RESUMEN
Certain precursor proteins (APP751 and APP770) of the amyloid beta-protein (AP) present in Alzheimer's disease contain a Kunitz-type serine protease inhibitor domain (APPI). In this study, the domain is obtained as a functional inhibitor through both recombinant (APPIr) and synthetic (APPIs) methodologies, and the solution structure of APPI is determined by 1H 2D NMR techniques. Complete sequence-specific resonance assignments (except for P13 and G37 NH) for both APPIr and APPIs are achieved using standard procedures. Ambiguities arising from degeneracies in the NMR resonances are resolved by varying sample conditions. Qualitative interpretation of short- and long-range NOEs reveals secondary structural features similar to those extensively documented by NMR for bovine pancreatic trypsin inhibitor (BPTI). A more rigorous interpretation of the NOESY spectra yields NOE-derived interresidue distance restraints which are used in conjunction with dynamic simulated annealing to generate a family of APPI structures. Within this family, the beta-sheet and helical regions are in good agreement with the crystal structure of BPTI, whereas portions of the protease-binding loops deviate from those in BPTI. These deviations are consistent with those recently described in the crystal structure of APPI (Hynes et al., 1990). Also supported in the NMR study is the hydrophobic patch in the protease-binding domain created by side chain-side chain NOE contacts between M17 and F34. In addition, the NMR spectra indicate that the rotation of the W21 ring in APPI is hindered, unlike Y21 in BPTI, showing a greater than 90% preference for one orientation in the hydrophobic groove.
Asunto(s)
Precursor de Proteína beta-Amiloide/química , Aprotinina/genética , Inhibidores de Serina Proteinasa/genética , Secuencia de Aminoácidos , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
A rapid sensitive method for the quantification of in vitro HIV-protease activity has been developed on the basis of the endoproteolytic conversion of N-Dns-SQ-NYPIV to N-Dns-SQNY. The use of the N-dansyl group as a fluorescence label was shown to not significantly alter the apparent kinetic parameters for the peptide-enzyme interaction. Using fluorescence detection, the dansylated product and unconverted substrate are detected in a single rapid (3 min) isocratic reverse-phase HPLC separation in quantities as low as 0.2 pmol. The method is highly reproducible and suited to a variety of applications including the analysis of large sample numbers and rigorous enzymological studies.
Asunto(s)
Cromatografía Líquida de Alta Presión , Endopeptidasas/análisis , Fluorometría , Productos del Gen pol/análisis , Proteínas de los Retroviridae/análisis , Secuencia de Aminoácidos , Compuestos de Dansilo , Colorantes Fluorescentes , Proteasa del VIH , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/análisis , Especificidad por SustratoRESUMEN
Flow cytometry was used to monitor chitin synthesis in regenerating protoplasts of the yeast Candida albicans. Comparisons of cells stained with Calcofluor White, a fluorochrome with known affinity for chitin, and cells incubated in the presence of N-[3H]-acetylglucosamine, the precursor substrate for chitin, showed a linear relationship between fluorescence and incorporation of label over time. Changes in both the fluorescence and light scatter of regenerating protoplasts treated with inhibitors of fungal chitin synthase were also quantitated by flow cytometry.
Asunto(s)
Aminoglicósidos , Candida albicans/fisiología , Quitina/biosíntesis , Protoplastos/fisiología , Antibacterianos/farmacología , Antifúngicos/farmacología , Bencenosulfonatos , Candida albicans/efectos de los fármacos , Citometría de Flujo , Colorantes Fluorescentes , Protoplastos/efectos de los fármacosRESUMEN
The major human rhinovirus receptor has been identified with monoclonal antibodies that inhibit rhinovirus infection. These monoclonal antibodies recognize a 95 kd cell surface glycoprotein on human cells and on mouse transfectants expressing a rhinovirus binding phenotype. Purified 95 kd protein binds to rhinovirus in vitro. Protein sequence from the 95 kd protein showed an identity with that of intercellular adhesion molecule-1 (ICAM-1); a cDNA clone obtained from mouse transfectants expressing the rhinovirus receptor had essentially the same sequence as ICAM-1. Thus, the major human rhinovirus receptor is ICAM-1. The gene for this receptor maps to human chromosome 19, which also contains the genes for a number of other picornavirus receptors.
Asunto(s)
Antígenos de Superficie/metabolismo , Cromosomas Humanos Par 19 , Receptores Virales/metabolismo , Rhinovirus/metabolismo , Alcaloides/farmacología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/genética , Moléculas de Adhesión Celular , Humanos , Glicoproteínas de Membrana/biosíntesis , Datos de Secuencia Molecular , Peso Molecular , Procesamiento Proteico-Postraduccional , Receptores Virales/genética , Swainsonina , Transfección , Tunicamicina/farmacologíaRESUMEN
The recent cloning of complete cDNAs encoding carcinoembryonic antigen (CEA) and non-specific cross-reacting antigen has revealed the existence of a new gene family belonging to the immunoglobulin gene superfamily. We have reported the isolation of a partial CEA cDNA and of L-cell transfectant cell lines that express human antigens cross-reactive with commercial antibodies directed to native CEA (Kamarck, M., J. Elting, J. Hart, S. Goebel, P. M. M. Rae, J. Nedwin, and T. Barnett. 1987. Proc. Natl. Acad. Sci. USA. 84:5350-5354). In this study, we describe the identification and cloning of 3.9-, 3.7-, 2.2-, and 1.8-kb cDNAs and a 23-kb genomic transcription unit, which code for new members of the CEA gene family. DNA sequence analysis of these cloned DNAs establishes the existence of a set of four alternatively spliced mRNAs which are expressed in several tumor cell lines, in human fetal liver, and in L-cell transfectants. Deduced amino acid sequences of the encoded isoantigens show extensive similarity to CEA and nonspecific cross-reacting antigens, but in addition demonstrate transmembrane and cytoplasmic domains. We designate members of this antigen family transmembrane CEAs. The transmembrane CEA isoantigens share general structural characteristics with members of the immunoglobulin gene superfamily and can be specifically compared to the cell adhesion molecules, N-CAM (neural cell adhesion molecule) and MAG (myelin-associated glycoprotein).
Asunto(s)
Antígeno Carcinoembrionario/genética , Empalme del ARN , ARN Mensajero/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Western Blotting , Clonación Molecular , ADN/aislamiento & purificación , Enzimas de Restricción del ADN , Exones , Regulación de la Expresión Génica , Humanos , Intrones , Isoantígenos/análisis , Isoantígenos/genética , Células L , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Plásmidos , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
Genomic DNA and mRNA from the adenocarcinoma cell line LoVo were used to generate L-cell transfectants and a bacteriophage lambda gt11 cDNA clone that express epitopes of carcinoembryonic antigen (CEA). Primary and secondary L-cell transfectants expressing CEA were selected with a fluorescence-activated cell sorter (FACS). These transfectants, including some clones that were selected for high-level CEA expression by multiple rounds of FACS sorting, express a surface protein of 150 kDa that reacts with all anti-CEA antibodies tested. In parallel, a cDNA library of LoVo poly(A)+ RNA was constructed in lambda gt11 and fusion proteins were screened with polyclonal antisera against CEA. One positive clone, lambda cLV7, was identified that hybridized specifically to transfectant DNA. The nucleic acid sequence of the cDNA insert (cLV7) contained two regions of extensive internal homology, with greater than 70% identity at the amino acid level. cLV7 hybridized to three mRNA species of LoVo cells and to a predominant mRNA of the CEA-expressing transfectants. Hybridization of cLV7 to restriction endonuclease-digested genomic DNA of colon carcinoma cells, normal human cells, and human-mouse somatic cell hybrids revealed the presence of multiple hybridizing bands, one of which was present in transfectant cells. These CEA-related sequences are not rearranged in tumors and, by somatic cell hybrid analysis, were mapped to human chromosome 19.
Asunto(s)
Bacteriófago lambda/genética , Antígeno Carcinoembrionario/genética , ADN/análisis , Regulación de la Expresión Génica , Transfección , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , RatonesRESUMEN
We describe the first isolation of a human creatine kinase M cDNA clone and its mapping of the gene to human chromosome 19. A human creatine kinase M cDNA clone, pJN2CK-M, harboring a 1,160-bp insert, was isolated by colony hybridization with a previously sequenced chicken creatine kinase M cDNA probe. The human cDNA was used as a probe in Southern transfers of TaqI-digested genomic DNA from mouse/human somatic-cell hybrids to localize the human creatine kinase-M gene to chromosome 19. In situ hybridization of the tritiated cDNA probe to metaphase chromosomes of peripheral blood lymphocytes from normal males revealed significant labeling to chromosome 19. These two independent methodologies assign the human creatine kinase-M gene to chromosome 19. Since greater than 69% of the grains of chromosome 19 label band q13, the human creatine kinase-M gene has been mapped to 19q13. On the basis of high-resolution G-banding, the predominant labeling site was 19q13.2-q13.3.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 19 , Creatina Quinasa/genética , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , Enzimas de Restricción del ADN , Humanos , Células Híbridas , Isoenzimas , Ratones , Hibridación de Ácido Nucleico , ConejosRESUMEN
Mouse cells containing human surface antigen genes introduced by cell hybridization or DNA transfection can be labeled by indirect immunofluorescence and isolated using the FACS. As illustrated in this chapter, this methodology facilitates genetic studies of the human cell surface ranging from the initial chromosome mapping of a surface antigen gene to its isolation and cloning.
Asunto(s)
Línea Celular , Células Híbridas/citología , Transfección , Animales , Separación Celular/métodos , ADN/genética , Citometría de Flujo/métodos , Genes , Antígenos HLA/genética , Humanos , Células L/citología , Complejo Mayor de Histocompatibilidad , Ratones , PlásmidosRESUMEN
We have described a transfection method for the isolation of surface antigen genes which requires no mRNA or protein purification. Application of this technique results in the recovery of the entire gene in a single step since selection for expression of genomic DNA forms the basis of the procedure. Based on our results with the transferrin receptor gene and other systems, it is evident that large transcription units can be transferred and expressed in mouse L-cells. This size consideration represents a major advantage over the use of cosmid shuttle vectors for genomic DNA expression. In the case of genes which code for very long mRNAs this method may also have advantages over cDNA expression systems. Although we have described methods for FACS isolation of transfectants based on the binding of species specific antibodies to surface antigens, other methods of identifying transfected cells could be employed. For example, in combination with an appropriate assay, sib selection of recipient cells could be used to identify genes encoding secreted products. Ligand binding assays could be used for receptors which are not expressed on the host cell. Finally, the development of cDNA expression vectors which produce membrane-associated products would extend this methodology to genes not normally expressed at the cell surface.
Asunto(s)
Clonación Molecular/métodos , Genes , Receptores de Superficie Celular/genética , Animales , Antígenos de Superficie/genética , ADN/genética , ADN/aislamiento & purificación , Células L/metabolismo , Ratones , Plásmidos , Transformación GenéticaRESUMEN
LFA-3 is expressed on a wide variety of human cell lines, including those which have been used as recipients for gene transfer of human class I gene products, whereas a murine counterpart is either absent or significantly different such that the anti-LFA-3 monoclonal antibody (MAb) does not bind. By using a somatic cell genetic approach, we demonstrate that LFA-3 is not a major histocompatibility complex-encoded molecule, and that its gene locus maps to human chromosome 1. When LFA-3 and HLA-A2 are coexpressed on the mouse cell surface, anti-LFA-3 MAb interfered with specific recognition and lysis of these target cells by human CTL capable of lysing HLA-A2-expressing mouse transfectants. A significant contribution of the LFA-3 molecule to CTL reactivity was not observed, however, because the presence of LFA-3 did not restore recognition by CTL clones previously found incapable of lysing HLA-A2-expressing mouse transfectants, nor was it required by those human CTL that could lyse mouse cell transfectants. Thus, we have used genetic techniques to demonstrate that LFA-3 may serve a role in CTL-target cell interactions at the target cell level, but is not a molecule absolutely required for human allospecific CTL recognition of HLA antigens expressed on mouse cells. We suggest that LFA-3 may not participate directly in CTL function under normal circumstances, but delivers a more general inhibitory signal only when provoked by bound MAb.
Asunto(s)
Antígenos de Superficie/genética , Mapeo Cromosómico , Citotoxicidad Inmunológica , Células Híbridas/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos de Superficie/análisis , Antígenos de Superficie/inmunología , Aotus trivirgatus , Comunicación Celular , Línea Celular , Chlorocebus aethiops , Antígenos HLA/análisis , Antígenos HLA/genética , Antígeno HLA-A2 , Humanos , Antígeno-1 Asociado a Función de Linfocito , Ratones , Especificidad de la EspecieRESUMEN
Antisera raised against a human X mouse hybrid cell line containing human chromosome 21 as its only human chromosome, block induction of an antiviral state by human alpha interferon (IFN-alpha), block induction of (2'-5')oligoisoadenylate synthetase [2'-5')A synthetase), and block binding of 125I-labeled and 35S-labeled recombinant, human IFN-alpha A, but not 125I-labeled IFN-gamma, to cell surface receptors. The data presented clearly demonstrate that the cell surface receptors for IFN-alpha and IFN-gamma are different, and provide independent evidence of the role of a chromosome 21 coded cell surface molecule in the pathway to the generation of the antiviral state.
Asunto(s)
Cromosomas Humanos 21-22 e Y , Interferón Tipo I/metabolismo , Interferón gamma/metabolismo , Receptores Inmunológicos/metabolismo , 2',5'-Oligoadenilato Sintetasa/biosíntesis , Animales , Complejo Antígeno-Anticuerpo , Inducción Enzimática , Humanos , Células Híbridas/inmunología , Sueros Inmunes , Interferón Tipo I/farmacología , Ratones , Ratones Endogámicos C3H , Receptores de InterferónRESUMEN
The recent advances in human gene mapping have been largely due to the development of interspecies cell hybrids containing human chromosomes and their fragments. The importance of characterized panels of these hybrid lines has grown exponentially with the application of recombinant DNA technologies to human genetics. In this article, we discuss current strategies employed in the construction of somatic cell hybrid mapping panels.
Asunto(s)
Mapeo Cromosómico/métodos , Genes , Células Híbridas , Animales , Fusión Celular , Línea Celular , Transformación Celular Neoplásica , Computadores , Prueba de Complementación Genética , Marcadores Genéticos , Humanos , Hibridación de Ácido Nucleico , Oncogenes , FenotipoRESUMEN
We have used a mouse cell transformant generated by human chromosome-mediated gene transfer (CMGT) to explore the use of cell surface antigens in the identification of fragments of human chromosomes retained by somatic cell hybrids. The transformed line, 21-30b, contained an intact rear-ranged human chromosome, and could be shown by isozyme analysis to contain genetic material from chromosomes 9 and X. By using the transformant as an immunogen in mice, it was also possible to produce antiserum to human-specific surface antigens. Using genetically characterized human X rodent hybrid lines, the genes controlling expression of these antigens could be localized to 11per----11p13, segregating concordantly with surface antigen S3. These conclusions were possible despite the fact that the presence of chromosome 11 in the transformant was not detectable by the presence of chromosome specific isozyme LDH-A or surface antigens W6/34 and 4F2. Finally, the fluorescence-activated cell sorter (FACS) was used to fractionate the transformant cells into antigen positive and negative subpopulations. This resulted in the isolation and characterization of four additional chromosome rearrangements involving interspecies chromosome translocations. This work demonstrates the value of chromosome-specific surface antigens and the FACS in the evaluation of human chromosome fragments retained by interspecies hybrids.
Asunto(s)
Antígenos de Superficie/genética , Mapeo Cromosómico/métodos , Células Híbridas/fisiología , Animales , Separación Celular , Citotoxicidad Inmunológica , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Células Híbridas/inmunología , Isoenzimas/genética , Cariotipificación , RatonesRESUMEN
The genes that code for the human lymphocyte cell surface determinants defined by monoclonal antibodies A- 1A5 and A- 3A4 have been genetically mapped. All human chromosomes, except Y, were included in a series of human less than mouse lymphocyte hybrid populations that retained expression of lymphocyte-specific surface markers. Expression of the A- 1A5 and A- 3A4 antigens was quantitated by indirect immunofluorescence and fluorescence-activated cell sorter (FACS) analysis. Hybrid populations heterogeneous for antigen expression were sorted to yield antigenically homogeneous subpopulations. Isozyme analysis indicated concordant segregation of the A- 1A5 determinant with chromosome 10, and the A- 3A4 determinant with chromosome 4. In contrast to the unhybridized human parent cell line (MOLT-4), from which A- 1A5 immunoprecipitated two proteins (160,000 and 125,000 Mr), A- 1A5 only immunoprecipitated a single band (125,000 Mr) from an A- 1A5 -expressing human less than mouse hybrid. The genetic disassociation of these two proteins from the A- 1A5 -reactive complex suggests that the appearance of the 160,000 Mr protein requires a gene locus that is unlinked to the locus for the 125,000 Mr protein on chromosome 10. A third component of the A- 1A5 -reactive protein complex (210,000 Mr), which is recognized by the monoclonal antibody TS2/7, was not expressed on the parent MOLT-4 cells, but was weakly expressed on MOLT-4 less than mouse BW5147 hybrids. This allowed preliminary mapping of that determinant to either chromosome 10 or 15. The A- 3A4 antigen (approximately 45,000 Mr) is a novel cell surface structure expressed on all hematopoietic cell lines tested, and represents the first cell surface marker mapped to chromosome 4.
Asunto(s)
Antígenos de Superficie/genética , Epítopos/genética , Células Madre Hematopoyéticas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/clasificación , Antígenos de Superficie/inmunología , Línea Celular , Mapeo Cromosómico , Cromosomas Humanos 4-5 , Cromosomas Humanos 6-12 y X , Epítopos/inmunología , Marcadores Genéticos , Humanos , Cariotipificación , Ratones , Peso Molecular , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
The human S11 surface antigens are expressed on fibroblasts and are coded by a gene on the X-chromosome. We have regionally mapped this gene by examining S11 expression on a panel of hybrid lines which had fragmented the X-chromosome either during chromosome-mediated gene transfer, or by interspecies translocation during hybrid cell expansion. using indirect immunofluorescence and the fluorescence-activated cell sorter (FACS), it was possible to isolate antigen-positive and -negative hybrid subpopulations for subsequent genetic analysis. The gene coding S11 could be localized to Xq27-28, between the loci for HPRT and G6PD where genes for the S10 and S12 antigens have been previously mapped. This work demonstrates the value of cell surface antigens and the FACS in somatic cell genetic analysis, and provides evidence for regional clustering of surface antigen loci on the human X-chromosome.
Asunto(s)
Antígenos de Superficie/genética , Genes , Cromosoma X , Animales , Mapeo Cromosómico , Femenino , Citometría de Flujo , Glucosafosfato Deshidrogenasa/genética , Humanos , Células Híbridas , Hipoxantina Fosforribosiltransferasa/genética , Cariotipificación , Ratones , Translocación GenéticaRESUMEN
We describe an approach to the cloning of cell surface proteins that is independent of messenger RNA isolation. Mouse Ltk- cells are cotransformed with the thymidine kinase gene from Herpes Simplex Virus and total human DNA. Transformants expressing the human surface antigens of interest are isolated by two selection steps, consisting of treatment with hypoxanthine/aminopterin/thymidine and fluorescence-activated cell sorting. Using this procedure, we isolated seven transformants expressing HLA-A,B,C antigens and 12 transformants expressing the 4F2 antigen. We have so far failed to identify any OKT-10 antigen expressing L-cell transformants. Three independent secondary 4F2 transformants were obtained after identical cotransformation of fresh Ltk- cells with DNA from primary transformants. Analysis of their genome by hybridization with human DNA revealed a shared set of human restriction fragments in all three cell lines. This 32 X 10(3) base-pair segment of DNA codes for the human 4F2 antigen, thereby offering the opportunity to clone the gene. To substantiate this hypothesis, we analyzed the seven HLA-expressing cell lines, and we found that all of them had acquired an HLA-coding sequence concomitant to its expression.
Asunto(s)
Antígenos de Superficie/genética , Antígenos HLA/genética , Animales , Separación Celular/métodos , Clonación Molecular , Citometría de Flujo , Regulación de la Expresión Génica , Ligamiento Genético , Cobayas , Humanos , Células L , Transformación GenéticaRESUMEN
Human genes coding cell surface molecules can be introduced into mouse host cells using a variety of somatic cell genetic techniques. Because these human gene products can be detected using indirect immunofluorescence on viable cells, the genes themselves can be monitored and manipulated using flow cytometry and sorting. In this paper, we review ways that we have used cell sorting to develop a somatic cell genetic analysis of the human cell surface.
Asunto(s)
Antígenos de Superficie/genética , Separación Celular , Citometría de Flujo , Genes , Técnicas Genéticas , Animales , Fusión Celular , Clonación Molecular , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Antígenos HLA/genética , Humanos , Células Híbridas , Interferón Tipo I/farmacología , Ratones , Transfección , Microglobulina beta-2/genéticaRESUMEN
Genetic mapping of differentiation specific surface antigens has been hampered by difficulty in preparation of interspecies hybrid cells which continue to express differentiated functions. A method has been developed for production of interspecies T cell hybrids which continue to express T cell specific cell surface molecules. Hybrids were constructed from either the human leukemic cell line MOLT-4 or freshly isolated human peripheral blood T cells and the mouse T lymphoma line BW5147. Optimal fusion efficiency resulted with pre-treatment of the human parental line with phytohemagglutinin followed by hybridization with 40% polyethylene glycol and plating without thymocyte feeder layers. Immortalization of hybrid lines was accomplished through addition of rat T cell growth factor to cultures.
Asunto(s)
Antígenos de Superficie/genética , Células Híbridas/inmunología , Leucemia Experimental/inmunología , Leucemia Linfoide/inmunología , Linfocitos T/inmunología , Animales , Diferenciación Celular , Fusión Celular , Línea Celular , Técnicas de Cultivo/métodos , Técnica del Anticuerpo Fluorescente , Humanos , Isoenzimas/genética , Ratones , Ratones Endogámicos AKRRESUMEN
We have mapped the gene which codes the species-specific determinant defined by monoclonal antibody 4F2 to human chromosome 11. All human chromosomes, except Y, were included in a group of four human-mouse hybrid lines. Hybrids heterogeneous for 4F2 antigen expression were sorted using the fluorescence-activated cell sorter (FACS) to yield populations homogeneous with respect to the presence or absence of this determinant. Isozyme analysis indicated corresponding genetic selection for or against human chromosome 11. This map assignment was confirmed using a hybrid line which contained only human chromosome 11. Immunoprecipitation of the 4F2 determinant from the 11 only hybrid resulted in a heavy subunit of molecular weight (Mr) = 100,000 and a light subunit of Mr = 41,000. This contrasts with results obtained from nonhybrid human cells of different lineages. These results demonstrate the importance of FACS techniques in the rapid mapping of genes which code human cell surface antigens.
Asunto(s)
Antígenos de Superficie/genética , Animales , Mapeo Cromosómico , Epítopos , Humanos , Células Híbridas/inmunología , RatonesRESUMEN
We have screened a large number of isolated human genomic clones that hybridize to a cloned HLA cDNA probe for their ability to direct the synthesis of HLA-A, -B, and -C surface antigens on mouse L cells following DNA-mediated gene transfer. The surface expression of human histocompatibility antigens, monitored by indirect immunofluorescence and the fluorescence-activated cell sorter, was examined at 60 hr after transfection and on hypoxanthine/aminopterin/thymidine-resistant (HATR) populations derived from cotransfer with the herpes simplex virus thymidine kinase gene. Two unique genomic clones designated JY B3.2 and JY 158, isolated from the human lymphoblastoid cell line JY (homozygous HLA-A2, -B7), were shown to contain gene sequences capable of directing expression of an HLA-A, -B, -C determinant. By using allo-specific antibodies, the gene products of these clones were identified as HLA-A2 and HLA-B7, respectively. HATR clonal populations isolated from cotransfections with these genomic clones displayed varying levels of surface HLA expression that correlated with the number of intact donor HLA sequences present in the cells. In general, these levels of expression were stable during 3 months in culture. This system provides a powerful tool for the study of human surface antigen gene structure, expression, and function on a mouse cell background.