Michael E. Kamarck

Genetic and biochemical characterization of a human surface determinant on somatic cell hybrids: the 4F2 antigen.

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 humanmouse 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.

Antibodies to chromosome 21 coded cell surface components block binding of human α interferon but not λ interferon to human cells

Abstract Antisera raised against a human × mouse hybrid cell line containing human chromosome 21 as its only human chromosome, block induction of an antiviral state by human α interferon (IFN-α), block induction of (2′-5′)oligoisoadenylate synthetase ((2′-5′)A synthetase), and block binding of 125 I-labeled and 35 S-labeled recombinant, human IFN-αA, but not 125 I-labeled IFN-ψ, to cell surface receptors. The data presented clearly demonstrate that the cell surface receptors for IFN-α and IFN-λ 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.

Somatic cell hybrid mapping panels

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.

Development of a protein A enzyme immunoassay for use in screening hybridomas.

A protein A enzyme immunoassay has been developed which is effective in the rapid screening of hybridomas. The detecting reagent is a stable alkaline phosphatase-protein A conjugate, prepared by a simple, inexpensive, single-step procedure. The assay requires small amounts of antigen to coat microtiter wells (less than 25 microgram/well) and can be evaluated visually or with a spectrophotometer. It compares favorably in sensitivity to a protein A-radioimmunoassay and enjoys considerably lower backgrounds than that popular screening procedure.

Somatic cell genetic analysis of HLA-A, B, C and humanβ2-microglobulin expression

We have examined the cell surface expression of the human histocompatibility antigens HLA-A, B, C and β2-microglobulin (β2m) on a human-mouse somatic cell hybrid line. Using specific antibodies and the fluorescence-activated cell sorter (FACS), we viably fractionated and characterized four separate hybrid subpopulations (HLA+,β2m+; HLA +,β2m−; HLA−,β2m+; HLA−,β2m−). Hybrid selection based on surface antigen expression resulted in corresponding genetic selection for and against human chromosomes 6 and 15. Studies of the homogeneous hybrid sublines revealed that the presence of human β2m in a hybrid cell dramatically increased the surface expression of human HLA-A, B, C and mouse H-2Kk antigens. The results demonstrate the importance of human chromosome-specific surface markers and the fluorescence-activated cell sorter in somatic cell genetic analysis.

[25] Molecular cloning of receptor genes by transfection

Summary 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.

Production of interspecies T cell hybrids which retain differentiation specific surface antigens

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.

DNA-Mediated Gene Transfer in Mammalian Gene Cloning

Mammalian gene cloning in prokaryotic host and vector systems provides insights over a broad spectrum of biological mechanisms. Conventional cloning methods based on messenger RNA isolation are highly effective in the isolation of genes with high transcriptional activity. An alternative means of gene cloning which is independent of the abundance of mRNA involves the use of DNA-mediated gene transfer and selection in mammalian cells. This approach has its basis in somatic cell genetics, and makes use of a large reservoir of knowledge concerning selection systems in mammalian somatic cell populations in vitro. It will be the aim of this article to review the technical basis, historical development, and recent progress in this area of mammalian gene cloning.

The gene coding the human S11 surface antigens maps between the loci for HPRT and G6PD on the X-chromosome

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.

Variation in Expression of Human Major Histocompatibility Genes in Mouse L Cells After DNA-Mediated Gene Transfer

Somatic cell genetics and gene mapping have contributed significantly to our understanding of the organization and expression of the large and complex genomes of higher eukaryotes (1). The random segregation of donor chromosomes in somatic cell hybrids has allowed genes to be mapped to particular chromosomes by correlating their expression with the presence or absence of particular chromosomes. Recently, the availability of cloned DNA sequences and the techniques of Southern blot hybridization has circumvented the need for expression for successful gene mapping (2). From this work, a number of multigene families, i.e., groups of distinct but homologous gene sequences having similar function and structure, have been identified and chromosomally located. These gene families can either be closely linked or dispersed throughout the genome (3).