Lecture 14 Unit 2

Selecting Mutant Cells: B. Indirect Selection (Prototroph, Auxotroph)

1. Prototroph-grows on a minimal media. Growing cells are susceptible to penicillin2. Auxotroph-the mutant cells. Auxotrophs are mutants that have additional nutrient requirements. The auxotrophs will require additional amino acids for growth on minimal media.

Genetic Recombination

the exchange of genes between two DNA molecules to form new combinations of genes on a chromosome. This process creates diversity like mutation and therefore also drives evolution. Mutation however, only involves a single gene. There are 3 general methods of genetic recombination available to bacteria. They are transformation, conjugation and transduction.

combating the effect of the U.V. light (1,2)

1. many species possess an enzyme induced by visible light which will cleave or break the covalent bonds joining these dimers. 2. In addition to this method of repair, a dark repair mechanism exists. In this process an enzyme first excises the dimer. A DNA polymerase (second enzyme) then inserts new thymines opposite to the existing adenines to reform the naturally bonded DNA.

Selecting Mutant Cells: A. Direct Selection

Grow organisms in the presence of an antibiotic. Those that survive will have the mutant gene that allows those cells to live in the presence of the antibiotic. Those that don't survive the presence of the antibiotic are prototroph (original cells that do not have the mutant gene). The selected cells growing in the presence of the antibiotic would represent the mutant population.

Genes will mutate independently of each other. i.e. the chance of a single cell having 2 separate mutations

The chance that a single cell will have 2 separate mutations is equal to the product of the single rates. -For example let's say that a cells chance for having a gene that allows it to be resistant to penicillin is 10^(-9). Let's also establish that a cells chance for having another mutant gene that allow for resistance to streptomycin is 10^(-5). The chance that a single cell will have both resistant genes is the product of the individual rates or 10^(-9) X 10^(-5) which equals 10^(-14).

prototroph

The word prototroph refers to the original cells that do not have the mutant gene. The prototroph population is also referred to as the wild type or normal or parent group of cells.

Experimental Procedure for Auxotroph Selection

a. Expose the prototroph population to UV light (intermediate dose). -1. Kills some -2. Mutates some (shocked survivors) -3. Some unaffected (shocked survivors) b. Grow the shocked survivors on a nutritionally complete media to allow for recovery c. To separate out the shocked survivors, place them on a minimal media that contains penicillin. d. The prototrophs will grow and then be killed by the penicillin. e. The auxotrophs cannot grow on a minimal media. They sit on media and are not killed. f. Transfer the cells to a complete media. All auxotrophs grow. g. Use replica plating technique to separate the different auxotrophs.

The Ames test

makes use of Salmonella histidine auxotrophs to efficiently screen for chemical carcinogens. Expose these auxotrophs to a suspected carcinogen and look for the presence of the original Salmonella prototroph. The prototroph does not need added histidine for growth and will grow on a minimal media. Growth and appearance of the prototroph indicates that the auxotroph experienced a reversion or a back mutation when exposed to the suspected carcinogen. There is a 90% chance that this mutagenic agent is carcinogenic. It is deemed a suspected carcinogen.

Varying rates of mutation caused by mutagens

mutagens can cause an increased rate of mutation for all genes in a genome. These are general mutagens. However, the mutation rate for different genes can be different. Genes will have different lengths and configurations increasing or decreasing their susceptibilities.

Causes of genetic mutations-physical mutagen: U.V. radiation, general mutagens

when bacterial cells are exposed to U.V. radiation adjacent thymines are unnaturally bonded to create thymine-thymine dimers (or just thymine dimers: A pair of abnormally chemically bonded adjacent thymine bases in DNA). Too much U.V. radiation can prevent repair. U.V. light is most damaging at wavelengths between 260 and 270 nanometer. -above mentioned mutagens will cause an increased rate of mutation for all genes in the genome.