Proc. Nat. Acad. Sci. USA Vol. 70, No. 5, pp. 1303-1307, May 1973

Genetic Model for the Rh -Group System (conjugated operons/repressors/quantitative blood typing) RICHARD E. ROSENFIELD*, FRED H. ALLEN, JR.t, AND PABLO RUBINSTEIN*t *Department of Pathology, Mount Sinai School of Medicine, 5th Ave. and 100th Street, New York, N.Y. 10029; tNew York Blood Center, 310 East 67th Street, New York, N.Y. 10021 Communicated by P. Levine, February 26, 1173

ABSTRACT Inherited quantitative aspects of the Rh No such studies have been possible with membrane anti- blood-group system and susceptibility of Rh to the effects gens, perhaps because their actual isolation from lipid mem- of independently segregating suppressor can be cause serious accounted for with a conjugated operon model. This brane constituents might degradation (17). assumes the existence of four operator or promotor (con- Recent studies of the microstructure of human erythrocyte trol) genes for these functions, while closely linked struc- membranes revealed moveable particles embedded in tural regions determine the qualitative characteristics of the lipid phase (18); they were associated with blood-group A Rh . Observed restriction of antigenic crossre- activity (18) and with receptors for both phytohemagglutinin activity to the products of adjacent genetic regions and data from blood typing of nonhuman primates both and influenza virus (19). The number of these particles was suggest that Rh complexity arose from a series of estimated to be 4200 (18)-4500 (19) per /Am2 of the surface of duplications and independent mutations. erythrocyte ghosts or about 6 X 105 for an average intact cell. The 33 qualitatively different antigenic specificities of the Rh Membrane protein particles are likely to consist of several blood-group system (1, 2) have become extraordinarily dif- polypeptides, some of which may be wholely or partly re- ficult to organize systematically. Each at the Rh locus sponsible for the expression of blood-group antigens. If such a determines a variable number of different antigens (3, 4), and polypeptide were directly determined by a single blood-group recombination has been observed in just one family (5). In gene, alloantigenic variation could be the result of single addition, quantitative differences in the expression of Rh anti- amino-acid substitutions similar to the situation for Gml of gens are also under the control of Rh genes (6). Accommoda- IgG heavy chains (20). However, tertiary structures arising tion of quantitative data by genetic theories devised to ac- from interaction between a polypeptide and either other poly- count only for qualitative alloantigenic variants (3, 4, 7, 8) peptides or other membrane structures could also give rise to results in a vast increase in an already overwhelmingly large blood-group antigens. For instance, Rh antigenic activity was number of complex . lost when ghosts were extracted with lipid solvents, but Rh In this report, Rh data have been arranged in a manner that distinguishes qualitative from quantitative information. From this arrangement, a consistent genetic model emerges that may TABLE 1. Glossary of Rh terminology* provide a biologically sounder conceptual basis for this com- (refs. 26 and 27, text) plex system. In the Rh system (see Table 1 for glossary of terms) Rh: wl - 12 - rhG or G 23 - Dw (Rh or DU) was the first quantitative variant found (9). I Rho or D A most interesting situation was shown to involve allelic 2 - rhI or C 13 a RhA 24 w ET interaction, with the R-",2 -3 (r' or dCe) gene being suppres- 3 - rh" or E 14 - RhB 26 a Deal sive of RI alleles (R or D) in trans position (10). Quantitative 4 a hr' or c 15 - RhC 27 - cis cE variants of RI were documented quantitatively by Silber et 5 - hr" or e 16 n Rh0 28 - hrH

al. (11) and Masouredis (12), but Gibbs and Rosenfield (6) 6 a hr or f or cis ce 17 a Hr0 29 - 'total Rh' found that the quantitative "degree of expression" was under 7 - hr1 or cis Ce 18 a Hr 30 u Goa the strict genetic control of Rh genes and so was the inter- 19 a hrs 31 - hrB allelic depressive effect of R-1"2 -3. Within an extensive pedi- 8 - rhWl or CW gree, identical were quantitatively identical for 9 - rhX or CX 20 a VS 32 a determined by RN four different Rh antigens, whereas qualitatively similar geno- 10 = hrt or V or eS 21 a CG 33 - determined by R0 Har types in the general population varied over a significant range 11 - rht or Ew 22 a cia CE 34 a Bas. (6). Using a different method, Berkman et al. (13) con- firmed these observations and extended the findings to the * Antigens shown as "Rh" followed by corresponding number. other blood groups. shown as "Rh: " followed by numbered antigens Studies of the chemistry of secreted human blood-group separated by commas for which tests were performed. Negative substances have provided detailed insight into the chemical or weak result of test shown by minus (-) or "w," respectively, of and Lewis antigens (14, 15), while evalua- preceding number. Alleles shown by R with antigens produced genetics ABO, H, or not produced given as for but in superscript. Rh25 tion of N-acetyl-galactosaminyl transferases established both (LW) is the main shared by human and rhesus eryth- the nature and the mechanism of production of quantitative rocytes. In man Rh25 is determined by genes that segregate variants of the A antigen through the Km value of the specific independently of the Rh locus. Rh34 has been assigned to the transferase (16). specificity of the total immune response of Mrs. Bas (32). 1303 Downloaded by guest on September 25, 2021 1304 Genetics: Rosenfield et al. Proc. Nat. Acad. Sci. USA 70 (1973)

activity of ghosts treated with n-butanol was restored by gens are expressed, while XQ/XQ depresses slightly less and addition of "nonspecific" phosphatidyl choline (21). In- produces "pseudo rhG" (34). Furthermore, all known ex- terestingly, F. A. Green found that Rh.,,1 cells reported in amples of either are associated with congenital ref. 22 were neither deficient in "restorative" phosphatidyl hemolytic characterized by cup-shaped erythrocytes choline nor could their ghosts be rendered Rh-antigenic by (stomatocytes) rather than normal discoid erythrocytes (35, addition of n-butanol extracts of normal cells (personal com- 36). Rhnull also arises from the homozygous state of "amor- munication to R.E.R.). Tertiary structure interactions appear phic" Rh genes (r or -) (37), and this, too, is associated with to underly the HI and AI determinants (3) and may explain stomatocytic (38). Thus, one adequately (see Rh25 of Table 1) the phenotypic association between Rh functioning Rh gene appears to be needed for production of and LW (3, 23). normal erythrocyte membranes. Whether the erythrocyte Strongly suggestive evidence that Rh is protein in nature changes associated with RhnuiI and "pseudo rhG" are a direct (24) includes its reversible inactivation by p-chloromercuri- consequence of the altered expression of Rh antigens or the benzoate (25), inactivation by N-ethylmaleimide (25), de- result of an epistatic effect of Rh genes is as yet unknown. struction by heating to 56' (26), susceptibility to proteolytic The expression of all Rh antigens from one Rh gene can be digestion (27), and denaturation by urea (25, 28) or exposure depressed by certain paired genes, especially those determin- to pH 3.0 (29). The role of phosphatidyl choline and the high ing Rh2 (rh' or C) (6). This effect, too, is more readily com- lability of Rh indicate that the tertiary structure of a protein- prehensible when a main point of regulation is assumed. lipid complex is essential to the formation, orientation, and/or The main regulatory locus of Rh may display allelic alterna- stabilization of the expression of Rh determinants. tives. If R29 is considered the "normal" allele, R-29 can be Table 1 gives the 33 antigenic specificities described in the assumed for cis Rhnun1 and RI' can be used for an abnormal Rh system. The genetics of Rh, however, is far more com- expression of all structurally specified antigens with coinciden- plicated because the qualitative combination of antigens tal emergence of a rare and otherwise unobserved antigen, within each allotype and the degree to which each antigen is Rh33 (39). These allelic possibilities, however, are not obliga- expressed are both under the control of the Rh locus. In fact, tory because similar effects would obtain with selected cis to account for the thousands of resulting Rh allotypes, no less conditions at other regions of the Rh system. These and other than four conjugated and coordinated systems, or operons problems relating to the main point of regulation will be con- (30), appear to be needed on (31). This is shown sidered later. in Fig. 1. The serology of the Rh blood-group system can be divided A main point of regulation, although not an absolute neces- into two parts, one concerning Rhl (Rh. or D) and the other sity, clearly indicates how all of Rh is susceptible to suppres- non-Rhl antigenic specificities. Indeed, this is the basis of sion, especially by independently segregating, partially reces- Wiener's Rh-Hr terminology (4). However, Rhl is not likely sive genes. The known independent suppressors are called to be a discrete antigenic determinant but, rather, a series of X~r (33) and XQ (34) although neither is associated with the distinct antigens inherited en bloc and all determined by X chromosome. Both have profound effects when homo- "normal" RI genes. Rare R' genes lack one or more of these zygous: X~r/X~r produces Rhnull (33) in which no Rh anti- antigens, and people who are type Rh:1 (Rh- or D-positive)

0 = Operator orpromotor; LII = Structural gene; - = Direction of control

IMPORTANT R933 RWI R(v),23 R32 R-4,21 pW3 R-3,5 ALLELES R29 Rp ,(V),30 qP17 also -518 also AT p(I,-) R4,±26 R-34 R3,tll,±24 EACH \ih/)R R21,t2,tB9 p3or5 R5,±10,28 LOCUS RI(')

Suppressors Irons of R29 (x°x0)(1?21 )

CROSSREACTIVITIES Cis PRODUCTS SPECIFICITY CROSSREACTION ANTIGEN ALLELE RhI2 Rhl/Rh2I Rh6 R4,5 or weokly, R-3,4 hr- Rh 3 / Rh4 Rh7 R2 45 hrjr Rh2I/Rh 5 Rh 22 P2,3 Rh2O RhlO/ Rh28 Rh27 34 FIG. 1. Proposed genetic model for the Rh blood-group system. Figure represents some described relationships of different portions of the Rh system and does not purport molecular assumptions in terms of mechanisms whereby, for example, R17 regulates both the Rh4 or 21 structural region and the control locus for the Rh3 or 5 region. Likewise, it does not attempt to discriminate as to whether the control region for the entire Rh system is a complete operon or merely an operator or promotor. Numbers used refer to recognized sero- logical specificities as defined in refs. 1 and 2, text, and Table 1. Roman numerals for categories of Rhl positivity (23) have been used in place of ill-defined combinations of R13, R14, R15, R16, and possibly others. Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Genetic Model for Rh 1305

by virtue of such "incomplete" genes can become immunized mines Rh2l (C') with or without Rh2. Rh: - 2,21 has been by, what are for them, foreign components. A study of the called rh' (46). R21 alleles also may (18R21) or may not produce specificity of different sources of anti-Rhl revealed strong re- Rh8, and may (R9,21) or may not produce Rh9. semblances between made by many Rh: -1 persons The Rh3 or 5 region in Fig. 1, in addition to being under and the immune response of a person carrying an "incom- control of both R29 and R17, has its own control gene as evi- plete" Rh:1 phenotype (40). Furthermore, the unusual im- denced by the rare R-8' - allotypes, DCP'- and Dc- (3), which munogenicity of Rhl (3, 4), in comparison with other non- arise from the "off" alternative. The "on" alleles control the ABO antigens, may well be due to its being a cluster of dif- degree of expression of Rh3 or 5 antigens in "normal" allo- ferent antigens that provide the Rh:1 recipient with a spec- types while additional alleles at this control locus determine trum of antigens toward which to respond. Rh: w3 (EU) (3) and both the Shabalala-Santiago (2, 47) and There is no evidence to suggest the simultaneous existence Bastiaan (2, 32) types. The latter two alternatives are des- of both Rhl as a discrete antigen and of component Rhi-like ignated, respectively, R-18 and R-"4. Rh34 has been assigned antigens. Alternatively, the Rh phenotype in reference to Rhl to the specificity of the total immune response of Mrs. Bas can be considered to be conditioned by a structural genetic (32). that specifies component antigens under control of a region The Rh3 or 5 structural region in Fig. 1 also cannot be re- closely linked regulatory gene. Such a control gene, as shown solved beyond treating its genes as a series of alternatives in Fig. 1, can be designated RI because its effects fit exactly (1, 2,32). Thus, RI produces Rh3 with (R1? ) or without Rhil, the operational definition of the Rhl antigen. RI switches and with (RI 24) or without Rh24. R5, the most inclusive allele "on" the structural region for components, but alleles at the of R3, may produce Rh5 with (R5 10) or without RhlO, and RI locus switch "on" with variable efficiency, giving rise to with (R5,21) or without Rh28 (48). Two antigens, Rhl9 and genetically controlled quantitative differences in "expression" Rh3l, are expressed by R5 alleles except when control genes of Rhl. At very weak levels of "on," RI can be called Rel (R are R-1'8-34, or R-34. or DU) because the majority of these people show depression R-18, of all studied components. The R-1 allele (r or d) provides Ascribing control functions to genetic determinants of anti- "off" to explain the Rh: -1 (Rh-negative) status. Further- genic specificities such as Rh29, Rhl7, Rhi8, and Rh34 is not more, as a regulatory gene, R-' could not have an antigenic necessarily contradictory. Such antigens could be properties structural product, d. There is no way to determine at present of structural products arising in consequence of each and all whether this closely linked control locus acts as "operator" or "on"l alleles at corresponding control loci. A new symbology is "promotor." For the purposes of this discussion, alleles at not needed immediately merely to denote this relationship. such regulatory loci will be referred to as control genes. Separate antigenic determinants interact in various ways. Tippett (23) resolved Rhl component polymorphism into (1) Some antigens alter the serological expression of others. six categories, whereas Wiener (4) described four "factors," Thus in Fig. 1, R21 in trans position partially represses the RhAB.CD. A more precise definition of this structural region products of all three structural regions (6). In cis position, is needed, but Tippett's classification accommodates all ob- however, R21 augments the expression of Rh3 while Rhl ap- servations efficiently, including the very important discoveries pears depressed when compared to its expression in Rh: 1,-21 that Class IV phenotypes have Rh3O (Goa) (41) and that phenotypes (3). Another example is RhlO from R45'60,, which persons in Class V have Rh23 (DW) (42). Neither Rh3O nor is often associated with reduced cis Rh6 (13). (2) Antigens Rh23 is observed under other conditions. Thus, we can assume may crossreact and thereby create additional specificities, as the existence, under the control of R', of structural alleles shown in Fig. 1. These include Rhl2 representing either Rhl t R R(), R~ R(i) 0, R(M23,2R(), and R(), with R(°) or Rh2l; "hri" (43) representing either Rh3 or Rh4; "hr-." designating absence of a class and thereby presence of all representing either Rh2l or Rh5; and Rh2O representing either components (Fig. 1). RhlO or Rh28 (47). (3) As shown in Fig. 1, antigenic deter- The "non-Rhl" part of Rh includes structural genes for minants may arise as cis products (1-3) of different structural either Rh4 or Rh2l, and for either Rh3 or Rh5. It appears to regions: Rh6 from R4,5 or, weakly, from R-3'4' 5 (cD-); Rh7 consist of two conjugated operons, but one control gene com- from R2'-4'5; Rh22 from R2'3; and Rh27 from R834. (4) Rh mands both operons and is defined (Fig. 1) by the rare "off" antigens may be essential for the expression of other blood- allele, R-'7 (R0 or D-) (3, 4). The homozygote R-1-'17/ group genes. Thus, all examples of Rhull are LW-negative R- '-7 (43) could also produce cis Rhull, hypothetically and show depressed S, s, and U (49). The mechanism of this indistinguishable from what would be produced by R-29/R-29. interaction is unknown but since LW-positive persons may Another rare alternative of R-17, R32 (RN) (44), depresses become phenotypically negative temporarily (50), it can be both Rh2l and Rh5 and leads to the expression, perhaps as a postulated that the corresponding structure (polypeptide?) is direct consequence, of the rare antigen, Rh32 (45). The allo- antigenic only when bound to Rh. This bond could be lost type designated D(C)-, carries partially depressed Rh2l and reversibly enabling such people to make anti-LW even though fully suppressed Rh5 without appearance of Rh32 (23), but is LW antigenicity eventually reappears (50). otherwise quite similar. Rare allotypes, Ad, rL, and rt (3), Assignation of control and structural Rh functions to sepa- could arise from other alleles at this control locus. rate loci may be considered as merely an updating of Fisher's The common alleles of R-'7, collectively termed R17, regulate CDE proposal (7, 8). The model now proposed, however, more directly the degree of expression of the Rh4 or 21 structural closely resembles the four conjugated operons responsible for region in which several alternative antigenic characteristics structural protein synthesis by A phage (51). Rh also appears are known (1, 2). Data are insufficient to further resolve this to be part of an important membrane protein. In both Rh and structural region. In Fig. 1, R4 produces Rh4 with or without X phage a main control locus commands three structural re- Rh26. The most inclusive alternative to R4 is R21, which deter- gions, each of which has its own control gene. Further, in both Downloaded by guest on September 25, 2021 1306 Genetics: Rosenfield et al. Proc. Nat. Acad. Sci. USA 70 (1973)

systems one of these secondary control genes influences two similar models, but there is insufficient data as yet to attempt distinct structural regions. this. Two systems in which we require more quantitative data The existence of specific control genes that determine for such consideration are Kell and Lutheran. Both have "degree of expression" while structural genes generate qual- complex alleles, each of which determines multiple antigens. itative differences has already been proposed for mammals: However, some of these alloantigens appear to be allelic to X-linked testicular feminization in the mouse is very likely others in the sense that they segregate as alternatives, and no the result of a noninducible regulatory mutation of the Jacob- gene complex carrying more than one has been observed (3). Monod type (52). Rare amorphic genes at both the Kell and Lutheran loci Hughes-Jones et al. (53) found evidence suggesting that the determine "null" types when homozygous (3). Lutheran genes total number of antigenic sites per cell, as determined by the are additionally subject to the repressive effect of a rare dom- three structural regions of both chromosomes, may be con- inant gene at an unmapped locus causing a "null"-like type stant for most Rh: 29 phenotypes. According to the model now (3). Kell genes, too, are repressed by what appear to be rare proposed, such a quantitative limitation could easily be im- recessive genes at an independent locus (3), apparently re- posed by a structural product of the main control (R29) region lated to the X chromosome (57). When hemizygous, this is (e.g., RNA polymerase) on all conjugated genes. associated with "McLeod," a very weak Kell phenotype, and Although conjugated systems are not characterized by high often with X-linked chronic granulomatous disease (57). crossover rates, and only one Rh recombination has actually This genetic model for membrane structural polypeptides been detected (5), some complex Rh alleles may have resulted that have not been isolated is, admittedly, entirely specula- from unequal crossing over. R-',2 -3 4,5,6 -7,21,28 (r'") and the tive. It does, however, provide a rational conceptual frame- very similar R1,w2,3,4,5,6,7,w21'28 (GU) are prime suspects work with which to view the complexity of Rh in its entirety. (1, 54). The difference between these alleles is likely to reside The model arose as a consequence of finding the strict genetic with R17 control genes for the "non-Rhl" part of Rh, some be- control exerted by the Rh locus on the quantitative expression ing more depressive of the Rh4 or 21 region than others. The of all Rh antigens, and it should be subject to critical study same sort of variation could underly the difference between from quantitative considerations. Human kinships involving R-1,2, -3, -4,5, -6,7,21, -28 (r') and R-1,w2, -3, -4,5, 6, -7,21, -28 (rG). large sibships and three generations should be assayed quan- From these examples, Rh7 is clearly a product of R2 -4,5 in titatively in search for crossover data involving control genes. which Rh2 must be reasonably well expressed. These loci, rather than those for qualitative structural data, The products of adjacent structural regions, as proposed, may provide new and significant information concerning the exhibit several important crossreactions. This finding suggests structure of genes that control the expression of Rh antigens. that these structural loci may have appeared during evolu- tion by a series of gene duplications resembling the evolution of Hp2 from Hp"F plus HplS (55). Serological crossreactions 1. Rosenfield, R. E., Allen, F. H., Swisher, S. N. & Kochwa, are the result of stereochemical similarities more likely to exist S. (1962) "A review of Rh serology and presentation of a between products of closely related genes than new terminology," Transfusion 2, 287-312. between prod- R. E. "Review of Rh ucts of genes separated by more mutational events. There- 2. Allen, F. H., Jr. & Rosenfield, (1972) serology. Eight new antigens in nine years," Haematologia fore, the Rh4 or 21 region should occupy a central position 6, 113-120. because its antigens crossreact with antigens of both the Rhl 3. Race, R. R. & Sanger, R. (1968) in Blood Groups in Man and the Rh3 or 5 regions and probably its first duplication, (F. A. Davis, Philadelphia), 5th ed., pp. 171-248 (Rh system), pp. 249-264 (Lutheran system), and pp. 265-290 through mutation, evolved into Rhl components. The dupli- (Kell system). cation that gave rise to Rh3 or 5 is likely to be more recent 4. Wiener, A. S. & Wexler, I. B. (1963) in An Rh Syllabus since many more crossreactions involve its products with those (Grune and Stratton, New York), 2nd ed., pp. 29-48. of Rh4 or 21. Crossreactions between Rhl and Rh3 have not 5. Steinberg, A. G. (1965) "Evidence for a mutation or cross- been observed. ing-over at the Rh locus," Vox Sang. 10,721-724. 6. Gibbs, M. B. & Rosenfield, R. E. (1966) "Immunochemical The phylogeny of Rh shows a parallel increase in com- studies of the Rh system. IV. Hemagglutination assay plexity. All gibbons tested (56) had only Rh4, while chim- of antigenic expression regulated by interaction between panzees, gorillas, and orangutans possessed both Rh4 and paired Rh genes," Transfusion 6, 462-474. Rhl (56). Neither Rh3 nor Rh5 determinants were found in 7. Fisher, R. A., cited by Race, R. R. (1944) "An 'incomplete' in human serum," Nature 153, 771-772. nonhuman primates (56), and hence this region's appearance 8. Fisher, R. A. & Race, R. R. (1946) "Rh gene frequencies in in man may have occurred later in evolution. The main point Britain," Nature 157, 48. of control (R29) either coexisted with an original Rh4 operon 9. Stratton, F. (1946) "A new Rh allelomorph," Nature 158, 25. or it arose from a duplication of the R17 control gene. The fact "An inter- of both and 10. Ceppellini, R., Dunn, L. C. & Turri, M. (1955) that the Rh3 or 5 operon is under the control R29 action between alleles at the Rh locus in man which weakens R'7 as well as R3 or further supports the idea of its more the reactivity of the Rho factor (Du)," Proc. Nat. Acad. recent divergence from Rh4 or 21. Sci. USA 41, 283-288. The status of Rh4 as the oldest Rh antigen may also 11. Silber, R., Gibbs, M. B., Jahn, E. F. & Akeroyd, J. H. explain in the Rh the interesting finding that, when present, Rh4 is associated (1961) "Quantitative hemagglutination studies blood group system. II. A study of the D(Rho) ag- with more antigenic sites per cell than any other Rh antigen glutinogen," Blood 17, 291-302. (53). 8 X 104 Rh4 sites per cell were found in the homozygote, 12. Masouredis, S. P. (1960) "Relationship between Rh.(D) bound to red very close to the value of 105 for Rhl sites on Rh:17 (Rh. or and quantity of I'll anti-Rh,(D) cells," J. Clin. Invest. 39, 1450-1462. D-) cells (53) in which there is no competitive production of 13. Berkman, E. M., Nusbacher, J., Kochwa, S. & Rosenfield, "non-Rhl" antigens. R. E. (1971) "Quantitative blood typing profiles of human Other blood-group systems may be explained on the basis of erythrocytes," Transfusion 11, 317-332. Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Genetic Model for Rh 1307

14. Watkins, W. M. (1966) "Blood group substances," Science heterozygous and when homozygous," Amer. J. Hum. 152, 178-181. Genet. 24, 623-637. 15. Lloyd, K. 0. & Kabat, E. A. (1968) "Immunochemical 35. Sturgeon, P. (1970) "Hematological observations on the studies on blood groups. XLI. Proposed structures for the anemia associated with blood type Rh~un1," Blood 36, carbohydrate portions of blood group A, B, H, Lewis-a, 310-320. and Lewis-b substances," Proc. Nat. Acad. Sci. USA 61, 36. Chown, B., Lewis, M., Kaita, H. & Lowen, B. (1971) "A 1470-1477. new cause of haemolytic anaemia?" Lancet i, 396. 16. Schacter, H., Michaels, M. A., Crookston, M. C., Tilley, 37. Ishimori, I. & Hasekura, H. (1967) "A Japanese with no C. A. & Crookston, J. H. (1971) "A quantitative difference detectable Rh blood group antigens due to silent Rh alleles in the activity of blood group A-specific N-acetylga- or deleted chromosome," Transfusion 7, 84-87. lactosaminyl transferase in serum from Al and A2 human 38. Hasekura, H., Ishimori, T., Furusawa, S., Kawaguchi, H., subjects," Biochem. Biophys. Res. Commun. 45, 1011-1018. Kawada, K., Shishido, H., Komiya, M., Fukuoka, Y. & 17. Singer, S. J. & Nicholson, G. L. (1972) "The fluid mosaic Miwa, S. (1971) "Hematological observations on the Ph model of the structure of cell membranes," Science 175, (---/---) propositus, the homozygote of amorphic Rh 720-731. blood group genes," Proc. Jap. Acad. 47, 579-583. 18. Pinto Da Silva, P., Douglas, S. D. & Branton, D. (1971) 39. Giles, C. M., Crossland, J. D., Haggas, W. K. & Longster, "Localization of A antigen sites on human erythrocyte G. (1971) "An Rh gene complex which results in a 'new' ghosts," Nature 232, 194-196. antigen detectable by a specific antibody, anti-Rh33," 19. Tillack, T. W., Scott, R. E. & Marchesi, V. T. (1972) Vox Sang. 21, 289-301. "The structure of erythrocyte membranes studied by 40. Rosenfield, R. E., Haber, G. & Gibbel, N. (1958) in "A freeze-etching. II. Localization of receptors for phytohemag- new Rh variant," Proc. 6th (1956) Congr. Int. Soc. Blood glutin and influenza virus to the intramembranous par- Trans. (Karger, Basel), pp. 90-95. ticles," J. Exp. Med. 135, 1209-1227. 41. Lewis, M., Chown, B., Kaita, H., Hahn, D., Kangelos, 20. Thorpe, N. 0. & Deutsch, H. F. (1966) "Studies on papain M., Shepeard, W. L. & Shackelton, K. (1967) "Blood group produced subunits of human gamma-G-globulins II. antigen Goa and the Rh system," Transfusion 7, 440- Structures of peptides related to the genetic Gm activity of 441. gamma G-globulin Fc-fragments," Immunochemistry 3, 42. 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