Conditional Lethal Mutants

The mutants discussed so far will grow if a required growth factor they cannot synthesize is added to the medium. Mutants,

Glucose-salts synthetic medium

Auxotrophs

Prototroph s::

+ Penicillin incubate ->

Penicillin kills actively multiplying cells

Most prototrophs are killed; auxotrophs survive because they cannot multiply in the medium.

+ Penicillinase

(destroys penicillin)

Enriched complex medium

Enriched complex medium

+ Penicillinase

(destroys penicillin)

Figure 8.10 Penicillin Enrichment of Mutants Auxotrophs require a growth factor to multiply; prototrophs do not.

Auxotroph

Prototroph

Figure 8.10 Penicillin Enrichment of Mutants Auxotrophs require a growth factor to multiply; prototrophs do not.

Bacteria plated on agar medium and incubated at 25°C

Conditional mutant

Bacteria plated on agar medium and incubated at 25°C

Conditional mutant

Replica plated onto two plates

Conditional mutant (temperature-sensitive)

Replica plated onto two plates

Conditional mutant (temperature-sensitive)

Incubate at 25"C

Incubate at 37"C

Only wild-type bacteria can grow

Only wild-type bacteria can grow

Figure 8.11 Isolation of Temperature-Sensitive (Conditional) Mutants This procedure selects for mutants that can grow only at their lower range of temperature as a result of defective proteins.

however, also may be defective in the enzymes required for macromolecule synthesis, such as factors involved in the translation of mRNA in protein synthesis or the enzymes of DNA replication. These mutations are ordinarily lethal to the cell, because their defects cannot be overcome by adding the macro-molecules RNA, DNA, or protein to the medium and have them function inside the cell. Therefore, they cannot be isolated by the techniques previously described. Mutants in essential genes, however, given the name conditional lethal mutants, can be isolated since mutant proteins often function at a low temperature such as 25°C but not at a higher temperature such as 37°C. They are called temperature-sensitive mutants, one class of conditional lethal mutants.

Temperature-sensitive mutants can be isolated by incubating a master plate at a low temperature (25°C) at which all cells, both normal and temperature-sensitive, will grow (figure 8.11). The master plate is then replica plated onto two plates containing an enriched medium. One is incubated at 37°C, the other at 25°C. Colonies growing at the low temperature but not the high temperature may be mutant in genes required for macromolecule synthesis or other growth functions such as membrane synthesis that cannot be overcome by adding growth factors to the medium.

8.6 Mutant Selection 201

effect of the potential carcinogen on DNA in a microbiological system. The first one was devised by Bruce Ames and his colleagues in the 1960s and illustrates the concept of such tests. This test takes only a few days and is based on three facts: (1) the reversion of a mutant gene in a biosynthetic pathway, such as histidine biosynthesis, can be readily measured; (2) the frequency of reversions is increased by mutagens; and (3) most carcinogens are mutagens. Specifically, the Ames test measures the effect of a test chemical on the rate of reversion of a specific Salmonella strain, a histidine requiring auxotroph, to one that no longer requires histidine (figure 8.12). If the chemical is mutagenic, it will increase the reversion rate of the strain relative to that observed when no chemical is added (the control). The test also gives some idea about how powerful the mutagen is, and therefore how potentially hazardous the chemical is by the number of revertants that arise.

The Ames test as just discussed fails to detect many carcinogens, because some substances are not carcinogenic themselves but can be converted to active carcinogens by a metabolic reaction that occurs in animals but not in bacteria. Therefore, an extract of ground-up rat liver, which has the enzymes to carry out these conversions, is added to the Petri plates containing the suspected mutagen (carcinogen) being analyzed by the Ames test. To increase the sensitivity of the test, a mutant tester strain which lacks the excision repair enzyme is often used. As a result reversions cannot be repaired.

Additional testing must be done on any mutagenic agent identified in the Ames test to confirm that it is actually carcinogenic in animals. Although data are not available on the percentage of mutagens that are carcinogens, it is clear that the Ames test is useful as a rapid screening test to identify those compounds that have a high probability of being carcinogenic. Thus far, no compound with a negative Ames test has been shown to be carcinogenic in animals.

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