAHNE, B. and K. JUNEJA, 1978 Post-albumin types in cattle, horse, pig and sheep plasma identified as vitamin D bicding protein (Gc protein). (Abstr.) XVI Internat. Conf. Anim. Blood Grps. Biochem. Polymorphism, Leningrad, p. 3. GROVE, C. P., 1974 Horses, Asses nnd Zebras. Ralph Curtis Books, Hollywood, Florida. HAMERTON, J L. and P. J. L. COOK, 1974 Report of the committee on the genetic constitution of chromosome I. Cytogenet. Cell Genet. 13: HIRSCHFELD, J., 1959 Immune-electrophoretic demonstration of qualitative differences in human sera and their relationship to the haptoglobins. Acta Path. Microbiol. Scand. 47: LALLEY, P. A., J. D. MINNA and U. FRANCKE, 1978 Conservation of autosomal gene synteny groups in mouse and man. Nature 274: MEO, T., T. DOUGLAS and A.-M. RIJNBECK, 1977 Glyoxalase I polymorphism in the mouse: A new genetic marker linked to H-2. Science 198: MORTON, N., 1955 Sequential tests for the detection of linkage. Amer. J. Hum. Genet. 7: OHNO, S., 1973 Ancient linkage groups and frozen accidents. Nature 244.: REINSKOU, T., 1968 The Gc system. Ser. Haematol. 1: SANDBERG, K., 1974 Linkage between the K blood group locus and the 6-PGD locus in horses. Anim. Blood Grps. Biochem. Genet 5: TROMMERSHAUSEN-SMITH, A., 1978 Linkage of tobiano coat spotting and albumin markers in a pony family. J. Heredity 69: WEITKAMP, L. R., 1977 Data on linkage disequilibrium in man. (Abstr.) Am. J. Hum. Genet. 29: 113A. --, 1978a Biochemical genetics of the Thoroughbred horse. Identification of variants of group-specific component (Gc). XIV Internat. Cong. Genet. MOSCOW. Abstracts Part I, p , Equine marker genes. Polymorphism for groupspecific component. Anim. Blood Grps. Biochem. Genet. 9: , 1978~ Comparative Gene Mapping. Linkage between the albumin and Gc loci in the horse. (Abstr.) Am. J. Hum. Genet. 3: 128a. WEITKAMP, L. R., P. Z. ALLEN and R. K. SELANDER, 1978 Polymxphism for the Gc protein in Equus and Mus. (Abstr.) XVI Internat. Conf. Anim. Blood Groups. Biochem. Poly morphism. Leningrad, p WEITKAMP, L. R. and U. FRANCKE, 1978 Report of the committee on the genetic constitution of chromosome 6. Cytogenet. Cell Gene. 22: WEITKAMP, L. R., J. H. RENWICK, J. BERGER, D. C. SHREFFLER,. DRACHMA, F. WUHRMANN, M. BRAEND and G. FRANGLEN, 197 Additional data and summary for albumin-gc linkage in man. Human Hered. 2: 1-7. WEITKAMP, L. R., P. L. RUCKNAGEL, and H. GERSHOWITZ, 1966 Genetic linkage between the structural loci for albumin and group-specific component (Gc). Am. J. Hum. Genet. 18: WRENSCH, F. A., 1948 Harness Horse Racing in the United Stares. van Nostrand, New York. Corresponding editor: D. BENNETT