Experimental Isolation of Mutations

The emergence of molecular biology has relied greatly on the analysis of mutations in a variety of organisms. Obviously, such analysis requires a supply of mutants to work with. These may be obtained by a wide range of approaches. Mutations may be spontaneous or artificially induced. Artificial mutagenesis may be carried out using living organisms (in vivo mutagenesis) or performed using isolated DNA (in vitro mutagen-esis). In addition, some means is needed for identifying mutants, whether they occur spontaneously or are made deliberately.

Since the frequency of spontaneous mutations is low, it is only possible to rely on this source of mutations when a population of millions of organisms can be surveyed in a reasonable time. Consequently, this approach is largely restricted to bacteria and single-celled eukaryotes, such as yeast. For a gene of average size (~1000 bp) the mutation rate is approximately 0.5 per million per generation in bacteria such as E. coli (see Table 13.01). Thus, a typical culture of several 1,000 million bacteria per ml that has resulted from several generations of growth may contain half a dozen spontaneous mutants per million cells (or several thousand mutants per ml of culture) affecting any particular gene of interest (assuming such mutations are not lethal).

The problem then becomes how to isolate these mutants. It is clearly impractical to examine millions of microorganisms individually. Therefore the isolation of spontaneous mutants relies on some form of direct selection. Usually, samples of the culture are spread on the surface of solid medium designed to allow only the desired mutants to grow. For example, mutations in DNA gyrase make bacterial cells resistant to quinolone antibiotics, such as nalidixic acid. Therefore medium containing nalidixic acid kills the vast majority of bacteria and can be used to isolate gyrase mutants. Sometimes bacterial cultures may be enriched for the required mutation by growth for several generations in liquid selective medium before transferring to solid selective medium for the final isolation. This increases the proportion of the required mutants in the population.

A large number of direct selections have been used to isolate bacterial mutants. The major categories of selection and the kinds of genes affected are as follows:

A. Resistance to antibiotics. Alterations in genes whose products are targets of the antibiotic or that are involved in entry of the antibiotic into the cell. For example, streptomycin resistance selects alterations in ribosomal protein S12, rifampicin resistance selects alterations in RNA polymerase, nalidixic acid resistance selects mutations in DNA gyrase.

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Bacterial mutants are often screened for their responses on a variety of specialized culture media.

B. Resistance to analogs of metabolites. Alterations in genes whose products are involved in synthesis, degradation or transport of the metabolite. For example, chlorate (an analog of nitrate) selects mutants defective in nitrate reductase, chloroethanol selects mutants defective in alcohol dehydrogenase, various selenium compounds select mutants with altered sulfur metabolism.

C. Resistance to bacteriophage. Alterations in genes encoding bacteriophage receptor or components needed for entry of viral DNA. For example, resistance to bacteriophage lambda selects for loss of LamB protein on cell surface, resistance to bacteriophage T1 selects for loss of TonB protein needed to energize viral DNA entry.

D. Growth in the absence of certain metabolic supplements. Usually used to select revertants from mutants defective in synthesis of amino acids, nucleotides, vitamins etc.

E. Growth on certain substances as carbon source. Usually used to select rever-tants from mutants defective in metabolism of sugars, organic acids by known pathways. Growth on novel compounds is sometimes selected. For example, selection for growth of E. coli on propanediol selects for aerobic expression of a pathway normally only expressed anaerobically during the fermentation of deoxysugars.

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