Journal of Reproductive Immunology, 18 (1990) 199--203
199
Elsevier Scientific Publishers Ireland Ltd.
JR100673
Brief c o m m u n i c a t i o n
Northern blot analysis of HLA-G expression by BeWo human choriocarcinoma cells J a n e t M. Risk a n d P e t e r M. J o h n s o n Pregnancy Immunology Group, Department of Immunology, University of Liverpool, p.O. Box 147, Liverpool L69 3BX (U. K.) (Accepted for publication 4 May 1990)
Summary The BeWo human choriocarcinoma cell line expresses an unusual 41 kDa/32microglobulin-associated class I MHC antigen. HLA-E, -F and -G are known to encode antigens of lower M than HLA-A, -B or -C proteins. Using locus-specific probes, we now show that RNA isolated from BeWo ceils contains markedly more HLA-G transcripts than peripheral blood lymphocytes (PBL), whilst the converse was noted for HLA-B. These results support recent cloning and sequencing data showing the presence of HLA-G transcripts in BeWo cells. Key words: choriocarcinoma cell line; class I M H C transcripts; HLA-G.
Trophoblastic expression of major histocompatibility complex (MHC) antigens has been of central interest to reproductive immunologists. Class I (HLA-A, -B, -C) and II (HLA-DR, -DQ, -DP) MHC antigens are not expressed by villous cytotrophoblast or syncytiotrOphoblast (Sunderland et al., 1981; Bulmer and Johnson, 1985). This is thought to be regulated at the level of gene transcription (Alberti and Herzenberg, 1986; Hunt et al., 1987). In contrast, a class I M H C antigen has been identified on invasive extravillous cytotrophoblast as well as cytotrophoblast columns and the maternal aspect of the chorion laeve (Hsi et al., 1984; Redman et al., 1984; Bulmer and Johnson, 1985). Serological and biochemical analyses have shown this unusual class I MHC antigen to be closely similar to that expressed also by baboon placental trophoblast and by the BeWo human choriocarcinoma (malignant trophoblast) cell line, but absent from the Jar human choriocarCorrespondence to: Prof. P.M. Johnson. ol65-0378/90/$03.50
@ 1990 Elsevier Scientific Publishers Ireland Ltd.
200
cinoma cell line (Anderson and Berkowitz, 1985; Ellis et al., 1986; Stern et al., 1986; Johnson and Stern, 1986). This was not a classical HLA-A or -B alloantigen but, instead, the fl2-microglobulin-associated class I MHC heavy chain had an M of 40--41 kDa and was non-polymorphic on isoelectric focusing. Initial Northern blot analysis and cDNA cloning from BeWo cells indicated the unusual class I MHC antigen to be an HLA-C locus product, but the sequence of the derived cDNA clone did not correspond exactly with any known single HLA-C locus sequence (Ellis et al., 1989). In addition, restriction fragment length polymorphism (RFLP) studies have shown that this cell line genomically does not carry the HLA-Cw4, 5, 7 or 8 alleles (Risk and Johnson, 1990). More recently, Ellis et al. (1990) have identified a different full-length class I MHC cDNA clone and analogous transcripts were demonstrated also in extravillous cytotrophoblast using the polymerase chain reaction; this sequence had a very high degree of homology with a genomic clone HLA-6.0 (now designated HLA-G) that is the product of a class I MHC locus other than HLA-A, -B or -C (Geraghty et al., 1987). This gene, in addition to two further non-HLA-A, -B, -C class I MHC gene loci (HLA-E and -F) identified from genomic cloning, have been shown to encode p~-microglobulin-associated class I MHC proteins of lower M than HLA-A, -B and -C proteins (Shimizu et al., 1988; Koller et al., 1988; Geraghty et al., 1990). We have investigated the identity of class I MHC transcripts in total RNA, prepared by the guanidinium isothiocynate/caesium chloride method of Chirgwin et al. (1979) from BeWo cells and unstimulated peripheral blood lymphocytes (PBL), using formaldehyde gel electrophoresis (20/~g/iane) and Northern blotting (Sambrook et al., 1989). The blots were hybridised at 42°C in the presence of 50o-/o formamide (Sambrook et ai., 1989) to the following probes radiolabelled with 32p by the random priming method: pB550, a 0.55 kb HLA-B locus-specific probe, gift of Dr. T. Strachan (Strachan et al., 1986, and unpublished data); p6.2p10 (HLA-Eo), a 0.24 kb HLA-E locusspecific probe (Koller et al., 1988); p5.4SH, a 0.75 kb HLA-F locus-specific probe (Geraghty et al., 1990); pHLA-6pl, a 0.45 kb HLA-G locus-specific probe (Geraghty et al., ~987; Orr et al., unpublished data); the HLA-E, -F and -G probes were obtained from Dr. H.T. Orr. All probes recognised sequences in the terminal exon or 3'-untranslated regions. The activity of all radiolabelled probes had been confirmed by Southern blot analysis on Hindlll-digested human PBL genomic DNA. Northern blots were washed at high stringency (0.1 x SSC/0.1°T0 SDS at 68°C) before autoradiography with intensifying screens (Risk and Johnson, 1990). Under these conditions, it can be clearly seen that BeWo cells contain significantly more HLA-G transcripts than PBL when identical amounts of total RNA were loaded into each lane (Fig. 1). Conversely, PBI_ appear to
201
H L A-B
HLA-G
HLA-E
HLA-F
1.95 k b i
P
B
P
B
P
B
P
B
Fig. 1. Northern blot analysis of peripheral blood lymphocyte (P) and BeWo cell (B) total RNA after hybridisation with uP-radiolabeUed DNA probes for HLA-B, -G, -E and -F, respectively.
transcribe very much more HLA-B RNA than do BeWo cells (Fig. 1). Although the pBs~0 probe is derived from the region of the gene showing lowest inter-locus homology, the most likely explanation for the BeWo cell signal with this probe centres on possible cross-reactivity notably with related HLA-C locus alleles (Stern et al., 1988; Ellis et al., 1989). RFLP analysis has shown BeWo cells to carry the HLA-B8 allele in their genome but they are serologically unreactive with anti-HLA-B8 tissue-typing alloantisera (Risk and Johnson, 1990), yet it is also known that these cells express low levels of a 45 kDa class I MHC protein as well as the unusual 40m41 kDa species (Johnson and Stern, 1986; Stern et al., 1988). There was no unequivocal hybridisation in repeated Northern blot analysis of BeWo or PBL RNA with the HLA-E and -F probes at the level of stringency employed (see Fig. 1), although these transcripts have previously been reported to be detected in resting T cells by Northern blot and RNase protection assays, respectively (Koller et al., 1988; Geraghty et al., 1990). No hybridisation with total RNA isolated from the Jar choriocarcinoma cell line was detected using any of the probes for HLA-B, -E, -F or -G (data not shown).
202
The HLA-G gene is thought to be a structural homologue of a murine Qaregion gene (Geraghty et al., 1987). There has been no report of any expression by non-trophoblastic normal cell types and its cellular function is unknown (Koller et al., 1989). Selective expression of this class I MHC antigen by cytotrophoblast cells (Ellis et al., 1990; and this report) therefore implies a specific function. Induction of immune suppression of maternal anti-fetal cytotoxic responses has been suggested. However, if this antigen is confirmed to be pauci- or non-polymorphic, it would then be unlikely to act as a transplantation antigen or restriction element guiding T cell responses. Rather, it might be speculated that HI_A-G may function as a specialised cellular recognitive molecule related to cytotrophoblast proliferation and invasion. Acknowlegements We are grateful to Dr. T. Strachan (Manchester) and Dr. H.T. Orr (Minneapolis) for the gifts of gene probes used in this study. Note added in proof A further recent report has also identified HLA-G expression by human trophoblast cells (Korats, S., Main, E.K., Librach, C., Stubbeline, M., Fisher, S.J. and DeMars, R. (1990) Science 248,220--223). References Alberti, S. and Herzenberg, L.A. (1986) Transfection of DNA from choriocarcinoma cell lines and sperm cells: DNA methylation prevents the expression of genes for the major histocompatibility complex (HLA) class I and the T cell differentiation antigen, Leu-2. In: Reproductive Immunology (Clark, D.A., Croy, B.A. eds.), pp. 60--66. Elsevier Science Publishers, Amsterdam. Anderson, D.J. and Berkowitz, R.S. (1985) y-Interferon enhances expression of class 1 MHC antigens in the weakly HLA" human choriocarcinoma cell line BeWo, but does not induce MHC expression in the H L A choriocarcinoma cell line Jar. J. Immunol. 135, 2498--2501. Bulmer, J.N. and Johnson, P.M. (1985) Antigen expression by trophoblast populations in the human placenta and their possible immunobiological relevance. Placenta 6, 127--140. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294--5299. Ellis, S.A., Sargent, I.L., Redman, C.W.G. and McMichael, A.J. (1986) Evidence for a novel HLA antigen found on human extravillous trophoblast and a human choriocarcinoma cell line. Immunology 59, 595--601. Ellis, S.A., Strachan T., Palmer, M.S. and McMichael, A.J. (1989) Complete nucleotide sequence of a unique HLA class I C locus product expressed on the human choriocarcinoma cell line BeWo. J. Immunol. 142, 3281--3285. Ellis, S.A., Palmer, M.S. and McMichael, A.J. (1990) Human trophoblast and the choriocarcinoma cell line BeWo express a truncated H L A class I molecule. J. Immunol. 144, 731--735. Geraghty, D.E., Koller, B.H. and Orr, H.T. (1987) A human MHC class 1 gene that encodes a protein with a shortened cytoplasmic segment. Proc. Natl. Acad. Sci. U.S.A. 84, 9145--9149.
203 Geraghty, D.E., Wet, X., Orr, H.T. and Koller, B.H. (1990) Human leukocyte antigen F (HLA-F): an expressed HLA gene composed of a class I coding sequence linked to a novel transcribed repetitive element. J. Exp, Med. 171, 1--18. Hsi, B.-L., Yeh, C.-J.G. and Faulk, W.P. (1984) Class 1 antigens of the major histocompatibility complex on cytotrophoblast of human chorion laeve. Immunology 52,621--629. Hunt, J.S., Andrews, G.K. and Wood, G.W. (1987) Normal trophoblast resist induction of class I HLA. J. Immunol. 138, 2481--2487. Johnson, P.M. and Stem, P.L. (1986) Antigen expression at human matemo-fetal interfaces. In: Progress in Immunology V! (Cinader, B. and Miller, R.G., eds.), pp. 1056--1069. Academic Press, Orlando. Koller, B.H., Geraghty, D.E., Shin~zu, Y., DeMars, R. and Orr, H.T. (1988) HLA-E: a novel HLA class I gene expressed, ih resting T Iymphocytes. J. Immunol. 141,897--904. KoUer, B.H., Geraghty, D.E., DeMars, R., Duvick, L., Rich, S.S. and Orr, H.T. (1989) Chromosomal organisation of the MHC class I gene family. J. Exp. Med. 169, 469--480. Risk, J.M. and Johnson, P.M. (1990) Genetic studies of the MHC region in human recurrent spontaneous abortion. In: Molecular and Cellular Nature of Maternal-Fetal Signalling (Gill, T.J. Ill and Wegmann, T.G. eds.)Oxford University Press, New York, in press. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd Edn. pp. 7.43--7.52, Cold Spring Harbor Laboratory Press, New York. Shimizu, Y., Geraghty, D.E., Koller, B.H., Orr, H.T. and DeMars, R. (1988) Transfer and expression of three cloned human non-HLA-A, B, C class I major histocompatibility complex genes in mutant lymphoblastoid cells. Proc. Natl. Acad. Sci. U.S.A. 85,227--231. Stern, P.L., Beresford, N., Friedman, C.I., Stevens, V.C., Risk, J.M. and Johnson, P.M. (1986) Class Ilike MHC molecules expressed by baboon placental syncytiotrophoblast. J. immunol. 138, 1088-1091. Stern, P.L., Morris, A., McMain, A., Risk, J.M. et al. 0988) MHC class ! expression by developmental tumors: teratocarcinoma ceils are TCA positive. Hum. Immunol. 22, 247--261. Strachan, T., Dodge, A.B., Smillie, D., Dyer, P.A., Sodoyer, R., Jordan, B.R. and Harris, R. (1986) An HLA-C specific DNA probe. Immunogenetics 23, 115--120.
199
Elsevier Scientific Publishers Ireland Ltd.
JR100673
Brief c o m m u n i c a t i o n
Northern blot analysis of HLA-G expression by BeWo human choriocarcinoma cells J a n e t M. Risk a n d P e t e r M. J o h n s o n Pregnancy Immunology Group, Department of Immunology, University of Liverpool, p.O. Box 147, Liverpool L69 3BX (U. K.) (Accepted for publication 4 May 1990)
Summary The BeWo human choriocarcinoma cell line expresses an unusual 41 kDa/32microglobulin-associated class I MHC antigen. HLA-E, -F and -G are known to encode antigens of lower M than HLA-A, -B or -C proteins. Using locus-specific probes, we now show that RNA isolated from BeWo ceils contains markedly more HLA-G transcripts than peripheral blood lymphocytes (PBL), whilst the converse was noted for HLA-B. These results support recent cloning and sequencing data showing the presence of HLA-G transcripts in BeWo cells. Key words: choriocarcinoma cell line; class I M H C transcripts; HLA-G.
Trophoblastic expression of major histocompatibility complex (MHC) antigens has been of central interest to reproductive immunologists. Class I (HLA-A, -B, -C) and II (HLA-DR, -DQ, -DP) MHC antigens are not expressed by villous cytotrophoblast or syncytiotrOphoblast (Sunderland et al., 1981; Bulmer and Johnson, 1985). This is thought to be regulated at the level of gene transcription (Alberti and Herzenberg, 1986; Hunt et al., 1987). In contrast, a class I M H C antigen has been identified on invasive extravillous cytotrophoblast as well as cytotrophoblast columns and the maternal aspect of the chorion laeve (Hsi et al., 1984; Redman et al., 1984; Bulmer and Johnson, 1985). Serological and biochemical analyses have shown this unusual class I MHC antigen to be closely similar to that expressed also by baboon placental trophoblast and by the BeWo human choriocarcinoma (malignant trophoblast) cell line, but absent from the Jar human choriocarCorrespondence to: Prof. P.M. Johnson. ol65-0378/90/$03.50
@ 1990 Elsevier Scientific Publishers Ireland Ltd.
200
cinoma cell line (Anderson and Berkowitz, 1985; Ellis et al., 1986; Stern et al., 1986; Johnson and Stern, 1986). This was not a classical HLA-A or -B alloantigen but, instead, the fl2-microglobulin-associated class I MHC heavy chain had an M of 40--41 kDa and was non-polymorphic on isoelectric focusing. Initial Northern blot analysis and cDNA cloning from BeWo cells indicated the unusual class I MHC antigen to be an HLA-C locus product, but the sequence of the derived cDNA clone did not correspond exactly with any known single HLA-C locus sequence (Ellis et al., 1989). In addition, restriction fragment length polymorphism (RFLP) studies have shown that this cell line genomically does not carry the HLA-Cw4, 5, 7 or 8 alleles (Risk and Johnson, 1990). More recently, Ellis et al. (1990) have identified a different full-length class I MHC cDNA clone and analogous transcripts were demonstrated also in extravillous cytotrophoblast using the polymerase chain reaction; this sequence had a very high degree of homology with a genomic clone HLA-6.0 (now designated HLA-G) that is the product of a class I MHC locus other than HLA-A, -B or -C (Geraghty et al., 1987). This gene, in addition to two further non-HLA-A, -B, -C class I MHC gene loci (HLA-E and -F) identified from genomic cloning, have been shown to encode p~-microglobulin-associated class I MHC proteins of lower M than HLA-A, -B and -C proteins (Shimizu et al., 1988; Koller et al., 1988; Geraghty et al., 1990). We have investigated the identity of class I MHC transcripts in total RNA, prepared by the guanidinium isothiocynate/caesium chloride method of Chirgwin et al. (1979) from BeWo cells and unstimulated peripheral blood lymphocytes (PBL), using formaldehyde gel electrophoresis (20/~g/iane) and Northern blotting (Sambrook et al., 1989). The blots were hybridised at 42°C in the presence of 50o-/o formamide (Sambrook et ai., 1989) to the following probes radiolabelled with 32p by the random priming method: pB550, a 0.55 kb HLA-B locus-specific probe, gift of Dr. T. Strachan (Strachan et al., 1986, and unpublished data); p6.2p10 (HLA-Eo), a 0.24 kb HLA-E locusspecific probe (Koller et al., 1988); p5.4SH, a 0.75 kb HLA-F locus-specific probe (Geraghty et al., 1990); pHLA-6pl, a 0.45 kb HLA-G locus-specific probe (Geraghty et al., ~987; Orr et al., unpublished data); the HLA-E, -F and -G probes were obtained from Dr. H.T. Orr. All probes recognised sequences in the terminal exon or 3'-untranslated regions. The activity of all radiolabelled probes had been confirmed by Southern blot analysis on Hindlll-digested human PBL genomic DNA. Northern blots were washed at high stringency (0.1 x SSC/0.1°T0 SDS at 68°C) before autoradiography with intensifying screens (Risk and Johnson, 1990). Under these conditions, it can be clearly seen that BeWo cells contain significantly more HLA-G transcripts than PBL when identical amounts of total RNA were loaded into each lane (Fig. 1). Conversely, PBI_ appear to
201
H L A-B
HLA-G
HLA-E
HLA-F
1.95 k b i
P
B
P
B
P
B
P
B
Fig. 1. Northern blot analysis of peripheral blood lymphocyte (P) and BeWo cell (B) total RNA after hybridisation with uP-radiolabeUed DNA probes for HLA-B, -G, -E and -F, respectively.
transcribe very much more HLA-B RNA than do BeWo cells (Fig. 1). Although the pBs~0 probe is derived from the region of the gene showing lowest inter-locus homology, the most likely explanation for the BeWo cell signal with this probe centres on possible cross-reactivity notably with related HLA-C locus alleles (Stern et al., 1988; Ellis et al., 1989). RFLP analysis has shown BeWo cells to carry the HLA-B8 allele in their genome but they are serologically unreactive with anti-HLA-B8 tissue-typing alloantisera (Risk and Johnson, 1990), yet it is also known that these cells express low levels of a 45 kDa class I MHC protein as well as the unusual 40m41 kDa species (Johnson and Stern, 1986; Stern et al., 1988). There was no unequivocal hybridisation in repeated Northern blot analysis of BeWo or PBL RNA with the HLA-E and -F probes at the level of stringency employed (see Fig. 1), although these transcripts have previously been reported to be detected in resting T cells by Northern blot and RNase protection assays, respectively (Koller et al., 1988; Geraghty et al., 1990). No hybridisation with total RNA isolated from the Jar choriocarcinoma cell line was detected using any of the probes for HLA-B, -E, -F or -G (data not shown).
202
The HLA-G gene is thought to be a structural homologue of a murine Qaregion gene (Geraghty et al., 1987). There has been no report of any expression by non-trophoblastic normal cell types and its cellular function is unknown (Koller et al., 1989). Selective expression of this class I MHC antigen by cytotrophoblast cells (Ellis et al., 1990; and this report) therefore implies a specific function. Induction of immune suppression of maternal anti-fetal cytotoxic responses has been suggested. However, if this antigen is confirmed to be pauci- or non-polymorphic, it would then be unlikely to act as a transplantation antigen or restriction element guiding T cell responses. Rather, it might be speculated that HI_A-G may function as a specialised cellular recognitive molecule related to cytotrophoblast proliferation and invasion. Acknowlegements We are grateful to Dr. T. Strachan (Manchester) and Dr. H.T. Orr (Minneapolis) for the gifts of gene probes used in this study. Note added in proof A further recent report has also identified HLA-G expression by human trophoblast cells (Korats, S., Main, E.K., Librach, C., Stubbeline, M., Fisher, S.J. and DeMars, R. (1990) Science 248,220--223). References Alberti, S. and Herzenberg, L.A. (1986) Transfection of DNA from choriocarcinoma cell lines and sperm cells: DNA methylation prevents the expression of genes for the major histocompatibility complex (HLA) class I and the T cell differentiation antigen, Leu-2. In: Reproductive Immunology (Clark, D.A., Croy, B.A. eds.), pp. 60--66. Elsevier Science Publishers, Amsterdam. Anderson, D.J. and Berkowitz, R.S. (1985) y-Interferon enhances expression of class 1 MHC antigens in the weakly HLA" human choriocarcinoma cell line BeWo, but does not induce MHC expression in the H L A choriocarcinoma cell line Jar. J. Immunol. 135, 2498--2501. Bulmer, J.N. and Johnson, P.M. (1985) Antigen expression by trophoblast populations in the human placenta and their possible immunobiological relevance. Placenta 6, 127--140. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294--5299. Ellis, S.A., Sargent, I.L., Redman, C.W.G. and McMichael, A.J. (1986) Evidence for a novel HLA antigen found on human extravillous trophoblast and a human choriocarcinoma cell line. Immunology 59, 595--601. Ellis, S.A., Strachan T., Palmer, M.S. and McMichael, A.J. (1989) Complete nucleotide sequence of a unique HLA class I C locus product expressed on the human choriocarcinoma cell line BeWo. J. Immunol. 142, 3281--3285. Ellis, S.A., Palmer, M.S. and McMichael, A.J. (1990) Human trophoblast and the choriocarcinoma cell line BeWo express a truncated H L A class I molecule. J. Immunol. 144, 731--735. Geraghty, D.E., Koller, B.H. and Orr, H.T. (1987) A human MHC class 1 gene that encodes a protein with a shortened cytoplasmic segment. Proc. Natl. Acad. Sci. U.S.A. 84, 9145--9149.
203 Geraghty, D.E., Wet, X., Orr, H.T. and Koller, B.H. (1990) Human leukocyte antigen F (HLA-F): an expressed HLA gene composed of a class I coding sequence linked to a novel transcribed repetitive element. J. Exp, Med. 171, 1--18. Hsi, B.-L., Yeh, C.-J.G. and Faulk, W.P. (1984) Class 1 antigens of the major histocompatibility complex on cytotrophoblast of human chorion laeve. Immunology 52,621--629. Hunt, J.S., Andrews, G.K. and Wood, G.W. (1987) Normal trophoblast resist induction of class I HLA. J. Immunol. 138, 2481--2487. Johnson, P.M. and Stem, P.L. (1986) Antigen expression at human matemo-fetal interfaces. In: Progress in Immunology V! (Cinader, B. and Miller, R.G., eds.), pp. 1056--1069. Academic Press, Orlando. Koller, B.H., Geraghty, D.E., Shin~zu, Y., DeMars, R. and Orr, H.T. (1988) HLA-E: a novel HLA class I gene expressed, ih resting T Iymphocytes. J. Immunol. 141,897--904. KoUer, B.H., Geraghty, D.E., DeMars, R., Duvick, L., Rich, S.S. and Orr, H.T. (1989) Chromosomal organisation of the MHC class I gene family. J. Exp. Med. 169, 469--480. Risk, J.M. and Johnson, P.M. (1990) Genetic studies of the MHC region in human recurrent spontaneous abortion. In: Molecular and Cellular Nature of Maternal-Fetal Signalling (Gill, T.J. Ill and Wegmann, T.G. eds.)Oxford University Press, New York, in press. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd Edn. pp. 7.43--7.52, Cold Spring Harbor Laboratory Press, New York. Shimizu, Y., Geraghty, D.E., Koller, B.H., Orr, H.T. and DeMars, R. (1988) Transfer and expression of three cloned human non-HLA-A, B, C class I major histocompatibility complex genes in mutant lymphoblastoid cells. Proc. Natl. Acad. Sci. U.S.A. 85,227--231. Stern, P.L., Beresford, N., Friedman, C.I., Stevens, V.C., Risk, J.M. and Johnson, P.M. (1986) Class Ilike MHC molecules expressed by baboon placental syncytiotrophoblast. J. immunol. 138, 1088-1091. Stern, P.L., Morris, A., McMain, A., Risk, J.M. et al. 0988) MHC class ! expression by developmental tumors: teratocarcinoma ceils are TCA positive. Hum. Immunol. 22, 247--261. Strachan, T., Dodge, A.B., Smillie, D., Dyer, P.A., Sodoyer, R., Jordan, B.R. and Harris, R. (1986) An HLA-C specific DNA probe. Immunogenetics 23, 115--120.
