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Duchenne muscular dystrophy is a disease that causes progressive weakening and degeneration of the muscles. From its X-linked pattern of inheritance, the mutated allele causing this disorder was known to be on the X chromosome, but its precise location was uncertain. Examination of a number of patients having Duchenne muscular dystrophy, who also possessed small deletions, allowed researchers to position the gene to a small segment of the short arm of the X chromosome.

Somatic-Cell Hybridization

Another method used for positioning genes on chromosomes is somatic cell hybridization, which requires the fusion of different types of cells. Most mature somatic (nonsex) cells can undergo only a limited number of divisions and therefore cannot be grown continuously. However, cells that have been altered by viruses or derived from tumors that have lost the normal constraints on cell division will divide indefinitely; these types of cells can be cultured in the laboratory and are referred to as a cell line.

Cells from two different cell lines can be fused by treating them with polyethylene glycol or other agents that alter their plasma membranes. After fusion, the cell possesses two nuclei and is called a heterokaryon. The two nuclei of a heterokaryon eventually also fuse, generating a hybrid cell that contains chromosomes from both cell lines. If human and mouse cells are mixed in the presence of polyethylene glycol, fusion results in human - mouse somatic-cell hybrids (FIGURE 7.18). The hybrid cells tend to lose chromosomes as they divide and, for reasons that are not understood, chromosomes from one of the species are lost preferentially. In human - mouse somatic-cell hybrids, the human chromosomes tend to be lost, whereas the mouse chromosomes are retained. Eventually, the chromosome number stabilizes when all but a few of the human chromosomes have been lost. Chromosome loss is random and differs among cell lines. The presence of these "extra" human chromosomes in the mouse genome makes it possible to assign human genes to specific chromosomes.

In the first step of this procedure, hybrid cells must be separated from original parental cells that have not undergone hybridization. This separation is accomplished by using a selection method that allows hybrid cells to grow while suppressing the growth of parental cells. The most commonly used method is called HAT selection ( FIGURE 7.19), which stands for hypoxanthine, aminopterin, and thymi-dine, three chemicals that are used to select for hybrid cells. In the presence of HAT medium, a cell must possess two enzymes to synthesize DNA: thymidine kinase (TK) and hy-poxanthine-guanine phosphoribosyl transferase (HPRT). Cells that are tk~ or hprr cannot synthesize DNA and will not grow on HAT medium. The mouse cells used in the hy-

Human fibroblast

Mouse tumor cell

Human fibroblast

Mouse tumor cell

...creating hybrid cells called heterokaryons.

Heterokaryon

Human and mouse nuclei in some hybrid cells fuse.

Hybrid cell with fused nucleus

[4 Human chromosomes are randomly lost from the nucleus during cell division; all but a few of the human chromosomes are eventually lost.

ffi Human fibroblasts and mouse tumor cells are mixed in the presence of polyethylene glycol, which facilitates fusion of their membranes,...

...creating hybrid cells called heterokaryons.

Heterokaryon

Human and mouse nuclei in some hybrid cells fuse.

Hybrid cell with fused nucleus

[4 Human chromosomes are randomly lost from the nucleus during cell division; all but a few of the human chromosomes are eventually lost.

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