Proc. Natl. Acad. Sci. USA Vol. 74, No. 3, pp. 1147-1151, March 1977
Genetics
Assignment of the major histocompatibility complex to a region of the short arm of human chromosome 6* [human reciprocal translocation t(l;6) (p3200;p21OO)/ mapping of human genes by using man-Chinese hamster somatic cell hybrids/ regional mapping of phosphoglucomutase-3 and malic enzyme-1/gene dose effect]
UTA FRANCKEt AND MICHELE A. PELLEGRINOf t Department of Pediatrics M-009, University of California, San Diego, School of Medicine, La Jolla,
La Jolla, California 92093
Calif. 92093; and fScripps Clinic and Research Foundation,
Communicated by Victor A. McKusick, November 30, 1976
ABSTRACT Interspecific cell hybrids containing defined parts of human chromosome 6 were used for regional mapping of gene loci previously assigned to chromosome 6: the human leukocyte antigens (HLA) region, phosphoglucomutase-3
(PGM3; a-D-glucose-1,6-bisphosphate:a--glucose-i-phosphate phosphotransferase, EC 2.7.5.1) and malic enzyme-i [malic dehydrogenase(decarboxylating) (NADP+), L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40]. Human fibroblasts containing a balanced reciprocal translocation between the short arms of chromosome 1 and chromosome 6-designated t(I;6) (p3200;p2100)-were fused with an established line of Chinese hamster cells. Hybrid clones with segregating human chromosomes were studied for the presence of the translocation chromosomes IT and 6T and their normal homologues 1 and 6. Six clones that had retained IT, three clones with 6T, and three clones with 6 and 6T as controls, were analyzed by a microabsorption test for expression of the allelic antigens HLA-A2 and HLA-A3, both of which were present on the human parental cells. HLA-A2 segregated with the 1T translocation chromosome and HLA-A3 with the normal chromosome 6. Hybrid clones with 6T did not possess an HLA-A gene. In contrast, human PGM3 and malic enzyme-i expression segregated with the 6T translocation chromosome. These results indicate the location of the major histocompatibility complex in region 6p2100 - 6pter of the short arm of chromosome 6. The genes for PGM3 and malic enzyme-i map in region 6p2100 - 6qter. The HLA.PGM3s genetic ma distance is 15 centimorgans in males, as established by famify studies. This allows rather precise mapping of both loci because HLA is distal and PGM3 proximal to the translocation breakpoint in band 6p2100. The findings are consistent with earlier conclusions that HLA is proximal to 6p22. Quantitative correlation between the density of HLA antigens on the hybrid cell surface and the number of copies of the respective HLA gene-bearing chromosome suggests a gene dose effect for cell surface molecules, as it exists for intracellular gene products. The major histocompatibility complex (MHC) comprising 10 distinct gene loci-among them the four human leukocyte antigens (HLA) loci A, B, C, and D-within a map distance of approximately 2 centimorgans (cM) has been assigned to chromosome 6 in man (1). The initial assignment of HLA was made indirectly by somatic cell genetic localization of the gene for phosphoglucomutase-3 (PGM3, a-D-glucose-1,6-bisphosphate:a-D-glucose-1-phosphate phosphotransferase, EC 2.7.5.1) on chromosome 6 by taking into account the previously estabAbbreviations: MHC, major histocompatibility complex; HLA, human leukocyte antigens; cM, centimorgan; PGM3, phosphoglucomutase-3; ME-1, malic enzyme (NADP-dependent malate oxidoreductase decarboxylating); AD50 value, number of cells in absorption test which reduce cytolytic activity of alloantisera on known target cells by 50%. * These findings were presented in part at the Fifth International Congress of Human Genetics at Mexico City, October 1976.
1147
lished linkage between HLA and PGM3 (2, 3). Direct confirmation was obtained through linkage studies of a family with an inverted chromosome 6 (4) and by studying HLA expression in Chinese hamster-human somatic cell hybrids (5). No regional assignments of genes on chromosome 6 have as yet been reported. To determine the intrachromosomal location of the HLA/ PGM3 linkage group and of other loci assigned to chromosome 6, we have hybridized Chinese hamster cells with human cells containing a balanced reciprocal translocation between the short arms of chromosomes 1 and 6: t(1;6) (p3200;p2100) (6, 7). Segregation of the rearranged chromosomes in hybrid clones was correlated with expression of human genes previously assigned to chromosome 6 [HLA, PGM3 and malic enzyme, ME-1, malic dehydrogenase (decarboxylating) (NADP+), Lmalate: NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40] (8). The results indicate that the t(1;6) translocation has separated the HLA region from the loci for PGM3 and ME-1. MHC maps distal and PGM3 proximal to the breakpoint in band 6p2100. The genetic distance between these loci is 15 cM in males (1). Correlating the genetic map distances with band patterns on early metaphase chromosomes-if a uniform distribution of meiotic crossovers is assumed-allows precise localization of the genes for HLA and PGM3 on chromosome 6. MATERIALS AND METHODS Production of Somatic Cell Hybrids. The human parental fibroblasts (strain TH-5) were mixed in a ratio of 1:50 with 380-6 cells, a clone of hypoxanthine phosphoribosyltransferase-deficient Chinese hamster cells (derived from cell line V79), and were fused by means of inactivated Sendai virus as described (9). Hybrids were selected in hypoxanthine/aminopterine/thymidine containing selection (HAT) medium and were transferred into nonselective medium after clonal isolation. Chromosome preparations were made from all hybrid clones at early passages and were screened for the presence of the relevant translocation chromosomes 1T and 6T, and for their normal homologues. Three primary clones (XV-15A, XV-18A, and XV-18B) that were informative but heterogeneous were subcloned. For the studies described in this paper, we have used 1 primary and 11 secondary clones that were derived from at least four independent fusion events. Chromosome Analyses. Hybrid cells were harvested for chromosome studies in conjunction with harvests for HLA and enzyme studies. The chromosomes were identified by standard quinacrine mustard and trypsin/Giemsa banding methods (9). Photographic prints of at least 25 banded metaphases from each hybrid were analyzed in detail.
1148
Prnc. Natl. Acad. Sci. USA 74 (1977)
Genetics: Francke and Pellegrino
1~~~~~~~~
Studies of HLA expression Microlymphocytotoxicity Test. The eosin exclusion method was performed as described for human peripheral lymphocytes and lymphoblastoid cells (10, 11). HLA (HLA-A and -B) adloantisera were obtained from the Transplantation Immunology Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health and from the laboratories of R. Ceppellini, G. Ferrara, P. Terasaki, and E. J. Yunis. Microabsorption Test. Each hybrid clone was studied three times at successive cell passages. Hybrid cells in logarithmic .growth phase were harvested by trypsinization, washed in 0.01 M Tnis'HCI buffer at pH 7.4 containing MgCl2 and KC1, counted in a Coulter counter and resuspended in phosphatebuffered saline. The coded samples were sent to M. A. Pellegrino's laboratory for HLA studies. Microabsorption tests were performed as previously described (12). To compare results obtained from the different quantitative absorptions, we used the absorption dosage/50 (AD5o) parameter, i.e., the number of cells required to reduce the cytotoxicity of a selected alloantiserum by 50%. Electrophoresis of Enzymes. Cells were harvested, stored, and prepared for electrophoretic analysis as described (9). Human and Chinese hamster PGM3 phenotypes were separated by starch gel electrophoresis according to Spencer and colleagues (13). ME-1 was analyzed by starch (14) and by cellogel electrophoresis (15). RESULTS Characterization of the Human TH-5 Cell Strain. The karyotype was that of a diploid human male with a balanced reciprocal translocation between the short arms of chromosomes 1 and 6: 46,XY,t(1;6) (p3200;p2100). As indicated by this designation (7), the points of breakage and rejoining were interpreted to lie at the border between bands ip31 and lpS2 on chromosome 1 and at the junction~of bands 6pl2 and 6p2l on chromosome 6. The chromosomal regions distal to these breakpoints had been exchanged (Fig. 1). The derivative chromosome 1T, therefore, contained region 6pter 6p2100 of chromosome 6 and region ip32fI0 -l Iqter of chromosome 1. The derivative chromosome 6T contained region lpter 1p3200 of chromosome 1 and region 6p2100 -- 6qter of chromosome 6. The HLA-A and HLA-B antigen profile of the human parental TH-5 fibroblasts has been determined by direct cytotoxicity assay: 15 HLA specificities at locus A and 17 specificities at locus B were tested. The results indicated that the TH-5 cells possessed the specificities HLA-A2 and -AS. The presence of these antigens on the fibroblast surface was confirmed by absorption experiments (Table 1). Although no specificity was found at the HLA-B locus, the presence of two alleles at the HLA-A locus provided us with a marker for each of the two chromosomes carrying the MHC. The Chinese hamster parental cells (clone 380-6) did not absorb any anti-HLA antisera because as many as 105 cells did not reduce the cytotoxic activity of these alloantisera (Table
1).
Chromosome Content of TH-5/380-6 Hybrids. All hybrid lines contained from 31 to 38 Chinese hamster (380-6) chromosomes corresponding to a reduced 2s complement. The ls complement consists of 23 + 1 highly rearranged Chinese hamster chromosomes (for karyotype, see ref. 16). The number of human chromosomes in hybrid karyotypes ranged from 25 to 34; almost every human chromosome was present in one or two copies. Several subclones derived from four independent
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FIG. 1. Partial karyotypes from four different metaphases of human cell strain TH-5 depicting the translocation chromosomes 1T and 6T and their normal homologues 1 and 6; Q-banding (top row) and G-banding (three lower rows); dotted lines indicate the points of breakage and rejoining. The karotype designation is 46,XY,t(1;6) (p3200;p2100) (see ref. 7).
primary hybrid lines were informative for regional mapping of chromsoine 6. We have studied six clones which had retained chromosome 1T and had lost 6 and 6T (Fig. 2), and three clones with chromosome 6T that had lost 1T and 6. Three clones with the normal 6 and 6T that had lost 1 and 1T were used as controls. Within each clone, a high percentage of cells contained the relevant chromosomes (Table 1). Expression of HLA-A Antigens on Hybrid Cells. The microabsorption test was used to study the expression of the HLA-A2 and HLA-A3 antigens in the TH-5/380-6 hybrid clones. The direct cytotoxicity assay was found to be unreliable, first, because no standard source of complement could be used for all the hybrid clones investigated, and second, because the alloantisera, characterized against panels of normal human peripheral lymphocytes, are only "operationally" monospecific and contain other undefined antibodies which can give false positive reactions when the alloantisera are tested against cells other than normal human peripheral lymphocytes. The absorption technique avoids these problems, because the absorbed antisera are tested against a standard target cell (human peripheral lymphocyte of known HLA type) with a standardized rabbit srum as source of complement. In fact, when absorption was carried out three times with coded samples the results were clear-cut and reproducible (Table 1). All the hybrid clones used in this study were tested for the two antigens (HLA-A2 and -A3) present and for one antigen (HLA-Ai) not present on the human parental cells. Presence of the HLA antigens on the cell surface was indicated by AD50
Proc. Nati. Acad. Sci. USA 74 (1977)
Genetics: Francke and Pellegrino
1149
Table 1. Segregation of relevant human chromosomes and expression of HLA-A antigens and of human enzymes PGM3 and ME-1 in hybrid clones
Average number of copies per cell
Percentage of metaphases with chromosome* Cell line
Parental lines TH-5 380-6 Hybrid clones XV-15A-4a XV-18A-5c XV-18A-6b
iT
1T,1T
100
Human
ADS0 values (x 10-)t
6
6T
iT
6
HLA-A2
HLA-A3
100
100
1.00
1.00
0.7 + 0.3
0.8 + 0.4 >100
>> 1-00
>100
>100 >>100 >100 >100 >>100 >> 100 5.0 + 2.0 >> 100 >> 100 1.7 +0.7 > 100 5.2 + 2.8
t Mean and range from three independent determinations; for HLA-A1, the AD5o values were >>100 X
103 for all cell lines.
XV-18A-8b XV-18A-10a XV-18A-11b XV-16B XV-182-la XV-18B-2a
XV-18B.3-6a XV-18B-7a XV-18B-8a
56 28 92 76 96 84
-
4 72 -
8
4 -
92 92 96*
100
80 92 92 96 84 100
0.64 1.72 0.92 0.84 0.94 0.92
-
1.88
»>100 >> 100
1.00
* Twenty-five metaphases per clone were analyzed.
I
14.3 + 2.3 2.5 0.5 9.7 +0.3 8.1 +1.5 12.0 + 6.0 -10.0 >>100 0.92 >> 100 >> 100
enzymes
PGM3 ME-1 + +
+ +
+ + +
+ + + + + + + +
+
+
+
+ +
+ + + + +
+ +
+
The second chromosome 6 was distinguished by an insertion in the short arm.
values between 103 and 2 X 104, i.e., I03 to 2 X 104 cells were necessary to reduce by 50% the cytolytic activity of the corresponding anti-HLA alloantiserum. In contradistinction, clones were considered devoid of HLA antigens on their surface when their AD50 value was higher than 105 which was the highest number of cells used during this study to absorb out anti-HLA alloantisera. No intermediate AD50 values were obtained (Table 1). It was evident that expression of HLA-A2 segregated with the IT derivative chromosome, while HLA-A3 was only expressed in those clones that had retained the normal chromosome 6. Clones containing only 6T were negative for both HLA-A alleles. All hybrid clones were negative, for HLA-Al. There was a large variation in AD5o values among the hybrid clones expressing HLA-A2 or HLA-A3 antigens oP their surface. In fact, some hybrids had AD50 values close to, while others had AD'o values 5 to 15 times higher than those obtained for the parental cells. Although there was a range of AD50 values obtained for the same clone on three different occasions, the relative differences in AD50 values between clones were consistent within individual experiments. The mean AD50 values could be roughly correlated with the average number of copies of the respective HLA bearing chromosome per cell (Table 1). Thus, clone XV-15A-4a with a relatively low frequency of 1T chromosomes had the highest AD50 value, while the lowest AD50 values were obtained for clones XV-18A-5c and XV-18B-6a which contained on the average nearly two copies per cell of the chromosome carrying the respective HLA allele! Regarding the regional localization of HLA-A the results indicated that the HLA-A2 allele is on chromosome 1T and the HLA-A3 allele on the normal chromosome 6, while 6T does not carry an HLA-A locus. The gene for HLA-A can thus be assigned to region 6p2100 -- 6pter. Expression of PGM3. The electrophoretic pattern of human PGM3 consisted of two bands (Fig. 3). Six hybrid clones containing 1T were negative for PGM3 while five clones containing
6T and three clones containing 6T and the normal 6 were positive. These results place the PGM3 locus in region 6p2100 6qter. Expression of ME-1. Human ME-I activity was present in TH-5 fibroblasts as a single electrophoretic band migrating faster than the Chinese hamster single band. Hybrids containing the human gene for ME-1 produced a five-band pattern including three intermediate bands as, described (15). In the TH-5/380-6 hybrids the human ME-i phenotype segregated concordantly with human PGM3. No exceptions were observed. The gene for ME-1, therefore, maps in region 6p2100 6qter. DISCUSSION Regional Mapping of MHC, PGM3, and ME-1 on Chromosome 6. Our studies of the TH-5/380-6 hybrid clones have determined that the break in band 6p2100 leading to the t(l;6) translocation has separated the HLA region from the loci for PGM3 and ME-1. Family linkage studies have established a map distance of 15 cM between HLA and PGM3 for males (1).
We have constructed a cytologic map of chromosome 6 based on measurements of bands on early metaphase chromosomes (in contrast to the Paris Nomenclature ideogram that represents a visual impression but is not based on measurements) (Fig. 4). To fit the linkage map to the cytologic map, the relative length of chromosome 6 was taken as 6% of the haploid autosomal complement and thq mean number of autosomal chiasmata in male meiosis as 50 (6). Thus, by assuming a uniform distribution of meiotic crossovers throughout all autosomes, the genetic length of chromosome 6 should be approximately 150 cM in human males. For construction of the map in Fig. 4, we have assumed a linear relationship between cytologic and genetic map distances, although this assumption may not be correct as cytologic studies of diakinesis have shown a higher frequency of chiasmata toward the ends of chromosome arms (17). Brackets in Fig. 4 indicate the cytologic equivalent of 15 map
1150
Genetics: Francke and Pellegriho
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Proc. Natl. Acad. Sci. USA 74 (1977)
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FIG. 2. G-banded karyotype of hybrid clone XV-18A-5c; all Chinese hamster chromosomes (CH) and human chromosomes (H) have been identified except for "mar", a symmetrical rearranged chromosome of unknown derivation. The translocation chromosome 1T is present, but 6T and 6 are absent.
units (cM) in both directions from the breakpoint. The MHC should be located in the distal and the PGM3 locus in the proximal region. Based on data from benign ovarian teratomas, Ott and colleagues have calculated a distance of 17 cM (95% confidence limit: 7-34 cM) between the PGM3 locus and the centromere (18). Because the recombination frequencies for all gene linkages have been found to be 1.55-1.74 times higher in human females than in human males (19), the centromerePGM3 distance in males should be less than 17 cM, most likely 10-11 cM. The equivalent of 10 cM from the centromere into 1
2
3
4
5
6
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the long arm of chromosome 6 is an unlikely location for PGM3 would be more than 15 cM from the breakpoint in 6p2100. Therefore, it appears more likely that PGM3 and MHC are both on the short arm. In this case, the precise position for PGM3 would be in band 6pl2 near the breakpoint. The MHC should be 25 cM from the centromere, within, or slightly distal from, the narrow faint-staining band which subdivides the light-staining region 6p21 (Fig. 4). The regional localization *of the MHC in the proximal half of band 6p2l is consistent with its exclusion from region 6pter 6p22 by deletion (or translocation) mapping (1, 20). The gene for ME-I has been assigned to chromosome 6 by somatic cell hybridization, and no linkage data are available for this locus. We have established its regional localization in as it
--
6p2100
PGM3
-
6qter.
Quantitative Aspects of Expression of HLA on Hybrid Cells. A linear relationship between gene dose and levels of gene product has been demonstrated for various intracellular
[
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(-)
FIG. 3. Starch gel stained for PGM. Bracket indicates site of migration of human PGM3 bands between Chinese hamster bands. There were no heteropolymeric bands. (1) Hybrid clone XV-18A-6b; (2) hybrid clone XV-16B; (3) human parental cell strain TH-5; (4) Chinese hamster parental cell line 380-6; (5) hybrid clone XV-18B-la; (6) hybrid clone XV-18B-8a; (7) mixture of lysates (3) and (4). The hybrid clone in channel 1 was considered negative for human PGM:3, while those in channels 2, 5, and 6 were positive.
enzymes in erythrocytes and fibroblasts from individuals with unbalanced chromosomes (for review see ref. 21) and for serum enzyme levels in heterozygotes for enzyme deficiency disorders. We have found a correlation between the number of HLA-A gene-containing chromosomes in the TH-5/380-6 hybrid cells and the density of HLA-A antigens on the cell surface as determined by the microabsorption test. Some of the parameters other than gene dose that might influence HLA-antigen density, such as cell size and total number of chromosomes, were apparently similar in these hybrid clones. These results suggest that in Chinese hamster/human hybrids the number of HLA
Genetics: Francke and Pellegrino
Proc. Natl. Acad. Sci. USA 74 (1977)
cM -
0
MHC
PGM3
M E-1
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6
FIG. 4. Photograph of G-banded human chromosome 6 and ideogram of the major and minor bands. Numbering of bands is consistent with the Paris Nomenclature (6), but their relative widths have been adjusted to results of measurements on early metaphase chromosomes. A genetic map in map units (cM), totalling 150 cM and assuming a linear relationship of cytologic and genetic distances, is placed to the right of the ideogram. The dotted line indicating the t(1;6) translocation breakpoint in band p2100 (7) intersects at map position 45. The centromere corresponds to map position 56. Regional localizations of MHC, PGM3, and ME-1 are shown by brackets. genes (i.e., the number of copies of the respective part of chromosome 6), may be an important factor in determining the density of the HLA antigens on the cell surface.
Note Added in Proof. (B2-Microglobin (j32M), an immunoglobulinrelated molecule, is associated with HLA antigens in a noncovalent fashion. The gene for human ,32M is not linked to HLA but has been assigned to chromosome 15 [Goodfellow, P. N., Jones, E. A., Heyningen, V. van, Solomon, E., Bobrow, M., Miggiano, V. & Bodner, W. F. (1975) Nature 254,267-268; Smith, M., Gold, P., Freedman, S. 0. & Schuster, J. (1975) Ann. Hum. Genet. 39,21-31; Faber, H. E. F., Kucherlapati, R. S., Poulik, M. D., Ruddle, F. H. & Smithies, 0. (1976) Somat. Cell Genet. 2, 141-154J. Because of a possible significance of the presence or absence of human /B2M for the quantitative expression of HLA, we have studied ,32M expression in the TH-5/380-6 hybrid clones by an absorption test using bovine anti-human fB2M. All hybrids were positive for human ,#2M although one of them did not contain chromosome 15.
1151
Excellent technical assistance was provided by N. Busby, M. G. Brown, S. Hansen, R. Gutell, A. Begovich, and A. Pellegrino. We thank Dr. Donna L. George for helpful discussions and critical reading of the manuscript. The work was supported by U.S. Public Health Service Research Grants GM 21110, GM 17702, CA 16071, and Al 10180 from the National Institutes of Health. This is Publication no. 1214 from the Department of Molecular Immunology of Scripps Clinic and Research Foundation. 1. Bodmer, W. F. (1976) "Human gene mapping 3," Birth Defects: Original Article Series (The National Foundation, New York), Vol. 12, No. 7, pp. 24-30. 2. Jongsma, A., van Someren, H., Westerveld, A., Hagemeijer, A. & Pearson, P. (1973) Humangenetik 20, 195-202. 3. Lamm, L. U., Svejgaard, A. & Kissmeyer-Nielsen, F. (1971) Nature New Biol. 231, 109-110. 4. Lamm, L. U., Friedrich, U., Peterson, G. B., Jorgensen, J., Nielsen, J., Therkelsen, A. J. & Kissmeyer-Nielsen, F. (1974) Hum. Hered. 24,273-284. 5. van Someren, H., Westerveld, A., Hagemeijer, A., Mees, J. R., Meera Khan, P. & Zaalberg, 0. B. (1974) Proc. Natl. Acad. Sci. USA 71, 962-965. 6. Paris Conference (1971) "Standardization in human cytogenetics," Birth Defects: Original Article Series (The National Foundation, New York), Vol. 8, No. 7. 7. Paris Conference (1971) Supplement (1975) "Standardization in human cytogenetics," Birth Defects: Original Article Series (The National Foundation, New York), Vol. 11, No. 9. 8. Chen, T. R., McMorris, F. A., Creagan, R., Ricciuti, F., Tischfield, J. & Ruddle, F. (1975) Am. J. Hum. Genet. 25,200-207. 9. Francke, U., Busby, N., Shaw, D., Hansen, S. & Brown, M. G. (1976) Somatic Cell Genet. 2, 27-42. 10. Pellegrino, M. A., Ferrone, S. & Pellegrino, A. (1972) in Transplantation Antigens, eds. Cahan, B. D. & Reisfeld, R. A. (Academic Press, New York), pp. 433-452. 11. Ferrone, S., Pellegrino, M. A. & Reisfeld, R. A. (1971) J. Immunol. 107, 613-615. 12. Pellegrino, M. A., Ferrone, S. & Pellegrino, A. (1972) Proc. Soc. Exp. Biol. Med. 139,484-488. 13. Spencer, N., Hopkinson, D. A. & Harris, H. (1964) Nature 204, 742-745. 14. Cohen, P. T. W. & Omenn, G. S. (1972) Biochem. Genet. 7, 303-311. 15. van Someren, H., van Henegouwen, H. B., Los, W., WurzerFigurelli, E., Doppert, B., Vervloet, M. & Meera Khan, P. (1974) Humangenetik 25, 189-201. 16. Francke, U. (1976) Am. J. Hum. Genet. 28,357-362. 17. Hulten, M. (1974) Hereditas 76,55-78. 18. Ott, J., Hecht, F., Linder, D., Lovrien, E. W. & Kaiser McCaw, B. (1976) "Human gene mapping 3," Birth Defects: Original Article Series (The National Foundation, New York), Vol. 12, No. 7, pp. 396-398. 19. Weitkamp, L. R., Guttormsen, S. A. & Greendyke, R. M. (1971) Am. J. Hum. Genet. 23,462-470. 20. Borgaonkar, D. S. & Bias, W. B. (1974) "Human gene mapping 1," Birth Defects: Original Article Series (The National Foundation, New York), Vol. 10, No. 3, pp. 67-68. 21. George, D. L. & Francke, U. (1976) Science 195, 851-852.
Genetics
Assignment of the major histocompatibility complex to a region of the short arm of human chromosome 6* [human reciprocal translocation t(l;6) (p3200;p21OO)/ mapping of human genes by using man-Chinese hamster somatic cell hybrids/ regional mapping of phosphoglucomutase-3 and malic enzyme-1/gene dose effect]
UTA FRANCKEt AND MICHELE A. PELLEGRINOf t Department of Pediatrics M-009, University of California, San Diego, School of Medicine, La Jolla,
La Jolla, California 92093
Calif. 92093; and fScripps Clinic and Research Foundation,
Communicated by Victor A. McKusick, November 30, 1976
ABSTRACT Interspecific cell hybrids containing defined parts of human chromosome 6 were used for regional mapping of gene loci previously assigned to chromosome 6: the human leukocyte antigens (HLA) region, phosphoglucomutase-3
(PGM3; a-D-glucose-1,6-bisphosphate:a--glucose-i-phosphate phosphotransferase, EC 2.7.5.1) and malic enzyme-i [malic dehydrogenase(decarboxylating) (NADP+), L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40]. Human fibroblasts containing a balanced reciprocal translocation between the short arms of chromosome 1 and chromosome 6-designated t(I;6) (p3200;p2100)-were fused with an established line of Chinese hamster cells. Hybrid clones with segregating human chromosomes were studied for the presence of the translocation chromosomes IT and 6T and their normal homologues 1 and 6. Six clones that had retained IT, three clones with 6T, and three clones with 6 and 6T as controls, were analyzed by a microabsorption test for expression of the allelic antigens HLA-A2 and HLA-A3, both of which were present on the human parental cells. HLA-A2 segregated with the 1T translocation chromosome and HLA-A3 with the normal chromosome 6. Hybrid clones with 6T did not possess an HLA-A gene. In contrast, human PGM3 and malic enzyme-i expression segregated with the 6T translocation chromosome. These results indicate the location of the major histocompatibility complex in region 6p2100 - 6pter of the short arm of chromosome 6. The genes for PGM3 and malic enzyme-i map in region 6p2100 - 6qter. The HLA.PGM3s genetic ma distance is 15 centimorgans in males, as established by famify studies. This allows rather precise mapping of both loci because HLA is distal and PGM3 proximal to the translocation breakpoint in band 6p2100. The findings are consistent with earlier conclusions that HLA is proximal to 6p22. Quantitative correlation between the density of HLA antigens on the hybrid cell surface and the number of copies of the respective HLA gene-bearing chromosome suggests a gene dose effect for cell surface molecules, as it exists for intracellular gene products. The major histocompatibility complex (MHC) comprising 10 distinct gene loci-among them the four human leukocyte antigens (HLA) loci A, B, C, and D-within a map distance of approximately 2 centimorgans (cM) has been assigned to chromosome 6 in man (1). The initial assignment of HLA was made indirectly by somatic cell genetic localization of the gene for phosphoglucomutase-3 (PGM3, a-D-glucose-1,6-bisphosphate:a-D-glucose-1-phosphate phosphotransferase, EC 2.7.5.1) on chromosome 6 by taking into account the previously estabAbbreviations: MHC, major histocompatibility complex; HLA, human leukocyte antigens; cM, centimorgan; PGM3, phosphoglucomutase-3; ME-1, malic enzyme (NADP-dependent malate oxidoreductase decarboxylating); AD50 value, number of cells in absorption test which reduce cytolytic activity of alloantisera on known target cells by 50%. * These findings were presented in part at the Fifth International Congress of Human Genetics at Mexico City, October 1976.
1147
lished linkage between HLA and PGM3 (2, 3). Direct confirmation was obtained through linkage studies of a family with an inverted chromosome 6 (4) and by studying HLA expression in Chinese hamster-human somatic cell hybrids (5). No regional assignments of genes on chromosome 6 have as yet been reported. To determine the intrachromosomal location of the HLA/ PGM3 linkage group and of other loci assigned to chromosome 6, we have hybridized Chinese hamster cells with human cells containing a balanced reciprocal translocation between the short arms of chromosomes 1 and 6: t(1;6) (p3200;p2100) (6, 7). Segregation of the rearranged chromosomes in hybrid clones was correlated with expression of human genes previously assigned to chromosome 6 [HLA, PGM3 and malic enzyme, ME-1, malic dehydrogenase (decarboxylating) (NADP+), Lmalate: NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40] (8). The results indicate that the t(1;6) translocation has separated the HLA region from the loci for PGM3 and ME-1. MHC maps distal and PGM3 proximal to the breakpoint in band 6p2100. The genetic distance between these loci is 15 cM in males (1). Correlating the genetic map distances with band patterns on early metaphase chromosomes-if a uniform distribution of meiotic crossovers is assumed-allows precise localization of the genes for HLA and PGM3 on chromosome 6. MATERIALS AND METHODS Production of Somatic Cell Hybrids. The human parental fibroblasts (strain TH-5) were mixed in a ratio of 1:50 with 380-6 cells, a clone of hypoxanthine phosphoribosyltransferase-deficient Chinese hamster cells (derived from cell line V79), and were fused by means of inactivated Sendai virus as described (9). Hybrids were selected in hypoxanthine/aminopterine/thymidine containing selection (HAT) medium and were transferred into nonselective medium after clonal isolation. Chromosome preparations were made from all hybrid clones at early passages and were screened for the presence of the relevant translocation chromosomes 1T and 6T, and for their normal homologues. Three primary clones (XV-15A, XV-18A, and XV-18B) that were informative but heterogeneous were subcloned. For the studies described in this paper, we have used 1 primary and 11 secondary clones that were derived from at least four independent fusion events. Chromosome Analyses. Hybrid cells were harvested for chromosome studies in conjunction with harvests for HLA and enzyme studies. The chromosomes were identified by standard quinacrine mustard and trypsin/Giemsa banding methods (9). Photographic prints of at least 25 banded metaphases from each hybrid were analyzed in detail.
1148
Prnc. Natl. Acad. Sci. USA 74 (1977)
Genetics: Francke and Pellegrino
1~~~~~~~~
Studies of HLA expression Microlymphocytotoxicity Test. The eosin exclusion method was performed as described for human peripheral lymphocytes and lymphoblastoid cells (10, 11). HLA (HLA-A and -B) adloantisera were obtained from the Transplantation Immunology Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health and from the laboratories of R. Ceppellini, G. Ferrara, P. Terasaki, and E. J. Yunis. Microabsorption Test. Each hybrid clone was studied three times at successive cell passages. Hybrid cells in logarithmic .growth phase were harvested by trypsinization, washed in 0.01 M Tnis'HCI buffer at pH 7.4 containing MgCl2 and KC1, counted in a Coulter counter and resuspended in phosphatebuffered saline. The coded samples were sent to M. A. Pellegrino's laboratory for HLA studies. Microabsorption tests were performed as previously described (12). To compare results obtained from the different quantitative absorptions, we used the absorption dosage/50 (AD5o) parameter, i.e., the number of cells required to reduce the cytotoxicity of a selected alloantiserum by 50%. Electrophoresis of Enzymes. Cells were harvested, stored, and prepared for electrophoretic analysis as described (9). Human and Chinese hamster PGM3 phenotypes were separated by starch gel electrophoresis according to Spencer and colleagues (13). ME-1 was analyzed by starch (14) and by cellogel electrophoresis (15). RESULTS Characterization of the Human TH-5 Cell Strain. The karyotype was that of a diploid human male with a balanced reciprocal translocation between the short arms of chromosomes 1 and 6: 46,XY,t(1;6) (p3200;p2100). As indicated by this designation (7), the points of breakage and rejoining were interpreted to lie at the border between bands ip31 and lpS2 on chromosome 1 and at the junction~of bands 6pl2 and 6p2l on chromosome 6. The chromosomal regions distal to these breakpoints had been exchanged (Fig. 1). The derivative chromosome 1T, therefore, contained region 6pter 6p2100 of chromosome 6 and region ip32fI0 -l Iqter of chromosome 1. The derivative chromosome 6T contained region lpter 1p3200 of chromosome 1 and region 6p2100 -- 6qter of chromosome 6. The HLA-A and HLA-B antigen profile of the human parental TH-5 fibroblasts has been determined by direct cytotoxicity assay: 15 HLA specificities at locus A and 17 specificities at locus B were tested. The results indicated that the TH-5 cells possessed the specificities HLA-A2 and -AS. The presence of these antigens on the fibroblast surface was confirmed by absorption experiments (Table 1). Although no specificity was found at the HLA-B locus, the presence of two alleles at the HLA-A locus provided us with a marker for each of the two chromosomes carrying the MHC. The Chinese hamster parental cells (clone 380-6) did not absorb any anti-HLA antisera because as many as 105 cells did not reduce the cytotoxic activity of these alloantisera (Table
1).
Chromosome Content of TH-5/380-6 Hybrids. All hybrid lines contained from 31 to 38 Chinese hamster (380-6) chromosomes corresponding to a reduced 2s complement. The ls complement consists of 23 + 1 highly rearranged Chinese hamster chromosomes (for karyotype, see ref. 16). The number of human chromosomes in hybrid karyotypes ranged from 25 to 34; almost every human chromosome was present in one or two copies. Several subclones derived from four independent
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FIG. 1. Partial karyotypes from four different metaphases of human cell strain TH-5 depicting the translocation chromosomes 1T and 6T and their normal homologues 1 and 6; Q-banding (top row) and G-banding (three lower rows); dotted lines indicate the points of breakage and rejoining. The karotype designation is 46,XY,t(1;6) (p3200;p2100) (see ref. 7).
primary hybrid lines were informative for regional mapping of chromsoine 6. We have studied six clones which had retained chromosome 1T and had lost 6 and 6T (Fig. 2), and three clones with chromosome 6T that had lost 1T and 6. Three clones with the normal 6 and 6T that had lost 1 and 1T were used as controls. Within each clone, a high percentage of cells contained the relevant chromosomes (Table 1). Expression of HLA-A Antigens on Hybrid Cells. The microabsorption test was used to study the expression of the HLA-A2 and HLA-A3 antigens in the TH-5/380-6 hybrid clones. The direct cytotoxicity assay was found to be unreliable, first, because no standard source of complement could be used for all the hybrid clones investigated, and second, because the alloantisera, characterized against panels of normal human peripheral lymphocytes, are only "operationally" monospecific and contain other undefined antibodies which can give false positive reactions when the alloantisera are tested against cells other than normal human peripheral lymphocytes. The absorption technique avoids these problems, because the absorbed antisera are tested against a standard target cell (human peripheral lymphocyte of known HLA type) with a standardized rabbit srum as source of complement. In fact, when absorption was carried out three times with coded samples the results were clear-cut and reproducible (Table 1). All the hybrid clones used in this study were tested for the two antigens (HLA-A2 and -A3) present and for one antigen (HLA-Ai) not present on the human parental cells. Presence of the HLA antigens on the cell surface was indicated by AD50
Proc. Nati. Acad. Sci. USA 74 (1977)
Genetics: Francke and Pellegrino
1149
Table 1. Segregation of relevant human chromosomes and expression of HLA-A antigens and of human enzymes PGM3 and ME-1 in hybrid clones
Average number of copies per cell
Percentage of metaphases with chromosome* Cell line
Parental lines TH-5 380-6 Hybrid clones XV-15A-4a XV-18A-5c XV-18A-6b
iT
1T,1T
100
Human
ADS0 values (x 10-)t
6
6T
iT
6
HLA-A2
HLA-A3
100
100
1.00
1.00
0.7 + 0.3
0.8 + 0.4 >100
>> 1-00
>100
>100 >>100 >100 >100 >>100 >> 100 5.0 + 2.0 >> 100 >> 100 1.7 +0.7 > 100 5.2 + 2.8
t Mean and range from three independent determinations; for HLA-A1, the AD5o values were >>100 X
103 for all cell lines.
XV-18A-8b XV-18A-10a XV-18A-11b XV-16B XV-182-la XV-18B-2a
XV-18B.3-6a XV-18B-7a XV-18B-8a
56 28 92 76 96 84
-
4 72 -
8
4 -
92 92 96*
100
80 92 92 96 84 100
0.64 1.72 0.92 0.84 0.94 0.92
-
1.88
»>100 >> 100
1.00
* Twenty-five metaphases per clone were analyzed.
I
14.3 + 2.3 2.5 0.5 9.7 +0.3 8.1 +1.5 12.0 + 6.0 -10.0 >>100 0.92 >> 100 >> 100
enzymes
PGM3 ME-1 + +
+ +
+ + +
+ + + + + + + +
+
+
+
+ +
+ + + + +
+ +
+
The second chromosome 6 was distinguished by an insertion in the short arm.
values between 103 and 2 X 104, i.e., I03 to 2 X 104 cells were necessary to reduce by 50% the cytolytic activity of the corresponding anti-HLA alloantiserum. In contradistinction, clones were considered devoid of HLA antigens on their surface when their AD50 value was higher than 105 which was the highest number of cells used during this study to absorb out anti-HLA alloantisera. No intermediate AD50 values were obtained (Table 1). It was evident that expression of HLA-A2 segregated with the IT derivative chromosome, while HLA-A3 was only expressed in those clones that had retained the normal chromosome 6. Clones containing only 6T were negative for both HLA-A alleles. All hybrid clones were negative, for HLA-Al. There was a large variation in AD5o values among the hybrid clones expressing HLA-A2 or HLA-A3 antigens oP their surface. In fact, some hybrids had AD50 values close to, while others had AD'o values 5 to 15 times higher than those obtained for the parental cells. Although there was a range of AD50 values obtained for the same clone on three different occasions, the relative differences in AD50 values between clones were consistent within individual experiments. The mean AD50 values could be roughly correlated with the average number of copies of the respective HLA bearing chromosome per cell (Table 1). Thus, clone XV-15A-4a with a relatively low frequency of 1T chromosomes had the highest AD50 value, while the lowest AD50 values were obtained for clones XV-18A-5c and XV-18B-6a which contained on the average nearly two copies per cell of the chromosome carrying the respective HLA allele! Regarding the regional localization of HLA-A the results indicated that the HLA-A2 allele is on chromosome 1T and the HLA-A3 allele on the normal chromosome 6, while 6T does not carry an HLA-A locus. The gene for HLA-A can thus be assigned to region 6p2100 -- 6pter. Expression of PGM3. The electrophoretic pattern of human PGM3 consisted of two bands (Fig. 3). Six hybrid clones containing 1T were negative for PGM3 while five clones containing
6T and three clones containing 6T and the normal 6 were positive. These results place the PGM3 locus in region 6p2100 6qter. Expression of ME-1. Human ME-I activity was present in TH-5 fibroblasts as a single electrophoretic band migrating faster than the Chinese hamster single band. Hybrids containing the human gene for ME-1 produced a five-band pattern including three intermediate bands as, described (15). In the TH-5/380-6 hybrids the human ME-i phenotype segregated concordantly with human PGM3. No exceptions were observed. The gene for ME-1, therefore, maps in region 6p2100 6qter. DISCUSSION Regional Mapping of MHC, PGM3, and ME-1 on Chromosome 6. Our studies of the TH-5/380-6 hybrid clones have determined that the break in band 6p2100 leading to the t(l;6) translocation has separated the HLA region from the loci for PGM3 and ME-1. Family linkage studies have established a map distance of 15 cM between HLA and PGM3 for males (1).
We have constructed a cytologic map of chromosome 6 based on measurements of bands on early metaphase chromosomes (in contrast to the Paris Nomenclature ideogram that represents a visual impression but is not based on measurements) (Fig. 4). To fit the linkage map to the cytologic map, the relative length of chromosome 6 was taken as 6% of the haploid autosomal complement and thq mean number of autosomal chiasmata in male meiosis as 50 (6). Thus, by assuming a uniform distribution of meiotic crossovers throughout all autosomes, the genetic length of chromosome 6 should be approximately 150 cM in human males. For construction of the map in Fig. 4, we have assumed a linear relationship between cytologic and genetic map distances, although this assumption may not be correct as cytologic studies of diakinesis have shown a higher frequency of chiasmata toward the ends of chromosome arms (17). Brackets in Fig. 4 indicate the cytologic equivalent of 15 map
1150
Genetics: Francke and Pellegriho
CH
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Proc. Natl. Acad. Sci. USA 74 (1977)
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9
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.
.,
FIG. 2. G-banded karyotype of hybrid clone XV-18A-5c; all Chinese hamster chromosomes (CH) and human chromosomes (H) have been identified except for "mar", a symmetrical rearranged chromosome of unknown derivation. The translocation chromosome 1T is present, but 6T and 6 are absent.
units (cM) in both directions from the breakpoint. The MHC should be located in the distal and the PGM3 locus in the proximal region. Based on data from benign ovarian teratomas, Ott and colleagues have calculated a distance of 17 cM (95% confidence limit: 7-34 cM) between the PGM3 locus and the centromere (18). Because the recombination frequencies for all gene linkages have been found to be 1.55-1.74 times higher in human females than in human males (19), the centromerePGM3 distance in males should be less than 17 cM, most likely 10-11 cM. The equivalent of 10 cM from the centromere into 1
2
3
4
5
6
7
Hi)
the long arm of chromosome 6 is an unlikely location for PGM3 would be more than 15 cM from the breakpoint in 6p2100. Therefore, it appears more likely that PGM3 and MHC are both on the short arm. In this case, the precise position for PGM3 would be in band 6pl2 near the breakpoint. The MHC should be 25 cM from the centromere, within, or slightly distal from, the narrow faint-staining band which subdivides the light-staining region 6p21 (Fig. 4). The regional localization *of the MHC in the proximal half of band 6p2l is consistent with its exclusion from region 6pter 6p22 by deletion (or translocation) mapping (1, 20). The gene for ME-I has been assigned to chromosome 6 by somatic cell hybridization, and no linkage data are available for this locus. We have established its regional localization in as it
--
6p2100
PGM3
-
6qter.
Quantitative Aspects of Expression of HLA on Hybrid Cells. A linear relationship between gene dose and levels of gene product has been demonstrated for various intracellular
[
MA09'r
(-)
FIG. 3. Starch gel stained for PGM. Bracket indicates site of migration of human PGM3 bands between Chinese hamster bands. There were no heteropolymeric bands. (1) Hybrid clone XV-18A-6b; (2) hybrid clone XV-16B; (3) human parental cell strain TH-5; (4) Chinese hamster parental cell line 380-6; (5) hybrid clone XV-18B-la; (6) hybrid clone XV-18B-8a; (7) mixture of lysates (3) and (4). The hybrid clone in channel 1 was considered negative for human PGM:3, while those in channels 2, 5, and 6 were positive.
enzymes in erythrocytes and fibroblasts from individuals with unbalanced chromosomes (for review see ref. 21) and for serum enzyme levels in heterozygotes for enzyme deficiency disorders. We have found a correlation between the number of HLA-A gene-containing chromosomes in the TH-5/380-6 hybrid cells and the density of HLA-A antigens on the cell surface as determined by the microabsorption test. Some of the parameters other than gene dose that might influence HLA-antigen density, such as cell size and total number of chromosomes, were apparently similar in these hybrid clones. These results suggest that in Chinese hamster/human hybrids the number of HLA
Genetics: Francke and Pellegrino
Proc. Natl. Acad. Sci. USA 74 (1977)
cM -
0
MHC
PGM3
M E-1
I
:, -,.:
r.'
..I
'K.'
6
FIG. 4. Photograph of G-banded human chromosome 6 and ideogram of the major and minor bands. Numbering of bands is consistent with the Paris Nomenclature (6), but their relative widths have been adjusted to results of measurements on early metaphase chromosomes. A genetic map in map units (cM), totalling 150 cM and assuming a linear relationship of cytologic and genetic distances, is placed to the right of the ideogram. The dotted line indicating the t(1;6) translocation breakpoint in band p2100 (7) intersects at map position 45. The centromere corresponds to map position 56. Regional localizations of MHC, PGM3, and ME-1 are shown by brackets. genes (i.e., the number of copies of the respective part of chromosome 6), may be an important factor in determining the density of the HLA antigens on the cell surface.
Note Added in Proof. (B2-Microglobin (j32M), an immunoglobulinrelated molecule, is associated with HLA antigens in a noncovalent fashion. The gene for human ,32M is not linked to HLA but has been assigned to chromosome 15 [Goodfellow, P. N., Jones, E. A., Heyningen, V. van, Solomon, E., Bobrow, M., Miggiano, V. & Bodner, W. F. (1975) Nature 254,267-268; Smith, M., Gold, P., Freedman, S. 0. & Schuster, J. (1975) Ann. Hum. Genet. 39,21-31; Faber, H. E. F., Kucherlapati, R. S., Poulik, M. D., Ruddle, F. H. & Smithies, 0. (1976) Somat. Cell Genet. 2, 141-154J. Because of a possible significance of the presence or absence of human /B2M for the quantitative expression of HLA, we have studied ,32M expression in the TH-5/380-6 hybrid clones by an absorption test using bovine anti-human fB2M. All hybrids were positive for human ,#2M although one of them did not contain chromosome 15.
1151
Excellent technical assistance was provided by N. Busby, M. G. Brown, S. Hansen, R. Gutell, A. Begovich, and A. Pellegrino. We thank Dr. Donna L. George for helpful discussions and critical reading of the manuscript. The work was supported by U.S. Public Health Service Research Grants GM 21110, GM 17702, CA 16071, and Al 10180 from the National Institutes of Health. This is Publication no. 1214 from the Department of Molecular Immunology of Scripps Clinic and Research Foundation. 1. Bodmer, W. F. (1976) "Human gene mapping 3," Birth Defects: Original Article Series (The National Foundation, New York), Vol. 12, No. 7, pp. 24-30. 2. Jongsma, A., van Someren, H., Westerveld, A., Hagemeijer, A. & Pearson, P. (1973) Humangenetik 20, 195-202. 3. Lamm, L. U., Svejgaard, A. & Kissmeyer-Nielsen, F. (1971) Nature New Biol. 231, 109-110. 4. Lamm, L. U., Friedrich, U., Peterson, G. B., Jorgensen, J., Nielsen, J., Therkelsen, A. J. & Kissmeyer-Nielsen, F. (1974) Hum. Hered. 24,273-284. 5. van Someren, H., Westerveld, A., Hagemeijer, A., Mees, J. R., Meera Khan, P. & Zaalberg, 0. B. (1974) Proc. Natl. Acad. Sci. USA 71, 962-965. 6. Paris Conference (1971) "Standardization in human cytogenetics," Birth Defects: Original Article Series (The National Foundation, New York), Vol. 8, No. 7. 7. Paris Conference (1971) Supplement (1975) "Standardization in human cytogenetics," Birth Defects: Original Article Series (The National Foundation, New York), Vol. 11, No. 9. 8. Chen, T. R., McMorris, F. A., Creagan, R., Ricciuti, F., Tischfield, J. & Ruddle, F. (1975) Am. J. Hum. Genet. 25,200-207. 9. Francke, U., Busby, N., Shaw, D., Hansen, S. & Brown, M. G. (1976) Somatic Cell Genet. 2, 27-42. 10. Pellegrino, M. A., Ferrone, S. & Pellegrino, A. (1972) in Transplantation Antigens, eds. Cahan, B. D. & Reisfeld, R. A. (Academic Press, New York), pp. 433-452. 11. Ferrone, S., Pellegrino, M. A. & Reisfeld, R. A. (1971) J. Immunol. 107, 613-615. 12. Pellegrino, M. A., Ferrone, S. & Pellegrino, A. (1972) Proc. Soc. Exp. Biol. Med. 139,484-488. 13. Spencer, N., Hopkinson, D. A. & Harris, H. (1964) Nature 204, 742-745. 14. Cohen, P. T. W. & Omenn, G. S. (1972) Biochem. Genet. 7, 303-311. 15. van Someren, H., van Henegouwen, H. B., Los, W., WurzerFigurelli, E., Doppert, B., Vervloet, M. & Meera Khan, P. (1974) Humangenetik 25, 189-201. 16. Francke, U. (1976) Am. J. Hum. Genet. 28,357-362. 17. Hulten, M. (1974) Hereditas 76,55-78. 18. Ott, J., Hecht, F., Linder, D., Lovrien, E. W. & Kaiser McCaw, B. (1976) "Human gene mapping 3," Birth Defects: Original Article Series (The National Foundation, New York), Vol. 12, No. 7, pp. 396-398. 19. Weitkamp, L. R., Guttormsen, S. A. & Greendyke, R. M. (1971) Am. J. Hum. Genet. 23,462-470. 20. Borgaonkar, D. S. & Bias, W. B. (1974) "Human gene mapping 1," Birth Defects: Original Article Series (The National Foundation, New York), Vol. 10, No. 3, pp. 67-68. 21. George, D. L. & Francke, U. (1976) Science 195, 851-852.
