Week 03 - Exp 5

Week 03 - Exp 5 - BIOL 519 Week # 3 - Exp. 5 BACKGROUND:...

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BIOL 519 Week # 3 - Exp. 5 1 BACKGROUND: Last week you isolated mutations from cultures that had not been exposed to a mutagen (Spontaneous mutations). One of the points that should have been illustrated by that experiment was that some mutant phenotypes occur at a higher frequency than others (Nal R should have been isolated at a high frequency, while Str R should have been isolated at a low frequency). Still other mutations that require very specific DNA changes will occur at an even lower frequency than Str R . The isolation of rare mutations may require that the culture be first exposed to a mutagen. The mutagen will increase the overall mutation frequency in the culture, and therefore decrease the number of cells that must be screened or selected to find the mutation of interest. Even if the mutation occurs at a relatively high frequency, if one must use a screen to find the mutant phenotype of interest, it is often advantageous to increase the mutation frequency in the culture by exposure to a mutagen. In Experiment 5, you will again isolate mutations that confer Naladixic acid resistance and Streptomycin resistance to E. coli cells, however this time the cultures will first be exposed to a mutagen. The mutagen that we will employ in this experiment is UV light. UV light as a mutagen - Exposure of cells (both E. coli and your cells!) to light in the ultraviolet range (UV light - lots of it in sunlight) causes damage to the DNA. The most common form of damage is the formation of Thymine dimers in which two adjacent thymine bases form abnormal covalent bonds between their ring structures (see fig.). E. coli has a specific repair mechanism to fix these thymine dimers called Photoreactivation repair. Photoreactivation repair uses the energy from visible light (usually present along with the UV light, such as in sunlight) to directly break the abnormal bonds between the adjacent thymines. When using UV to deliberately induce mutations, the extent of Photoreactivation repair can be limited by avoiding much exposure of the cells to visible light. In sunlight (with both UV and visible light), exposure to too much UV can overload the repair system. Thymine dimers that are not repaired can form a block to DNA replication - DNA polymerase (both I and III) can not replicate past the thymine dimers. This type of block in
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This note was uploaded on 01/22/2011 for the course BIO 519 taught by Professor Ega during the Spring '10 term at Kansas.

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Week 03 - Exp 5 - BIOL 519 Week # 3 - Exp. 5 BACKGROUND:...

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