Cell Hybridization and the 24 Homan Gene Maps

2 CELL HYBRIDIZATION AND THE 24 HOMAN GENE MAPS Thomas B. Shows 1. INTRODUCTION The transfer of genes is an ancient phenomenon in the biology and evolution of organisms, as is evidenced by a variety of microorganisms. In bacteria during their sexual reproductive phase, cell-to-cell transfer of single-stranded DNA has been demonstrated (Lederberg, 1947; Wollman et ai., 1956). In addition, DNA isolated from one genotype could stably change the phenotype of another bac terium (Avery et al., 1944). Similarly, the infection of bacteria by a certain bacteriophage involves transfer of phage DNA into a bacterium and integration into the bacterial genome. When phage replication takes place, the phage genome sometimes acquires a bacterial gene that was closely linked to the inserted phage genome. Subsequent bacterial infections by this phage can transfer the bacterial gene to another bacterial genome (Zinder and Lederberg, 1952; Lennox, 1955). In the life cycle offungi, the entire haploid genome is transferred by the formation of heterokaryons after fusion of hyphae from different genomes (Fincham, 1966). In eukaryotes, fertilization of an egg could be considered a form of gene transfer, since an entire haploid genome is transferred. Thus, there are biological mech anisms for cells to accept new genetic material and propagate it through cell division. Understanding the principles of gene transfer in mammals, especially Homo sapiens, will teach us much about genetic mechanisms, gene expression, cell growth and differentiation, gene mapping, mutagenesis, and both normal and abnormal human biology. This information promises to be essential for understanding and possibly treating disease. Cell fusion resulting in multinucleated mammalian cells, termed "polykar yocytes," was first observed over 100 years ago after viral infection and was thought to be the first step in tumorigenesis (Langhans, 1868). This fusion Thomas B. Shows • Department of Human Genetics, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14263. 5 R. Kucherlapati (ed.), Gene Transfer © Plenum Press, New York 1986 6 THOMAS B. SHOWS represents the transfer of whole genomes into a single cell. It was Barski and Ephrussi and their colleagues who demonstrated, by fusing certain rodent cells in culture, that full complements of two genomes were represented in a single cell (cf. Ephrussi, 1972). Weiss and Green (1967) later showed that in a fusion of human and mouse cells in culture, only a partial complement of human chromosomes was retained after hybrid cells were cloned. This was the key observation that revolutionized and stimulated interest in human gene mapping. By the coupling of these findings with current technology in cytogenetics, en zymology, immunology, and recombinant DNA methodology, detailed human gene maps have been generated for each of the 24 human chromosomes (see Table I, p. 36). The ability to transfer mammalian genes parasexually has opened new pos sibilities for gene mapping, fine-structure mapping, and genetic dissection of disease. The DNA transferred has ranged from whole genomes to single genes and smaller segments of DNA. The transfer of whole genomes by cell fusion produces cell hybrids and has promoted the extensive mapping of human and mouse genes. Transfer by cell fusion of rearranged chromosomes has contributed significantly to determining close linkage and the assignment of genes to specific, small chromosomal regions. Transfer of single chromosomes has been achieved utilizing microcells fused to recipient cells. Metaphase chromosomes have been isolated and used to transfer single-to-multigenic DNA segments. DNA-mediated gene transfer, simulating bacterial transformation, has achieved transfer of single copy genes. Single genes and DNA segments have been cloned into eukaryotic and prokaryotic vectors for transfer into mammalian cells, their fate and chro mosome mapping being examined at the molecular level using sequence-specific probes. In fact, recombinant libraries in which entire mammalian genomes are represented collectively are an extensive source of transferable genes. Thus, virtually all gene-transfer methodology lends itself to mapping genes. This review will focus on the transfer of whole mammalian genomes and rearranged chro mosomes through cell fusion and the mapping of the human genome stimulated by this technology and other complementing technology. Other chapters in thIS book will describe the transfer of single chromosomes, segments of chromo somes, cloned genes, and defined fragments of DNA, all of which contribute to the mapping and linear order of genes and to fine-structure gene mapping. 2. SOMATIC-CELL HYBRIDIZATION AND THE TRANSFER OF WHOLE GENOMES Entire mammalian genomes are transferred routinely by fusing individual cells. A large array of mammalian cells and cell types have been fused principally by two fusing agents: Sendai virus (Okada and Tadokaro, 1963; Klebe et al., CELL HYBRIDIZATION AND THE 24 HUMAN GENE MAPS 7 1970) and polyethylene glycol (PEG) (Davidson and Gerald, 1976). Cultured somatic cells growing as monolayers or in suspension, single cells from living organisms (e.g., leukocytes, erythrocytes, and macrophages), and cancer cells in suspension are examples of cells that have been used for cell fusion (Ephrussi, 1972; Sell and Krooth, 1973; Ringertz and Savage, 1976; Shay, 1982). The genetics, state of differentiation, and chromosomal composition of parental cells participating in cell hybridization have been diverse; for example, diploid, sub diploid, or heteroploid cells and cells with gene mutations, differentiated func tions, and chromosome translocations and deletions have been used (Ephrussi, 1972; Sell and Krooth, 1973; Davidson, 1974; Ringertz and Savage, 1976; Shows, 1979; Shows et at., 1982). When growing cell hybrids are desired, at least one of the parental cells must be able to proliferate in cell-culture conditions, and an appropriate selection system must be available to isolate the hybrid cells from parental cells. Most cells can be fused using either inactivated Sendai virus or PEG of different molecular weights (Klebe et at., 1970; Davidson and Gerald, 1976). Homokaryons or heterokaryons constitute the multinucleated-cell hybrid population that is formed at concentrations depending on several criteria, such as concentration of the fusing agent, time, pH, temperature, ions, cell type, and cell cycle (Klebe et at., 1970; Poste, 1970; Croce et at., 1972; Davidson and Gerald, 1976; Ringertz and Savage, 1976; Shay, 1982; Mueller et at., 1983). Heterokaryons can be isolated mechanically, usually by size (Karig Hohmann and Shows, 1979), or by growth as clones in a selection medium that prohibits propagation of parental cells with enzyme deficiencies, drug sensitivities, or temperature dependencies (Littlefield, 1964; Shows, 1972; Ringertz and Savage, 1976; Shay, 1982). Heterokaryons, the parental cells of which have selectable markers, survive after nuclear fusion in a selection medium by complementation of the genetic defects of one or both parental cells (Ruddle, 1972; Shay, 1982; Shows et at., 1982). Thus, a hybrid cell survives that has retained chromosome complements from both genomes. Independent hybrid clones are isolated from proliferating cell hybrids by established cell-cloning techniques (Puck et at., 1956). Details of whole-cell hybridization have been described (Ringertz and Savage, 1976; Shay, 1982). Two genomes in the same cell were initially used for studying control of expression of differentiated products and for investigating genetic complemen tation when the two parental cells are deficient for the same phenotype but have mutations at different genes (Fincham, 1966; Davidson, 1974; Karig Hohmann and Shows, 1979; Honey et at .. 1982; Mueller et at., 1983). However, use of these genome-transfer techniques for gene-mapping purposes proved to be an additional advantage. Generally, cell hybrids formed between parental cells of the same species or two established tissue-culture lines do not lose large numbers of chromosomes (Ephrussi, 1972; Ringertz and Savage, 1976). However, it was observed that in some combinations of cell hybrids, interspecific hybrids in particular, large numbers of chromosomes from one cell type are lost by unknown 8 THOMAS B. SHOWS mechanisms. This makes it possible to map those genes of the parental type that loses chromosomes; the genes must be distinguishable between parental types. The observation of chromosome loss from only one of the parental cell types, described first by Weiss and Green (1967), has evolved into the dogma that in interspecific cell fusions, chromosomes from one of the species will be lost when the parental cells of that species are not derived from an established, infinitely growing cell line. In these combinations, chromosomes from the established cell line will be retained, but chromosomes from the other parental cell obtained from cells in vivo or from a finite growing cell-culture line will be lost. The coupling of gene-transfer and gene-mapping techniques has made it possible to map the genome of the species, the chromosomes of which are lost in cell hybrids. This parasexual strategy for mapping genes in mammals approximates classic parasexual procedures described for microorganisms. 3. SOMATIC-CELL HYBRIDS FOR GENE MAPPING Procedures for obtaining man-rodent cell hybrids, the mapping of human genes, and the genetic dissection of complex enzyme and protein systems in

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