Molecular Analysis of the Chicken Major Histocompatibility Complex Gene and Gene Products 1

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1 Molecular Analysis of the Chicken Major Histocompatibility Complex Gene and Gene Products 1 S. J. LAMONT, 2 C. M. WARNER, 3 and A. W. NORDSKOG 2 Departments of Animal Science and Biochemistry and Biophysics, Iowa State University, Ames, Iowa (Received for publication January 7, 1987) ABSTRACT New technologies are enabling researchers to conduct analysis of the molecular structure of the genes and gene products of the major histocompatibility complex (MHC). Two of these new technologies are the production of monoclonal antibodies by hybridomas and analysis of deoxyribonucleic acid (DNA) by restriction fragment length polymorphisms (RFLP) on Southern blots. Monoclonal antibodies, because of their exquisite specificity, can be used to isolate purified MHC antigens for further study. The RFLP analysis can be used to examine differences in the MHC at the DNA level. This paper describes the two aforementioned technologies and their application to molecular analysis of the chicken MHC. (Key words: major histocompatibility complex, restriction fragment length polymorphism, monoclonal antibodies) INTRODUCTION Many technologies are available to analyze the genes and gene products of the major histocompatibility complex (MHC). The fortuitous location of the genes encoding the erythrocyte antigen B (Ea-B) within the chicken MHC (Schierman and Nordskog, 1961) has allowed the use of blood typing by hemagglutination with alloantisera as a method for identifying the chicken MHC (Briles et al 1950). The MHC typing by hemagglutination with polyclonal antisera, while it has proved historically useful, is of somewhat limited analytical value because of recombinations within the MHC and because of the location of genes controlling several important traits in the B-L subregion which does not code for antigens located on erythrocytes (Nordskog et al., MONOCLONAL ANTIBODIES TO MAJOR HISTOCOMPATIBILITY COMPLEX ANTIGENS Monoclonal antibodies (McAb) have several advantages over polyclonal alloantisera for the study of chicken MHC antigens. The McAb are specific for one antigenic determinant, but polyclonal antibodies (PcAb) potentially recognize all antigenic determinants of the immuniz- 1 Journal Paper Number J of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA; Project Numbers 2237 and Animal Science Department. 'Biochemistry and Biophysics Department Poultry Science 66: ing material. The reproducibility of an individual McAb is constant, but PcAb vary from animal to animal and from one collection time to the next. The McAb are of a single immunoglobulin class, but PcAb usually contain all classes. Also, McAb have a much higher useful to irrelevant antibody ratio than PcAb. The protocol for McAb production, in theory, is quite simple (Figure 1). The technique was described by Kohler and Milstein in First, the antigen of interest is injected into a mouse. Because MHC antigens are cell surface proteins, either purified MHC antigens or cells expressing MHC antigens can be used as the immunogen. Immune cells of the mouse will then produce antibodies to the MHC. The spleen is removed and antibody-producing cells are isolated from it. Unfortunately, these cells produce specific antibodies to MHC proteins in only very small quantities, and the cells do not live long in culture. To overcome these limitations, the antibody-secreting cells are fused (by chemical, viral, or electrical methods) with myeloma (tumor) cells from another mouse. A small percentage of the cells resulting from that fusion (hybridoma cells) possesses the necessary combination of properties: production of the specific desired antibody and the ability to survive and function well in long-term cultures. The cells are cultured in limiting dilutions to ensure that all cells in a well or vessel are descendents of a single progenitor cell; thus, the antibody produced by them is monoclonal. The secretions of the hybridoma cells into their 819

2 820 LAMONT ET AL. Myeloma-Bearing Mouse V Myeloma Cells i n Culture Antigen Spleen Myeloma Cell ; Very Large Amounts of Specific Antibody Antibody-Producing Cell HYBRIDOMA FIG. 1. Diagram of procedure for production of antibody-secreting hybridomas. culture supernatants are screened by appropriate assays to select the clones producing McAb to MHC antigens. Relatively few McAb have been produced to chicken MHC antigens, compared with the numbers produced for MHC of mouse or human. However, McAb have been produced to antigens of each of the three subregions of the chicken MHC (B-G: Longenecker et al., 1979; Miller et al, 1982; B-F: Pink et al, 1985; B-L: Ewert et al, 1984; Guillemot et al, 1984; Hala et al, 1984; Crone et al, 1985). These McAb have been extremely useful in isolating highly purified MHC antigens for biochemical characterizations and analysis of molecular structure. The B-G antigens isolated from erythrocyte membranes by immunoprecipitation with McAb have been shown to exist as polymers of a 47 kda monomer (Salomonsen, personal communication). Two-dimensional gel electrophoresis of B-G antigens immunoprecipitated Specific Antibody in Very Small Quantity with anti-b-g McAb revealed extensive polymorphism of B-G antigens, hypothesized to be due to multiple loci in the B-G region (Miller et al, 1984). McAb to class I (B-F) antigens precipitate a 40 to 45 kd cell surface molecule, along with a 12 kda B 2 microglobulin (Pink et al, 1985). Molecular weight differences between haplotypes have been reported, with B >5 /B 15 yielding two a chains of 40 and 42 kda and B [9 /B 19 yielding only one a chain of 39 kda (Verland, personal communication). Crone et al (1985) used sequential immunoprecipitation by McAb of different specificities to demonstrate two distinct B-F products from B homozygous chickens, which indicates the existence of at least two Class I MHC gene products. Molecular analysis of class II (B-L) antigens isolated with McAb showed a monomorphic a chain of kda noncovalently bound to a polymorphic B chain of approximately kda (Ewert et al, 1984; Guil-

3 SYMPOSIUM: AVIAN MAJOR HISTOCOMPATIBILITY COMPLEX 821 lemot et al., 1986). Thus, use of McAb has shown the avian MHC to bear many similarities to the better known MHC of mouse and man and also to be more complex than originally imagined, with multiple loci in each subregion. RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF THE CHICKEN MAJOR HISTOCOMPATIBILITY COMPLEX A method for direct analysis of the genes of the MHC is the detection of restriction fragment Chicken Bursa, RBC, PBL, Liver, or Spermatozoa Isolate DNA, Cleave with Restriction Enzyme Separate Fragments by Electrophoresis Transfer Fragments to Nitrocellulose Paper Incubate Paper with Radioactively Labeled Probe, which Binds to Desired Gene iii Autoradiograph to Locate Labeled DNA twmumnw «s#rrthf#k FIG. 2. Diagram of procedure for producing Southern blots to examine deoxyribonucleic acid (DNA) banding patterns for restriction fragment length polymorphisms. RBC = Red blood cell. PBL = Peripheral blood leucocyte.

4 822 LAMONT ET AL. length polymorphisms (RFLP) on Southern blots. Soller and Beckman (1986) have described the theory of application of RFLP analysis to poultry breeding. The MHC, with its well-established role in immune response and disease resistance, should show RFLP with direct effects on economic values and, therefore, be of potential use in practical breeding programs (Warner, 1986). The basic protocol for RFLP analysis is presented in Figure 2. Deoxyribonucleic acid (DNA) is isolated from each individual. In chickens, nucleated erythrocytes or spermatozoa are convenient sources of DNA. The DNA is kb : w gl m M g^m "$&%' HBP m K ^P^» IfflSt m 3 ygi Hp. Sa:!r ISP : ^ ^ W. ^ ^ Mmjjjk kb ij! " 6 ** *#Hi» FIG. 3. Restriction fragment length polymorphism Southern blot analysis of the Iowa State University inbred chicken lines. Sperm deoxyribonucleic acid was digested with the restriction enzyme Pvull. Blots were probed with "P-labelled p!4, a chicken class II major histocompatibility complex probe (kindly provided by C. Auffray, CNRS, France). Inbred line designation and B haplotypes are: 1. M (B 152 ): 2. Sp {B 2 7.GH(B I3 );8. 19(B I3 );9.HN(B 15 ); 10. GH(B 15 '): (B r \ marker) are designated in the left margin. '); 3. GH (B'y, 4. G-B2 (B 6 ); 5. HN (B 12 ); 6. G-Bl (B 13 ); '); (B 15 '); (B 15 '). Fragment lengths (from

5 SYMPOSIUM: AVIAN MAJOR HISTOCOMPATIBILITY COMPLEX 823 digested into smaller fragments by use of restriction enzymes, which recognize and cleave certain nucleotide sequences. The DNA fragments are then separated according to size by agarose gel electrophoresis. The DNA is transferred from the gel to nitrocellulose filter paper by the procedure of Southern blotting (Southern, 1975). The Southern blot is then hybridized with a radiolabeled probe. The probe consists of a DNA sequence homologous to the gene of interest, which will specifically bind to its homolog on the filter. The filter is then dried and subjected to autoradiography. The visible bands indicate areas in which the probe recognized homologous sequences. A rough estimate, usually an underestimate, of the number of genes can be made by simply counting the bands (Wijsman, 1984). Polymorphisms in the relative position of the bands, a function of the size of the DNA fragments cleaved with the restriction enzymes, can be compared among individuals. An RFLP analysis of the B-L genes was conducted with Iowa State University inbred lines of chickens (Figure 3). Several observations regarding the MHC Class II B-L genes can be made. The number of genes seems to be in the order of 10 to 12. A single enzyme digestion may not yield a completely informative RFLP analysis. For example, some birds known to differ for the serological B haplotype do not have different RFLP patterns. This does not preclude, however, the possibility that these birds do have identical B-L genes (detected by the probe) but different B-G and B-F genes (detected by serological methods). Birds representing genetically divergent lines (Lane 1, Egyptian Fayoumi; Lane 2, Spanish) exhibit unique patterns when compared to the other lines (Lanes 3 to 13, Leghorn origin). Not only are the patterns unique, but specific bands within the pattern are unique, and therefore diagnostic for that genetic line (Lane 1, band 1.1 kb; Lane 2, band 1.3 kb). Lanes 4 and 6 and Lanes 5 and 9 represent two pairs of MHC-congenic lines that are identical for background genes but differ at the MHC. They show differences in RFLP pattern within each pair. In another congenic set, however (Lanes 3, 7, and 10), no polymorphisms are evident. Perhaps they share B-L, but not B-G and B-F genes. Use of additional restriction enzymes, combinations of enzymes, and different DNA probes will allow development of a comprehensive RFLP "fingerprint" profile of the MHC genes of these lines. CONCLUSIONS The application of biotechnological procedures provides researchers with valuable new tools for analyses of the chicken MHC. Studies at the protein and gene level indicate that there is more homology with murine and human systems and more complexity in the chicken MHC than was previously thought to exist. Cloning and sequencing of the avian MHC genes, currently underway in several laboratories, including our own, will provide additional crucial insight into the molecular biology of the chicken MHC. ACKNOWLEDGMENTS We thank Bradley Gerndt for excellent technical assistance and Charles Auffray for the Class II chicken cdna probe. This work was supported, in part, by a grant from the United States Department of Agriculture. REFERENCES Briles, W. E., W. H. McGibbon, and M. R. Irwin, On multiple alleles affecting cellular antigens in the chicken. Genetics 35: Crone, M., M. Simonsen, K. Skjodt, K. Linnet, and L. Olson, Mouse monoclonal antibodies to Class I and Class II antigens of the chicken MHC: evidence for at least two Class I products of the B complex. Immunogenetics 21: Ewert, D. L., M. S. Munchus, C.-L.H. Chen, and M. D. Cooper, Analysis of structural properties and cellular distribution of avian la antigen by using monoclonal antibody to monomorphic determinants. J. Immunol. 132: Guillemot, F. P., P. D. Oliver, B. M. Peault, and N. M. LeDouarin, Cells expressing la antigens in the avian thymus. J. Exp. Med. 160: Guillemot, F., P. Turmel, D. Charron, N. LeDouarin, and C. Auffray, Structure, biosynthesis, and polymorphism of chicken MHC Class II (B-L) antigens and associated molecules. J. Immunol. 137: Hala, K., G. Wick, R. L. Boyd, H. Wolf, G. Bock, and D. L. Ewert, The B-L (la-like) antigens of the chicken. Lymphocyte plasma membrane distribution and tissue localization. Dev. Comp. Immunol. 8: Kohler, G., and C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: Longenecker, B. M., T. R. Mosmann, and C. Shioyawa, A strong preferential response of mice to polymorphic antigenic determinants of the chicken MHC, analyzed with mouse hybridoma (monoclonal) antibodies. Immunogenetics 9: Miller, M. M., R. Goto, and H. Abplanalp, Analysis of the B-G antigens of the chicken MHC by two-dimensional gel electrophoresis. Immunogenetics 20:

6 824 LAMONT ET AL. Miller, M. M., R. Goto, and S. D. Clark, Structural characterization of developmentally expressed antigenic markers on chicken erythrocytes using monoclonal antibodies. Dev. Biol. 94: Nordskog, A.W., I.Y. Pevzner, and S.J. Lamont, Subregions and functions of the chicken major histocompatability complex. Poultry Sci. 66: Pink, J.R.L., M. W. Kieran, A. M. Rijnbeck, and B. M. Longenecker, A monoclonal antibody against chicken MHC Class I (B-F) antigens. Immunogenetics 21: Schierman, L. W., and A. W. Nordskog, Relationship of blood type to histocompatibility in chickens. Science 134: Soller, M., and J. S. Beckman, Restriction fragment length polymorphisms in poultry breeding. Poultry Sci. 65: Southern, E. M., Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: Warner, C. M., Genetic manipulation of the major histocompatibility complex. J. Anim. Sci. 63: Wijsman, E. M., Optimizing selection of restriction enzymes in the search for DNA variants. Nucleic Acids Res. 12: