Genetic Culture
It is possible to select for genetically distinct cultured animal cells since some somatic cells from animals can be cultured from single cells in a well defined medium, just like bacterial and yeast cells. The chromosomes in the animal cells are larger and highly visible after staining, making it easier to distinguish individual chromosomes. Genetic studies of cultured animal cells are called somatic cell genetics to distinguish them from classical genetics, which deals with the whole organisms derived from germ cells, sperm and eggs.
Cultured cells from different mammals can be fused to produce inter-specific hybrids, which have been widely used in somatic cell genetics. For instance, hybrids can be prepared from human cells and mutant mouse cells that lack an enzyme required for synthesis of a particular essential metabolite. As the human mouse hybrid cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. In a medium that can support growth of both the human cells and mutant mouse cells, the hybrids eventually lose all human chromosomes. However, in a medium lacking the essential metabolite that the mouse cells cannot produce, the one human chromosome that contains the gene encoding the needed enzyme will be retained, because any hybrid cells that lose it following mitosis will die. All other human chromosomes eventually are lost. Using different mutant mouse cells and media in which they cannot grow, researchers have prepared various panels of hybrid cell lines.
Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes. Because each chromosome can be identified visually under a light microscope, such hybrid cells provide a means for assigning, or mapping individual genes to specific chromosomes. For example, suppose a hybrid cell line is shown microscopically to contain a particular human chromosome. That hybrid cell line can then be tested biochemically for the presence of various human enzymes, exposed to specific antibodies to detect human surface antigens, or subjected to DNA hybridization and cloning techniques to locate particular human DNA sequences.
The genes encoding a human protein or containing a human DNA sequence detected in such tests must be located on the particular human chromosome carried by the cell line being tested. Panels of hybrids between normal mouse and mutant hamster cells also have been established; in these hybrid cells, the majority of mouse chromosomes are lost, allowing mouse genes to be mapped to specific mouse chromosomes.
One metabolic pathway has been useful in cell fusion experiments. Most animal cells can synthesize the purine and pyrimidine nucleotides de novo from simpler carbon and nitrogen compounds, rather than from already formed purines and pyrimidines. The folic acid antagonists amethopterin and aminopterin interfere with the donation of methyl and formyl groups by tetrahydrofolic acid in the early stages of de novo synthesis of glycine, purine nucleoside monophosphates, and thymidine monophosphate. These drugs are called antifolates, since they block reactions involving tetrahydrofolate, an active form of folic acid. Many cells, however, contain enzymes that can synthesize the necessary nucleotides from purine bases and thymidine if they are provided in the medium, these salvage pathways bypass the metabolic blocks imposed by antifolates .
A number of mutant cell lines lacking the enzyme needed to catalyze one of the steps in a salvage pathway have been isolated. For example, cell lines lacking thymidine kinase or TK can be selected because such cells are resistant to the otherwise toxic thymidine analog 5 bromodeoxyuridine. Cells containing TK convert 5 bromodeoxyuridine into 5 bromodeoxyuridine monophosphate. This nucleoside mono- phosphate is then converted into a nucleoside triphosphate by other enzymes and is incorporated by DNA polymerase into DNA, where it exerts its toxic effects. This pathway is blocked in cells with a TK mutation that prevents production of functional TK enzyme.
