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Molecular biology of class II genes in man: An introduction
Molecular Biology of Class II Genes in Man: An Introduction Bertrand R. Jordan
ABSTRACT: This introduction attempts to set the stage for the presentation of results on HLA-DR gene cloning and to briefly indicate the basic approaches used and their associated difficulties.
AIMS Obtaining cDNA and genomic clones coding for human class II histocompatibility antigens has been the subject of a major research effort in many laboratories over the past two or three years. There were many reasons for this large investment. First, sequence analysis of cDNA clones should rapidly yield information on the structure of the various types of ~ and/3 chains, supplementing direct protein sequencing which has been very difficult in this system. Second, once authentic cDNA clones were obtained the number and diversity of class II coding sequences could begin to be analyzed by "Southern" blotting. Third, genomic clones could be isolated from phage or cosmid D N A libraries and the organization of these genes could be analyzed by restriction enzyme maps and D N A sequencing. Finally, chromosome walking studies should allow mapping of the complete class II region and indicate precisely the number and arrangement of coding sequences contained in this region. DIFFICULTIES Obtaining class II-specific cDNA clones has proved to be a difficult task, as shown by the two-year lag between the characterization of the first class I [1] and the first class II cDNA clones [2]. Let us briefly review the reasons for this difficulty, as they are relevant to an understanding of the potentialities and limitations of gene cloning. cDNA cloning starts with messenger RNA. An assay for the specific m R N A sought is necessary and is usually provided by in vitro translation of m R N A fractions followed by immunoprecipitation of the in vitro synthesized proteins with specific polyclonal or monoclonal antibodies. This assay makes it possible to follow the purification of the m R N A (usually by size fractionation on sucrose gradients or agarose gels) until a purified fraction is obtained. This purified m R N A (which may still contain as little as 1% of specific mRNA) is then copied into D N A which after suitable manipulation is inserted into a plasmid vector. A
From the Centre d'Immunologie, INSERM-CNRS de Marseille-Luminy, Marseille Cedex 9. France. Address requests for reprints to Dr. Bertrand R. Jordan, Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, 1.3288 Marseille Cedex 9. France. Recei;'ed April 26, 198.3: accepted April 28, I983. Human Immunology 8, 83-87 (1983) © Elsevier Science Publishing Co., Inc., 1983 52 VanderbiIt Ave., New York, NY 10017
83 0198-8859/83/$ 3.00
84
B.R. Jordan collection of cDNA clones is thus obtained in which one or a few clones per thousand may contain the desired sequence. The clones in this "cDNA library" are then usually analyzed by in vitro translation assays. Briefly, the DNA of a particular cDNA clone is prepared and immobilized on a solid support. The mRNA preparation enriched for a particular mRNA sequence is hybridized to this immobilized cDNA; only the mRNA species corresponding to the cDNA insert is retained. This hybridized mRNA is then eluted from the immobilized DNA and tested in the in vitro translation system to see whether it directs the synthesis of the relevant protein. This procedure is repeated on every clone (or on small pools of clones) from the cDNA library until positive clones are obtained. These are then verified by DNA sequencing and comparison with the amino acid sequence of the protein. It is easy to realize that this procedure is long, difficult, and fraught with possible artefacts. cDNA cloning thus requires a plentiful supply of cells synthesizing the relevant protein at a high level (so that the corresponding mRNA will not be too rare), excellent serological reagents to detect the in vitro synthesized protein (bearing in mind that this may not be identical to the protein found in vivo in terms of processing, glycosylation, and number of chains), and as much sequence data as possible so that the cDNA sequences can be compared to something. Most of these requirements were not filled in the case of class II histocompatibility antigens. These molecules are produced in very low amount in cell lines. Even in "overproducing" human cell lines (which do not exist in the murine system and are responsible for the fact that all the initial cDNA cloning work was done in the human system) class II mRNA represents less than 0.1% of the total message. The problems with serology were multiple: almost all the reagents available recognized only the "native" dimer of c~ and fl chains and thus could not be used in standard in vitro assays in which only one chain was synthesized. Finally, very little sequence data was available and was only representative of the NH2 terminus of the protein, whereas cDNA cloning nearly always yielded the sequence of the 3' part of the mRNA (because the DNA copy is initiated at the poly-A tail of the mRNA). For class I cDNA cloning, in contrast, although the corresponding mRNA is also rare, almost complete protein sequences were known for several members of this family and monoclonal antibodies which could precipitate isolated heavy chains (without associated fl2-microglobulin) were available. New approaches are making cDNA (or genomic) cloning of sequences corresponding to proteins expressed at low level less difficult. Some of them (immunopurification of polysomes, use of synthetic oligonucleotide probes) are outlined below. Others, such as cloning through expression, [3] hold great promise.
SUCCESSES IN CLASS II cDNA C L O N I N G In spite of these difficulties several groups recently succeeded in obtaining authentic cDNA clones for human class II antigens. Janet Lee and co-workers [2] used a polyspecific hetero anti-DR serum which could precipitate isolated c~ (heavy) chains from an in vitro translation system containing dog pancreatic microsomes (which process and glycosylate the synthesized proteins). One of approximately 100 clones prepared from purified mRNA was positive in the selection assay; a clone with a larger insert was obtained by screening the cDNA library with this first clone. Sequencing of this second clone showed that it extended close enough to the NH2 terminal position of the protein to permit identification by comparison with published and unpublished protein sequence data. A similar approach was used by Wiman and co-workers [4] with an antiserum against fl (light) chains obtained by immunization of a rabbit with highly purified /3 chains. Information on the protein sequence of the DR-fl chain thereby allowing
Molecular Biology of Class II Genes in Man
85
identification of the c D N A clones was obtained simultaneously in the same laboratory. A very elegant approach was used by A. Korman and co-workers [5] who succeeded in applying immunopurification methods to polysomes (thus purifying the specific m R N A contained in these polysomes). This method had been widely investigated but had not been effective for purification ofpolysomes corresponding to proteins of low abundance. Crucial modifications were the use of a pure preparation of a monoclonal antibody against DR-or (heavy) chains to bind to polysomes and fractionation of the polysome-antibody complex on a column of protein A-Sepharose. Extremely efficient (about 500-fold) purification was obtained in this way. Using such a highly enriched m R N A preparation, subsequent cloning steps proved relatively straight forward, being achieved either by c D N A cloning o f the pure m R N A (although the amounts are very low) or by use of the fraction as a probe to screen a c D N A library made from total poly-A ÷ mRNA. Several c D N A clones for the c~ (heavy) chains were thus obtained. Stetler and COoworkers [6] used a different approach that eliminates the requirement for in vitro translation and single chain-specific antibodies. Based on the known N H , terminal sequence of the DR-c~ (heavy chain), they synthesized a set of oligonucleotides complementary for codons 11-14. Primer extension of these oligonucleotides on B cell membrane-bound poly-A ÷ m R N A templates (expected to be enriched in mRNAs for class II molecules) showed that one of them was complementary to the a chain m R N A and provided additional sequence information that allowed the synthesis of a longer oligonucleotide that was then used to screen a c D N A library constructed with membrane-bound mRNA. D N A sequence analysis of one c D N A clone thus identified confirmed that it coded for the DR-c* chain. E. Long and co-workers [7] succeeded in developing a sophisticated assay system, namely the injection of m R N A into Xenopus laevis oocytes. In this system, total m R N A directed the synthesis of the complete DR antigen (a + /3) which was readily recognized by sera raised against the native molecule. This system was also used in a complementation assay in which oe and/3 mRNAs could be injected separately (i.e., from mRNA hybrids selected using different c D N A clones or pools) and allowed the identification of both ce, /3, and also possible "invariant chain"-specific c D N A clones. S T U D I E S W I T H CLASS II c D N A C L O N E S Much further progress was made after the initial isolation of class II c D N A clones. These studies are reported in detail in the following papers, and only a brief summary o f the highlight of this ongoing work will be presented here. In a rather short time, sequence studies on c D N A clones have greatly increased our knowledge of the primary structures of class II polypeptides. Indeed, this is probably the first case in which D N A sequencing has provided the major part of data on the primary structure of a family of proteins. Whole genome Southern blots have been probed with the various class II cDNAs in an effort to determine how many related coding sequences exist. These blots generally show few hybridizing fragments, indicating that the family of genomic sequences closely homologous to any one probe is small (between one and three). This is in striking contrast to results with class I probes which reveal up to 15-20 fragments. It is however apparent that the different class II families may be quite different from each other so that o n e / 3 (light chain) probe for example may not reveal all the /3-related coding sequences. It is evident that we do not yet know how many ce, /3 and invariant chain coding sequences are present in the human genome.
86
B.R. Jordan Genomic clones have been isolated from either phage or cosmid libraries of human DNA. Some of these clones have been sequenced and have revealed the organization of class II genes which is in many ways analogous to that of the class I genes (separate signal exon, one exon for each of the two extracellular domains) and strengthens the case for an evolutionary relationship between these two gene families. It has proved possible to use human class II c D N A probes to isolate the corresponding mouse clones (usually genomic). The organization of the murine class II region has already been largely worked out, thanks to cosmid cloning and chromosome walking studies on one hand, and to analysis of mouse recombinants (sorely lacking in the human system) by Southern blots.
WHAT NEXT? The long-range organization of the class II region on chromosome 6 is being actively studied. Much of the information will probably come from comparisons between mouse and man and from the use of cross-hybridizing probes which may allow transposition of the (already largely known) murine class II D N A map onto the corresponding human region. Partial deletion mutants of human cell lines will certainly be very useful in this work since they supplement the nearabsence of genetically defined recombinants in the human system. Expression of cloned class II genes in vitro is a major goal of many laboratories. Expression of class I genes in murine L cells after calcium phosphate transformation [ 8 - 1 0 ] is at present an important tool in the analysis of class I genes: it allows the recognition of active genes, the serological characterization of the expressed protein, and thus the assignment of a given specificity to the cloned gene, and, in conjunction with functional assays like class I-restriction T cell lysis and manipulation (by exon shuffling or site directed mutagenesis) of the cloned genes, an approach to the definition of functional domains of the class I molecule. Transformation of cells with class II genes is proving more difficult. Fibroblast L cells may not be able to express these genes, and transformation of lymphocytes is technically difficult. Simultaneous transformation with both c~and/3 genes (plus possibly invariant chain) clones may be necessary, or suitably deleted recipient lines must be developed. These problems will surely be overcome in the near future and transformation studies will then provide a powerful approach to the function of class II genes.
REFERENCES 1. Ploegh HL, Orr HT, Strominger JL: Proc Natl Acad Sci USA 77:6081, 1980. 2. Lee JS, Trowsdale J, Bodmer WF: Proc Natl Acad Sci USA 79:545, 1982. 3. Kawathas P, Hertzenberg H: Proc Natl Acad Sci USA, in press. 4. Wiman K, Larham~ar D, Claesson L, Gustafsson K, Schenning L,. Bill P, B6hme J, Denaro M, Dobberstein B, H~immerling L, Kvist S, Servenius B, Sundelin J, Peterson PA, Rask L: Proc Natl Acad Sci USA 79:1703, 1982. 5. Korman AJ, Auffray C, Schambock A, Strominger JL: Proc Natl Acad Sci USA 79:6013 1982. 6. Stetler D, Das H, Nunberg JH, Saiki R, Sheng-Dong R, Mullis KB, Weissman SM, Ehrlich HA: Proc Natl Acad Sci USA 79:5966, 1982. 7. Long EO, Wake CT, Strubin M, Gross N, Accolla RS, Carrel S, Mach B: Proc Natl Acad Sci USA, in press.
Molecular Biology of Class lI Genes in Man
87
8. Goodenow RS, McMillan M, Orn A, Nicholson M, Davidson N, FrelingerJA, Hood L: Science 215:677, 1982. 9. BarbosaJA, Kamarck ME, Biro PA, Weissman SM, Ruddle FH: Proc Natl Acad Sci USA 79:6327, 1982. 10. Lemonnier FA, Malissen M, Gostein P, Le Bouteiller P, Rebai N, Damotte M, Birnbaum D, Caillol D, Trucy J, Jordan BR: Immunogenetics 16:355, 1982.
ABSTRACT: This introduction attempts to set the stage for the presentation of results on HLA-DR gene cloning and to briefly indicate the basic approaches used and their associated difficulties.
AIMS Obtaining cDNA and genomic clones coding for human class II histocompatibility antigens has been the subject of a major research effort in many laboratories over the past two or three years. There were many reasons for this large investment. First, sequence analysis of cDNA clones should rapidly yield information on the structure of the various types of ~ and/3 chains, supplementing direct protein sequencing which has been very difficult in this system. Second, once authentic cDNA clones were obtained the number and diversity of class II coding sequences could begin to be analyzed by "Southern" blotting. Third, genomic clones could be isolated from phage or cosmid D N A libraries and the organization of these genes could be analyzed by restriction enzyme maps and D N A sequencing. Finally, chromosome walking studies should allow mapping of the complete class II region and indicate precisely the number and arrangement of coding sequences contained in this region. DIFFICULTIES Obtaining class II-specific cDNA clones has proved to be a difficult task, as shown by the two-year lag between the characterization of the first class I [1] and the first class II cDNA clones [2]. Let us briefly review the reasons for this difficulty, as they are relevant to an understanding of the potentialities and limitations of gene cloning. cDNA cloning starts with messenger RNA. An assay for the specific m R N A sought is necessary and is usually provided by in vitro translation of m R N A fractions followed by immunoprecipitation of the in vitro synthesized proteins with specific polyclonal or monoclonal antibodies. This assay makes it possible to follow the purification of the m R N A (usually by size fractionation on sucrose gradients or agarose gels) until a purified fraction is obtained. This purified m R N A (which may still contain as little as 1% of specific mRNA) is then copied into D N A which after suitable manipulation is inserted into a plasmid vector. A
From the Centre d'Immunologie, INSERM-CNRS de Marseille-Luminy, Marseille Cedex 9. France. Address requests for reprints to Dr. Bertrand R. Jordan, Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, 1.3288 Marseille Cedex 9. France. Recei;'ed April 26, 198.3: accepted April 28, I983. Human Immunology 8, 83-87 (1983) © Elsevier Science Publishing Co., Inc., 1983 52 VanderbiIt Ave., New York, NY 10017
83 0198-8859/83/$ 3.00
84
B.R. Jordan collection of cDNA clones is thus obtained in which one or a few clones per thousand may contain the desired sequence. The clones in this "cDNA library" are then usually analyzed by in vitro translation assays. Briefly, the DNA of a particular cDNA clone is prepared and immobilized on a solid support. The mRNA preparation enriched for a particular mRNA sequence is hybridized to this immobilized cDNA; only the mRNA species corresponding to the cDNA insert is retained. This hybridized mRNA is then eluted from the immobilized DNA and tested in the in vitro translation system to see whether it directs the synthesis of the relevant protein. This procedure is repeated on every clone (or on small pools of clones) from the cDNA library until positive clones are obtained. These are then verified by DNA sequencing and comparison with the amino acid sequence of the protein. It is easy to realize that this procedure is long, difficult, and fraught with possible artefacts. cDNA cloning thus requires a plentiful supply of cells synthesizing the relevant protein at a high level (so that the corresponding mRNA will not be too rare), excellent serological reagents to detect the in vitro synthesized protein (bearing in mind that this may not be identical to the protein found in vivo in terms of processing, glycosylation, and number of chains), and as much sequence data as possible so that the cDNA sequences can be compared to something. Most of these requirements were not filled in the case of class II histocompatibility antigens. These molecules are produced in very low amount in cell lines. Even in "overproducing" human cell lines (which do not exist in the murine system and are responsible for the fact that all the initial cDNA cloning work was done in the human system) class II mRNA represents less than 0.1% of the total message. The problems with serology were multiple: almost all the reagents available recognized only the "native" dimer of c~ and fl chains and thus could not be used in standard in vitro assays in which only one chain was synthesized. Finally, very little sequence data was available and was only representative of the NH2 terminus of the protein, whereas cDNA cloning nearly always yielded the sequence of the 3' part of the mRNA (because the DNA copy is initiated at the poly-A tail of the mRNA). For class I cDNA cloning, in contrast, although the corresponding mRNA is also rare, almost complete protein sequences were known for several members of this family and monoclonal antibodies which could precipitate isolated heavy chains (without associated fl2-microglobulin) were available. New approaches are making cDNA (or genomic) cloning of sequences corresponding to proteins expressed at low level less difficult. Some of them (immunopurification of polysomes, use of synthetic oligonucleotide probes) are outlined below. Others, such as cloning through expression, [3] hold great promise.
SUCCESSES IN CLASS II cDNA C L O N I N G In spite of these difficulties several groups recently succeeded in obtaining authentic cDNA clones for human class II antigens. Janet Lee and co-workers [2] used a polyspecific hetero anti-DR serum which could precipitate isolated c~ (heavy) chains from an in vitro translation system containing dog pancreatic microsomes (which process and glycosylate the synthesized proteins). One of approximately 100 clones prepared from purified mRNA was positive in the selection assay; a clone with a larger insert was obtained by screening the cDNA library with this first clone. Sequencing of this second clone showed that it extended close enough to the NH2 terminal position of the protein to permit identification by comparison with published and unpublished protein sequence data. A similar approach was used by Wiman and co-workers [4] with an antiserum against fl (light) chains obtained by immunization of a rabbit with highly purified /3 chains. Information on the protein sequence of the DR-fl chain thereby allowing
Molecular Biology of Class II Genes in Man
85
identification of the c D N A clones was obtained simultaneously in the same laboratory. A very elegant approach was used by A. Korman and co-workers [5] who succeeded in applying immunopurification methods to polysomes (thus purifying the specific m R N A contained in these polysomes). This method had been widely investigated but had not been effective for purification ofpolysomes corresponding to proteins of low abundance. Crucial modifications were the use of a pure preparation of a monoclonal antibody against DR-or (heavy) chains to bind to polysomes and fractionation of the polysome-antibody complex on a column of protein A-Sepharose. Extremely efficient (about 500-fold) purification was obtained in this way. Using such a highly enriched m R N A preparation, subsequent cloning steps proved relatively straight forward, being achieved either by c D N A cloning o f the pure m R N A (although the amounts are very low) or by use of the fraction as a probe to screen a c D N A library made from total poly-A ÷ mRNA. Several c D N A clones for the c~ (heavy) chains were thus obtained. Stetler and COoworkers [6] used a different approach that eliminates the requirement for in vitro translation and single chain-specific antibodies. Based on the known N H , terminal sequence of the DR-c~ (heavy chain), they synthesized a set of oligonucleotides complementary for codons 11-14. Primer extension of these oligonucleotides on B cell membrane-bound poly-A ÷ m R N A templates (expected to be enriched in mRNAs for class II molecules) showed that one of them was complementary to the a chain m R N A and provided additional sequence information that allowed the synthesis of a longer oligonucleotide that was then used to screen a c D N A library constructed with membrane-bound mRNA. D N A sequence analysis of one c D N A clone thus identified confirmed that it coded for the DR-c* chain. E. Long and co-workers [7] succeeded in developing a sophisticated assay system, namely the injection of m R N A into Xenopus laevis oocytes. In this system, total m R N A directed the synthesis of the complete DR antigen (a + /3) which was readily recognized by sera raised against the native molecule. This system was also used in a complementation assay in which oe and/3 mRNAs could be injected separately (i.e., from mRNA hybrids selected using different c D N A clones or pools) and allowed the identification of both ce, /3, and also possible "invariant chain"-specific c D N A clones. S T U D I E S W I T H CLASS II c D N A C L O N E S Much further progress was made after the initial isolation of class II c D N A clones. These studies are reported in detail in the following papers, and only a brief summary o f the highlight of this ongoing work will be presented here. In a rather short time, sequence studies on c D N A clones have greatly increased our knowledge of the primary structures of class II polypeptides. Indeed, this is probably the first case in which D N A sequencing has provided the major part of data on the primary structure of a family of proteins. Whole genome Southern blots have been probed with the various class II cDNAs in an effort to determine how many related coding sequences exist. These blots generally show few hybridizing fragments, indicating that the family of genomic sequences closely homologous to any one probe is small (between one and three). This is in striking contrast to results with class I probes which reveal up to 15-20 fragments. It is however apparent that the different class II families may be quite different from each other so that o n e / 3 (light chain) probe for example may not reveal all the /3-related coding sequences. It is evident that we do not yet know how many ce, /3 and invariant chain coding sequences are present in the human genome.
86
B.R. Jordan Genomic clones have been isolated from either phage or cosmid libraries of human DNA. Some of these clones have been sequenced and have revealed the organization of class II genes which is in many ways analogous to that of the class I genes (separate signal exon, one exon for each of the two extracellular domains) and strengthens the case for an evolutionary relationship between these two gene families. It has proved possible to use human class II c D N A probes to isolate the corresponding mouse clones (usually genomic). The organization of the murine class II region has already been largely worked out, thanks to cosmid cloning and chromosome walking studies on one hand, and to analysis of mouse recombinants (sorely lacking in the human system) by Southern blots.
WHAT NEXT? The long-range organization of the class II region on chromosome 6 is being actively studied. Much of the information will probably come from comparisons between mouse and man and from the use of cross-hybridizing probes which may allow transposition of the (already largely known) murine class II D N A map onto the corresponding human region. Partial deletion mutants of human cell lines will certainly be very useful in this work since they supplement the nearabsence of genetically defined recombinants in the human system. Expression of cloned class II genes in vitro is a major goal of many laboratories. Expression of class I genes in murine L cells after calcium phosphate transformation [ 8 - 1 0 ] is at present an important tool in the analysis of class I genes: it allows the recognition of active genes, the serological characterization of the expressed protein, and thus the assignment of a given specificity to the cloned gene, and, in conjunction with functional assays like class I-restriction T cell lysis and manipulation (by exon shuffling or site directed mutagenesis) of the cloned genes, an approach to the definition of functional domains of the class I molecule. Transformation of cells with class II genes is proving more difficult. Fibroblast L cells may not be able to express these genes, and transformation of lymphocytes is technically difficult. Simultaneous transformation with both c~and/3 genes (plus possibly invariant chain) clones may be necessary, or suitably deleted recipient lines must be developed. These problems will surely be overcome in the near future and transformation studies will then provide a powerful approach to the function of class II genes.
REFERENCES 1. Ploegh HL, Orr HT, Strominger JL: Proc Natl Acad Sci USA 77:6081, 1980. 2. Lee JS, Trowsdale J, Bodmer WF: Proc Natl Acad Sci USA 79:545, 1982. 3. Kawathas P, Hertzenberg H: Proc Natl Acad Sci USA, in press. 4. Wiman K, Larham~ar D, Claesson L, Gustafsson K, Schenning L,. Bill P, B6hme J, Denaro M, Dobberstein B, H~immerling L, Kvist S, Servenius B, Sundelin J, Peterson PA, Rask L: Proc Natl Acad Sci USA 79:1703, 1982. 5. Korman AJ, Auffray C, Schambock A, Strominger JL: Proc Natl Acad Sci USA 79:6013 1982. 6. Stetler D, Das H, Nunberg JH, Saiki R, Sheng-Dong R, Mullis KB, Weissman SM, Ehrlich HA: Proc Natl Acad Sci USA 79:5966, 1982. 7. Long EO, Wake CT, Strubin M, Gross N, Accolla RS, Carrel S, Mach B: Proc Natl Acad Sci USA, in press.
Molecular Biology of Class lI Genes in Man
87
8. Goodenow RS, McMillan M, Orn A, Nicholson M, Davidson N, FrelingerJA, Hood L: Science 215:677, 1982. 9. BarbosaJA, Kamarck ME, Biro PA, Weissman SM, Ruddle FH: Proc Natl Acad Sci USA 79:6327, 1982. 10. Lemonnier FA, Malissen M, Gostein P, Le Bouteiller P, Rebai N, Damotte M, Birnbaum D, Caillol D, Trucy J, Jordan BR: Immunogenetics 16:355, 1982.