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Transfection and expression of human class II cDNA in human EBV transformed cells
Abstracts
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the 790 bp Sac I-Hind III fragment of the DR/~ cDNA probe. Clones thus identified were digested with a variety of restriction enzymes both individually and in combination, electrophoresed on a 1% agarose gel, Southern blotted, and then hybridized using the DR3 and DQ3 cDNA probes. A Pst 1 600 bp fragment which was identified as hybridizing to the DQ/3 probe but not the DR/3 probe was subcloned into the plasmid vector pUC 118, sequenced, and used to probe Southern blots of Taq I digested genomic DNA. Of 40 individuals thus far examined, 20 of which were HLA serotyped as DQw2, five persons possessing the DQw2 phenotype were observed to have fragments which hybridized to this probe (odds ratio = 6.7). These data suggest that there may be subsets of DQw2 or homologous sequences in linkage disequilibrium with the DQ locus.
DNA SEQUENCE POLYMORPHISM OF DR/3 CHAINS IN HLA-DR4, -7, AND -9 HAPLOTYPES. P.K Gregersen, T. Moriuchi, R. Karr, F. Obata, J. Maccari, D. Goldberg, R. Winchester, and J. Silver; Hospitalfor Joint Diseases, New York, NY We have isolated and sequenced cDNA clones corresponding to the DR/31 and DR32 loci from two homozygous B-cell lines typed as DR7 (Burkhart) and DR9 (ISK). These nucleotide sequences were compared to DR/31 and DRfl2 chains of other DR haplotypes. The first domain sequences of DR/32 chains are identical in DR4 and DR7 haplotypes. In addition, there is strong sequence homology within the 3' untranslated regions of DR/31 genes from DR4, - 7 , and - 9 haplotypes, thus establishing the close evolutionary relationship among these three haplotypes. In contrast, the first domain sequences of DR31 molecules from these haplotypes do not reflect the DR4, - 7 , - 9 family relationship; the DR4, - 7, and - 9 DR/31 sequences differ as much from each other as they do from the DR31 of any unrelated DR type. The differences in degree of diversity between DR/~I and DR/32 chains may be due to selection pressures; this implies functional differences for products of the DR/31 and DR/32 loci. Alternatively, we suggest that closely linked seqments of the human class II region may differ in their underlying rates of variation, independent of selection pressures, and that this may in part account for the extraordinary diversity found in the/31 first domain. TRANSFECTION AND EXPRESSION OF HUMAN CLASS II cDNA IN HUMAN EBV TRANSFORMED CELLS. K.G. Becker, S.H. Hsu, and W.B. Bias; The Johns Hopkins University School of Medicine, Baltimore, MD Functional studies of the products of human MHC genes are now possible through the manipulation of those genes. For such studies we have transfected a class I MHC gene into normal human T cells (Becker KG et al.,J. Immunol, manuscript submitted) and as described here class II cDNA into a human EBV transformed homozygous typing cell line (EF A 1,B8,Bw6,DR3,DQ2). Class II cDNA (DR31, DQ31, DP3, DO3), recently isolated and characterized (Tonnelle C et al., EMBO J. 4(11)) were modified by the ligation of the neomycin gene into the CLA I site found on the Okayama-Berg expression vectors carrying these DNA sequences, thus allowing dominant selection of transfected cells by Geneticin (G418). These plasmids were transfected into the transformed cell line by liposomal fusion. Stable lines bearing the gene products of the introduced genes were selected by G418 resistance. Preliminary analysis by cytofluorometry documents the stable expression of DQ1, while expression of other class II specificities will be deter-
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Abstracts mined when adequate cell density is achieved. These cell lines will be used for in vitro stimulation studies, specific screening and absorbtion of class II sera, and for antigen structural studies.
RAPID AND SPECIFIC ISOLATION OF MEMBERS OF MULTIGENE FAMILIES FROM LAMBDA DNA LIBRARIES. C. T. Lutz, W. C. Hollifield, B. Seed, J. M. Davie, and H. V. Huang; Washington Univ. Schoolof Med., St. Louis, MO The Syrinx 2A phage and 7rAN13 plasmid were designed for screening of DNA libraries by in vivo recombination, a technique that can distinguish between closely related genes. We have developed strategies to screen one or multiple libraries, isolate and expand phage-plasmid cointegrates, and obtain plasmid subclones, all in as little as 2 days. Syrinx 2A possesses multiple cloning sites (BamHI, EcoRI, Sall, SstI, and XbaI) and carries a newly described A gene, rap (recombination adept with plasmid), which increases homologous recombination 100-1000fold but which has been deleted from many A cloning vectors (the nin5 deletion). 7rAN13 is an 895 bp plasmid with multiple cloning sites and a supF gene. Homologous recombination between Syrinx 2A library phage and 7rAN13 probe plasmid produces phage-plasmid cointegrates which are isolated by selection for the supF gene. Using mouse K immunoglobulin genes as a model system, we tested how efficiently and specifically Syrinx 2A libraries could be screened by recombination. When we screened an aliquot of a hybridoma DNA library with a 3'Jk probe, six phage-plasmid cointegrates appeared, five of which hybridized with a specific 5'Vk probe and therefore contained the productively rearranged Vk/Jk gene segment. The five desired clones were easily distinguished from one aberrant cointegrate because homologous recombination produces a characteristic restriction endonuclease pattern. We screened aliquots of the same library with two 5'VK probes, the one derived from the Vk/Jk gene segment rearranged in the hybridoma (100% homologous) and the other derived from a closely related Vk gene segment (ca. 90% homologous). Both probe plasmids cointegrated with phage containing the rearranged Vk/Jk gene segment, but the 100% homologous probe did so 38 times more frequently than did the 90% homologous probe. Thus, recombination screening is highly selective and may be useful to rapidly isolate specific HLA genes or other genes which are members of a closely related multigene family. ABSTRACT SESSION III: SEROLOGY, MONOCLONAL ANTIBODIES, A N D CLASSICAL GENETICS POLYMORPHIC CLASSII MHC ANTIGENS IN MACAQUES DETECTED BY MOUSE ANTIHLA MONOCLONAL ANTIBODIES. Lakshmi K. Gaur, Brandt Schneider, Mary Valentine, Karen A. Nelson, John Pesando, Peter Parham, John A. Hansen, and Edward A. Clark; Regional Primate Research Center, University of Washington, Seattle, WA A panel of 38 mouse anti-HLA class II specific monoclonal antibodies (MoAbs) were tested for reactivity against an outbred population of pigtailed macaques (Macaca nemestrina) using a cellular ELISA assay. The antibodies detected in humans were either (a) monomorphic class II epitopes (4 Class II, 5 DR monomorphic) or (b) polymorphic epitopes (29 antibodies to private or broadly distributed specificities of DR, DQ, and/or DP). All 38 MoAbs tested reacted with some macaques with phenotypic frequencies (PF) ranging from 0.08 to 0.87.
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the 790 bp Sac I-Hind III fragment of the DR/~ cDNA probe. Clones thus identified were digested with a variety of restriction enzymes both individually and in combination, electrophoresed on a 1% agarose gel, Southern blotted, and then hybridized using the DR3 and DQ3 cDNA probes. A Pst 1 600 bp fragment which was identified as hybridizing to the DQ/3 probe but not the DR/3 probe was subcloned into the plasmid vector pUC 118, sequenced, and used to probe Southern blots of Taq I digested genomic DNA. Of 40 individuals thus far examined, 20 of which were HLA serotyped as DQw2, five persons possessing the DQw2 phenotype were observed to have fragments which hybridized to this probe (odds ratio = 6.7). These data suggest that there may be subsets of DQw2 or homologous sequences in linkage disequilibrium with the DQ locus.
DNA SEQUENCE POLYMORPHISM OF DR/3 CHAINS IN HLA-DR4, -7, AND -9 HAPLOTYPES. P.K Gregersen, T. Moriuchi, R. Karr, F. Obata, J. Maccari, D. Goldberg, R. Winchester, and J. Silver; Hospitalfor Joint Diseases, New York, NY We have isolated and sequenced cDNA clones corresponding to the DR/31 and DR32 loci from two homozygous B-cell lines typed as DR7 (Burkhart) and DR9 (ISK). These nucleotide sequences were compared to DR/31 and DRfl2 chains of other DR haplotypes. The first domain sequences of DR/32 chains are identical in DR4 and DR7 haplotypes. In addition, there is strong sequence homology within the 3' untranslated regions of DR/31 genes from DR4, - 7 , and - 9 haplotypes, thus establishing the close evolutionary relationship among these three haplotypes. In contrast, the first domain sequences of DR31 molecules from these haplotypes do not reflect the DR4, - 7 , - 9 family relationship; the DR4, - 7, and - 9 DR/31 sequences differ as much from each other as they do from the DR31 of any unrelated DR type. The differences in degree of diversity between DR/~I and DR/32 chains may be due to selection pressures; this implies functional differences for products of the DR/31 and DR/32 loci. Alternatively, we suggest that closely linked seqments of the human class II region may differ in their underlying rates of variation, independent of selection pressures, and that this may in part account for the extraordinary diversity found in the/31 first domain. TRANSFECTION AND EXPRESSION OF HUMAN CLASS II cDNA IN HUMAN EBV TRANSFORMED CELLS. K.G. Becker, S.H. Hsu, and W.B. Bias; The Johns Hopkins University School of Medicine, Baltimore, MD Functional studies of the products of human MHC genes are now possible through the manipulation of those genes. For such studies we have transfected a class I MHC gene into normal human T cells (Becker KG et al.,J. Immunol, manuscript submitted) and as described here class II cDNA into a human EBV transformed homozygous typing cell line (EF A 1,B8,Bw6,DR3,DQ2). Class II cDNA (DR31, DQ31, DP3, DO3), recently isolated and characterized (Tonnelle C et al., EMBO J. 4(11)) were modified by the ligation of the neomycin gene into the CLA I site found on the Okayama-Berg expression vectors carrying these DNA sequences, thus allowing dominant selection of transfected cells by Geneticin (G418). These plasmids were transfected into the transformed cell line by liposomal fusion. Stable lines bearing the gene products of the introduced genes were selected by G418 resistance. Preliminary analysis by cytofluorometry documents the stable expression of DQ1, while expression of other class II specificities will be deter-
132
Abstracts mined when adequate cell density is achieved. These cell lines will be used for in vitro stimulation studies, specific screening and absorbtion of class II sera, and for antigen structural studies.
RAPID AND SPECIFIC ISOLATION OF MEMBERS OF MULTIGENE FAMILIES FROM LAMBDA DNA LIBRARIES. C. T. Lutz, W. C. Hollifield, B. Seed, J. M. Davie, and H. V. Huang; Washington Univ. Schoolof Med., St. Louis, MO The Syrinx 2A phage and 7rAN13 plasmid were designed for screening of DNA libraries by in vivo recombination, a technique that can distinguish between closely related genes. We have developed strategies to screen one or multiple libraries, isolate and expand phage-plasmid cointegrates, and obtain plasmid subclones, all in as little as 2 days. Syrinx 2A possesses multiple cloning sites (BamHI, EcoRI, Sall, SstI, and XbaI) and carries a newly described A gene, rap (recombination adept with plasmid), which increases homologous recombination 100-1000fold but which has been deleted from many A cloning vectors (the nin5 deletion). 7rAN13 is an 895 bp plasmid with multiple cloning sites and a supF gene. Homologous recombination between Syrinx 2A library phage and 7rAN13 probe plasmid produces phage-plasmid cointegrates which are isolated by selection for the supF gene. Using mouse K immunoglobulin genes as a model system, we tested how efficiently and specifically Syrinx 2A libraries could be screened by recombination. When we screened an aliquot of a hybridoma DNA library with a 3'Jk probe, six phage-plasmid cointegrates appeared, five of which hybridized with a specific 5'Vk probe and therefore contained the productively rearranged Vk/Jk gene segment. The five desired clones were easily distinguished from one aberrant cointegrate because homologous recombination produces a characteristic restriction endonuclease pattern. We screened aliquots of the same library with two 5'VK probes, the one derived from the Vk/Jk gene segment rearranged in the hybridoma (100% homologous) and the other derived from a closely related Vk gene segment (ca. 90% homologous). Both probe plasmids cointegrated with phage containing the rearranged Vk/Jk gene segment, but the 100% homologous probe did so 38 times more frequently than did the 90% homologous probe. Thus, recombination screening is highly selective and may be useful to rapidly isolate specific HLA genes or other genes which are members of a closely related multigene family. ABSTRACT SESSION III: SEROLOGY, MONOCLONAL ANTIBODIES, A N D CLASSICAL GENETICS POLYMORPHIC CLASSII MHC ANTIGENS IN MACAQUES DETECTED BY MOUSE ANTIHLA MONOCLONAL ANTIBODIES. Lakshmi K. Gaur, Brandt Schneider, Mary Valentine, Karen A. Nelson, John Pesando, Peter Parham, John A. Hansen, and Edward A. Clark; Regional Primate Research Center, University of Washington, Seattle, WA A panel of 38 mouse anti-HLA class II specific monoclonal antibodies (MoAbs) were tested for reactivity against an outbred population of pigtailed macaques (Macaca nemestrina) using a cellular ELISA assay. The antibodies detected in humans were either (a) monomorphic class II epitopes (4 Class II, 5 DR monomorphic) or (b) polymorphic epitopes (29 antibodies to private or broadly distributed specificities of DR, DQ, and/or DP). All 38 MoAbs tested reacted with some macaques with phenotypic frequencies (PF) ranging from 0.08 to 0.87.