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Ontogeny and role of the MHC in mouse embryonic development
Abstracts
129
P703
P704
CHARACTERIZATION OF HLA-DRBI*040I MOLECULES EXPRESSED IN DROSOPHILA SCHNEIDER CELLS
ANALYSIS OF THE MHC CLASS I IN JAPANESE QUAIL
Hansen Bjarke, Andersson Christina, Madsen Lars, Sendergaard Leif, Fugger Lars, Svejgaard Arne. Dept. Clinical Immunology, The State University Hospital, Copenhagen, Denmark.
T.Shiina', A.Ando', H.Kawala', K.Hanzawa', SWalanabe', H.lnoko' Mol. Life. Sei., Tokai Univ. SOO. Meel., lsahara, Kanagawa, 'Dept. Zootech. Sei., Tokyo Unlv. Agri., Selagayaku, Tokyo, Japan
We have expressed the human MHC class II molecule, HLADRB 1*040 I, in Drosophila melanogaster Schneider cells under the control of a metallothionein promoter. Upon induction, DRBI*0401 molecules was expressed on the surface of the Schneider cells at high levels, where they could present peptides to DRB 1*0401 restricted T cells, indicating correct assembly into functional aI~ heterodimers. SDS-PAGE characterization of affinity purified DRB 1*0401 from Schneider cells molecules revealed that the ex and ~ chains had faster mobility than DRBI*0401 purified from a human cell line (Priess), but trimming of N-linked glycosylations resulted in bands with identical mobility. Affinity purified recombinant DRBI*0401 molecules were unstable in a SDS-PAGE, but could be stabilized with a peptide known to bind DRBI*0401, indicating that the recombinant molecules were devoid of endogenous peptides. This assumption was further substantiated by the fact that the recombinant class II molecules had a much higher peptide binding capacity than Priess DRB 1*0401 molecules. The purified recombinant DRB1*0401 molecules also showed biological activity because preformed DRB I *0401: peptide complexes immobilized on EUSA plates specifically stimulated DRB 1*0401 restricted T cells.
Seventeen cosmid clones corresponding to the MHC class I genes In the Japanese quail (Cotumix jsponica) were Isolated by screening a cosmld libraly with the Japanese qUail class I cDNA (QF41) as a probe. The Japanese quail was found to contain at least twelVe MHC class I loci (Ll-12) Within 170kb of two clusters (cluster 1; 9Okb, cluster 2; BOIcb), based on the contlg map which was drawn by the Southern hybridization analysis using QF41 as a probe and by cosmid mapping. Southern hybridization analysis using the chicken class II cDNA (B-L P) as a probe didn' reveal any positive band, suggesting that the Japanese quail etass I region is physically separated from the class II region and ~s genomic organization is dmerent from that of the chicken class I and class II regions. Investigation of the PeR amplified products and their nucleotide sequences using each DNA fragment as a template With the QF41, QF63, QF103 and OF108 loci speclllc primers suggested the presence of one locus (l1) for the 0F41 lsotype, one locus (L.6) lor the OF63 isotype, live loci (L2, 3, 8, 10, 12) for the OF103 like isotype, tour loci (L4, 5, 9, 11) for the OF108 like isotype. But no ampIWied product was produced from the L7 loous w~h any of QF41, OF63, OF103 or OF108 speclfic primers. The nucleotide sequence of the DNA tregment (3735 bp) containing the L7 locus was composed of the promoter region and all of the coding sequences, the GT/AG rule was conSSlVed, and the homology with each of the OF clone sequence was found to be only 88-89%. Taken together, these reSlAts suggest that the Japanese quail has at least five distinct expreased MHC class I loci.
P705 AN INTEGRATED PHYSICAL, GENETIC AND GENIC MAP OF THE
P706
CHICKEN MHC GENES IN THE BAND Rfp-Y COMPLEXES Zoorob Rimal, Forget Sebastienl, Billault AIain 2, Faille Annlckl, S~v~rac V~roniquel, Auffray Charlesl, ! Gen~tique MoJeculaire et Biologie de Developpement, CNRS UPR 420, Villejuif, France Fondatinn lean Dausset-CEPH, Paris, France The chicken major histocompatibility complex genes are located nn two complexes (B and Rfp-Y) which assort independently although theyare on the same microchromosome. In order to clarify which genes in these complexes are involved in the control of autoimmune diseases such as thyroiditis and the resistance to the development nf tumors induced by viruses such as the Rous sarcoma retrovirus and the Marek lymphome herpes virus, we have generated a first generation molecular map of these complexes using cosmid vectors. Systematic sequencing has revealed the presence of many novel genes closely associated with the class I (B-F) and class II (B-F) genes, including those encoding chicken homologs of C4, 21hydroxylase, TAP, butyrophilin, Ret finger protein and BAT-2. A second generation molecular map has been generated using collection of large DNA fragments cloned in PAC and BAC vectors, and microsatellite identified in the cloned regions to serve as genetic markers for genolyping. The polymorphism and expression patterns of these genes is under investigation in relation with the biological parameters controlled by the B and Rfp-Vcomplexes.
P707
ARCHAEOLOGICAL GENETICS: HLA-DQAl MEDIEVAL HUMAN RDlAINS IN RUSSIA
GENOTYPING
FROM
OvtchilIDikova Olga, Druzina Ekaterina, Buzhllova Alexandra, Makarov Nikolay, OVch1nnikov Igor, Genetic Identification Center, Institute of Archaeology, Moscow, Russia HLA-DQA1 gene polymorphism was typed from 25 human remains dated to XI-XIV centuries Trom the isolated Slavic necropolises of Russian North. The ancient DNA was isolated by phenol/chlorofonn method with isopropanol precipitation and the silica method. Polymorphic region of exon 2 of HLA-DQAl gene was amplified with primer !lairs GH26 and GH27, GH26 and GHB4, GH64 and GH84 (242, 166, 82 bp frapents). PCR of 242 bp frapent was carried ouf successfully in 32::1; specimens. In other samples pattern of alleles DQAl*0101, *0201, *0301, *0401 defined by sequences between codons 47 and 56 was amplified wi t1l primers GH64 and GHa4 and pattern of alleles DQAhOl 01 , *0102, *0103 based on sequence polymorphism between codons 30 and 42 was ~lified with primers GH26 and GHa4. Different alleles of HLA-DQAl gene were detected by restriction digestion and SSCP analysis, Seven genotypes were observed and the frequency of each allele was calculated. HLA anthropological markers (-DQ, -B) will be used for detection of outside gene flow and pathological condition.
REGION 'Dept. Japan;
ONTOGENY AND ROLE OF THE MHC IN MOUSE EMBRYONIC DEVELOPMENT CooperJoanne CLaire, Fernandez Nelson,DealtryGillian Departmentof Biologicaland Chemical Sciences,Central Campus,Universityof Essex, WivenhoePark, Colchester,C04 3SQ, England The embryos of both human and the mouse follow similar patterns during the early stagesof embryogenesis. Humanpre-implantation embryonic tissueis difficultto obtain for both ethicaland practicalreasons. For thesereasonsthe mouse provides an accessible model for studyingthe ontogenyand role of immunological receptors in development. Regulationof the expressionof the MHCcomplexduring the development is said to be a crucial factor in the maternal toleranceof the foetalallograft and plays an important role in embryonicdevelopment I , Using a highly sensitive RT-peR system- combined with confocal scanning laser microscopic imrnunodetection we have studiedthe expressionof MHC class I, non-classical class I (Qa), p,-microgiobulill, and the transporters associated with antigen processing (TAP) in mouse pre-implantation embryos. MHC class I transcriptshave been reportedbeforethe first cleavagedivision' Here we report the early expression ~rmicroglobulin and other class I relatedmolecules and proposea developmental modelfor the role of these molecules in embryogenesis. I Fernandez N, et aI. (1995) Embryonicdevelopmentand the major histocompatibility complex. HumanReproductiveImmunology. BiosScientific Publications, Oxford.. a Coopor JC, et al. (1994) A highly sensitive RT-PCR system for analysing gene expressionin embryosand singlecell lines. Immunology. 83 (I): 5L 3 Sprink. MT, et al. (1993) Pre-implantationmouseembryosexpressMhc class I genes beforethe first cleavagedivision, Immunogenetics38: 35-40.
P708
HUMAN {J-globin DNA EXTRACTED
GENE FROM
POLYHORPHISHS CHARACTERIZED IN FOSSIL BONES 12",000 YEARS OLD
Eliane Beraud-Colomb&£, Regine Roubin$, josiane Marrin§, Nicolas Maroc£, ArmeIIe Gardeisen&, Guy Trabucher II and Michel Goosseass£ INSERM U406, Genetique Medicale et Developpement, Faculte de Medecine, 27 Boulevard Jean Moulin, BOOS Marseille . s INSERM U119, 13009 Marseille. § l.aboratoire de Genetique Mcleculaite 94010 Creteil & l.aboratoire d'Anthropologie,Marseille Cedex IS 11 Centre de Genetique Moleculaire et Cellulaire 69622 Villeurba.uie Cedex France. Analyzing the nudear DNA from humans fossil bones would be an essential step to the understanding of genetic diversity in current populations provldlng such systematic studies are experimentally feasible.We report the successful extraction and amplification of nuclear DNA from the p-gtobin region from five out of ten bone specimens up to 12,000 years old. These have been typed for p-globtn frameworks by sequencing through two variable postttons and for a polymorphic (AT)x(T)y microsatellite 500 hp upstream of the p-g!ohin gene, These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably .older than those used to study high copy-number human mitochondrial DNA. These resuits show that the H LA typing of such specimens should be possible.
129
P703
P704
CHARACTERIZATION OF HLA-DRBI*040I MOLECULES EXPRESSED IN DROSOPHILA SCHNEIDER CELLS
ANALYSIS OF THE MHC CLASS I IN JAPANESE QUAIL
Hansen Bjarke, Andersson Christina, Madsen Lars, Sendergaard Leif, Fugger Lars, Svejgaard Arne. Dept. Clinical Immunology, The State University Hospital, Copenhagen, Denmark.
T.Shiina', A.Ando', H.Kawala', K.Hanzawa', SWalanabe', H.lnoko' Mol. Life. Sei., Tokai Univ. SOO. Meel., lsahara, Kanagawa, 'Dept. Zootech. Sei., Tokyo Unlv. Agri., Selagayaku, Tokyo, Japan
We have expressed the human MHC class II molecule, HLADRB 1*040 I, in Drosophila melanogaster Schneider cells under the control of a metallothionein promoter. Upon induction, DRBI*0401 molecules was expressed on the surface of the Schneider cells at high levels, where they could present peptides to DRB 1*0401 restricted T cells, indicating correct assembly into functional aI~ heterodimers. SDS-PAGE characterization of affinity purified DRB 1*0401 from Schneider cells molecules revealed that the ex and ~ chains had faster mobility than DRBI*0401 purified from a human cell line (Priess), but trimming of N-linked glycosylations resulted in bands with identical mobility. Affinity purified recombinant DRBI*0401 molecules were unstable in a SDS-PAGE, but could be stabilized with a peptide known to bind DRBI*0401, indicating that the recombinant molecules were devoid of endogenous peptides. This assumption was further substantiated by the fact that the recombinant class II molecules had a much higher peptide binding capacity than Priess DRB 1*0401 molecules. The purified recombinant DRB1*0401 molecules also showed biological activity because preformed DRB I *0401: peptide complexes immobilized on EUSA plates specifically stimulated DRB 1*0401 restricted T cells.
Seventeen cosmid clones corresponding to the MHC class I genes In the Japanese quail (Cotumix jsponica) were Isolated by screening a cosmld libraly with the Japanese qUail class I cDNA (QF41) as a probe. The Japanese quail was found to contain at least twelVe MHC class I loci (Ll-12) Within 170kb of two clusters (cluster 1; 9Okb, cluster 2; BOIcb), based on the contlg map which was drawn by the Southern hybridization analysis using QF41 as a probe and by cosmid mapping. Southern hybridization analysis using the chicken class II cDNA (B-L P) as a probe didn' reveal any positive band, suggesting that the Japanese quail etass I region is physically separated from the class II region and ~s genomic organization is dmerent from that of the chicken class I and class II regions. Investigation of the PeR amplified products and their nucleotide sequences using each DNA fragment as a template With the QF41, QF63, QF103 and OF108 loci speclllc primers suggested the presence of one locus (l1) for the 0F41 lsotype, one locus (L.6) lor the OF63 isotype, live loci (L2, 3, 8, 10, 12) for the OF103 like isotype, tour loci (L4, 5, 9, 11) for the OF108 like isotype. But no ampIWied product was produced from the L7 loous w~h any of QF41, OF63, OF103 or OF108 speclfic primers. The nucleotide sequence of the DNA tregment (3735 bp) containing the L7 locus was composed of the promoter region and all of the coding sequences, the GT/AG rule was conSSlVed, and the homology with each of the OF clone sequence was found to be only 88-89%. Taken together, these reSlAts suggest that the Japanese quail has at least five distinct expreased MHC class I loci.
P705 AN INTEGRATED PHYSICAL, GENETIC AND GENIC MAP OF THE
P706
CHICKEN MHC GENES IN THE BAND Rfp-Y COMPLEXES Zoorob Rimal, Forget Sebastienl, Billault AIain 2, Faille Annlckl, S~v~rac V~roniquel, Auffray Charlesl, ! Gen~tique MoJeculaire et Biologie de Developpement, CNRS UPR 420, Villejuif, France Fondatinn lean Dausset-CEPH, Paris, France The chicken major histocompatibility complex genes are located nn two complexes (B and Rfp-Y) which assort independently although theyare on the same microchromosome. In order to clarify which genes in these complexes are involved in the control of autoimmune diseases such as thyroiditis and the resistance to the development nf tumors induced by viruses such as the Rous sarcoma retrovirus and the Marek lymphome herpes virus, we have generated a first generation molecular map of these complexes using cosmid vectors. Systematic sequencing has revealed the presence of many novel genes closely associated with the class I (B-F) and class II (B-F) genes, including those encoding chicken homologs of C4, 21hydroxylase, TAP, butyrophilin, Ret finger protein and BAT-2. A second generation molecular map has been generated using collection of large DNA fragments cloned in PAC and BAC vectors, and microsatellite identified in the cloned regions to serve as genetic markers for genolyping. The polymorphism and expression patterns of these genes is under investigation in relation with the biological parameters controlled by the B and Rfp-Vcomplexes.
P707
ARCHAEOLOGICAL GENETICS: HLA-DQAl MEDIEVAL HUMAN RDlAINS IN RUSSIA
GENOTYPING
FROM
OvtchilIDikova Olga, Druzina Ekaterina, Buzhllova Alexandra, Makarov Nikolay, OVch1nnikov Igor, Genetic Identification Center, Institute of Archaeology, Moscow, Russia HLA-DQA1 gene polymorphism was typed from 25 human remains dated to XI-XIV centuries Trom the isolated Slavic necropolises of Russian North. The ancient DNA was isolated by phenol/chlorofonn method with isopropanol precipitation and the silica method. Polymorphic region of exon 2 of HLA-DQAl gene was amplified with primer !lairs GH26 and GH27, GH26 and GHB4, GH64 and GH84 (242, 166, 82 bp frapents). PCR of 242 bp frapent was carried ouf successfully in 32::1; specimens. In other samples pattern of alleles DQAl*0101, *0201, *0301, *0401 defined by sequences between codons 47 and 56 was amplified wi t1l primers GH64 and GHa4 and pattern of alleles DQAhOl 01 , *0102, *0103 based on sequence polymorphism between codons 30 and 42 was ~lified with primers GH26 and GHa4. Different alleles of HLA-DQAl gene were detected by restriction digestion and SSCP analysis, Seven genotypes were observed and the frequency of each allele was calculated. HLA anthropological markers (-DQ, -B) will be used for detection of outside gene flow and pathological condition.
REGION 'Dept. Japan;
ONTOGENY AND ROLE OF THE MHC IN MOUSE EMBRYONIC DEVELOPMENT CooperJoanne CLaire, Fernandez Nelson,DealtryGillian Departmentof Biologicaland Chemical Sciences,Central Campus,Universityof Essex, WivenhoePark, Colchester,C04 3SQ, England The embryos of both human and the mouse follow similar patterns during the early stagesof embryogenesis. Humanpre-implantation embryonic tissueis difficultto obtain for both ethicaland practicalreasons. For thesereasonsthe mouse provides an accessible model for studyingthe ontogenyand role of immunological receptors in development. Regulationof the expressionof the MHCcomplexduring the development is said to be a crucial factor in the maternal toleranceof the foetalallograft and plays an important role in embryonicdevelopment I , Using a highly sensitive RT-peR system- combined with confocal scanning laser microscopic imrnunodetection we have studiedthe expressionof MHC class I, non-classical class I (Qa), p,-microgiobulill, and the transporters associated with antigen processing (TAP) in mouse pre-implantation embryos. MHC class I transcriptshave been reportedbeforethe first cleavagedivision' Here we report the early expression ~rmicroglobulin and other class I relatedmolecules and proposea developmental modelfor the role of these molecules in embryogenesis. I Fernandez N, et aI. (1995) Embryonicdevelopmentand the major histocompatibility complex. HumanReproductiveImmunology. BiosScientific Publications, Oxford.. a Coopor JC, et al. (1994) A highly sensitive RT-PCR system for analysing gene expressionin embryosand singlecell lines. Immunology. 83 (I): 5L 3 Sprink. MT, et al. (1993) Pre-implantationmouseembryosexpressMhc class I genes beforethe first cleavagedivision, Immunogenetics38: 35-40.
P708
HUMAN {J-globin DNA EXTRACTED
GENE FROM
POLYHORPHISHS CHARACTERIZED IN FOSSIL BONES 12",000 YEARS OLD
Eliane Beraud-Colomb&£, Regine Roubin$, josiane Marrin§, Nicolas Maroc£, ArmeIIe Gardeisen&, Guy Trabucher II and Michel Goosseass£ INSERM U406, Genetique Medicale et Developpement, Faculte de Medecine, 27 Boulevard Jean Moulin, BOOS Marseille . s INSERM U119, 13009 Marseille. § l.aboratoire de Genetique Mcleculaite 94010 Creteil & l.aboratoire d'Anthropologie,Marseille Cedex IS 11 Centre de Genetique Moleculaire et Cellulaire 69622 Villeurba.uie Cedex France. Analyzing the nudear DNA from humans fossil bones would be an essential step to the understanding of genetic diversity in current populations provldlng such systematic studies are experimentally feasible.We report the successful extraction and amplification of nuclear DNA from the p-gtobin region from five out of ten bone specimens up to 12,000 years old. These have been typed for p-globtn frameworks by sequencing through two variable postttons and for a polymorphic (AT)x(T)y microsatellite 500 hp upstream of the p-g!ohin gene, These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably .older than those used to study high copy-number human mitochondrial DNA. These resuits show that the H LA typing of such specimens should be possible.