UV-Induced Killing and Mutagenesis

Transcription

1 UV-Induced Killing and Mutagenesis Why are we using ultraviolet light to induce mutations? The most advantageous aspect of UV mutagenesis in a teaching laboratory is that we avoid the potential hazard of exposure to a chemical mutagen, and the expense of waste disposal. Also, UV raises the rate of several different kinds of DNA change; transitions, transversions, as well as small additions and deletions. This can be an advantage for some projects. The downside of using UV is that the mechanism of mutagenesis is indirect, and complicated. The molecular mechanism of UV killing, UV mutagenesis, and repair of UV photo-damage is explained in a separate document listed as reading for this experiment. What kinds of mutants are we going to isolate, and how will we isolate them? We will isolate mutants that have regained the ability to metabolize lactose and to synthesize arginine, starting with strains SC 107 an SC 108, which lack those abilities. i.e. We say that these types of mutants are "revertants" because a second mutation causes them to revert back to a wildtype phenotype.(see the previous exercise.) Why do we isolate revertants? Isolation of revertants is often a relative slam/dunk because we can get them by direct selection. Suppose that we start with a population of cells that are Arg - (like strain SC 108) and that our mutagenesis procedure induces mutations in a small number of cells that make them Arg+ (i.e. they have reverted to the wild-type). When we plate such mixed population on Minimal Glucose medium (without arginine) only the rare revertants grow into colonies. On the other hand, if we start with a population of Arg + cells and induce a few Arg - mutants, there is no obvious way to select directly for the mutants and against the wild-type. 1 of 5

2 Chemical Mutagenesis 4/7/11 What kind of changes in DNA sequence will we need to induce with UV in order to get the revertants? The Lac - phenotype of strain SC 107 is due to the insertion of a single base pair in the lacz gene sequence. This causes a frameshift leading to a non-functional gene product. We can expect that deletion of a single base pair at or near the site of the original insertion would restore the correct reading frame so that a functional form of the enzyme would be produced. So in this case we most likely need to induce single base pair insertions. Bothe the arge and lacz mutations in strain SC 108 resulted from base pair substitutions that covert amino acid codons to a UAG termination codon (i.e. they are nonsense mutations). Therefore we can expect that base pair substitutions could change those amber codons back into sense codons. 2

3 Experimental Strategy The procedure below is intended to provide the data we need to address the following issues: 1. Increasing exposure to UV should produce more cell killing. 2. Increasing exposure to UV should produce more Lac+ revertants in BOTH strain SC 107 and SC 108 because UV mutagenesis raises the frequency of insertion/deletion mutations as well as the frequency of base pair substitutions. 3. We should be able to establish an optimum UV dose where the frequency of mutants relative to the parent cell population is maximized. 4. We should be able to show that mutational reversion is gene specific. i.e. Lac + revertants of SC 108 should still be Arg -, and vice versa. UV mutagenesis can be readily carried out using one of the widely available germicidal lamps. These are mercury vapor lamps whose operating principle is similar to common fluorescent tubes. Hg vapor has a strong emission line near 254 nm, well within the absorption band of DNA. In an ordinary fluorescent lamp this potentially harmful UV is entirely absorbed by the glass tube. Germicidal lamp tubes are constructed of a material that transmits short wave UV, and anything exposed to the lamp emission gets a hefty dose of UV around 254 nm. 3 of 5

4 Chemical Mutagenesis 4/7/11 Procedure We provide cultures of strains SC 107 and SC 108. The cell concentration in these cultures will be determined (by us) and this information will be provided. Spread 50 ul of strain SC 107 on a single Min Lac Arg plate. Spread 50 ul of strain SC 108 each of 3 plates as indicated below. i.e. You will have 4 plates total according to the table below. Be sure that you clearly understand what each of these plates is intended to accomplish. SC107 SC108 Minimal Glucose Arginine - + Minimal Glucose - + Minimal Lactose Arginine + + The time points for UV exposure will be set at 30" intervals beginning with 0". Each pair of student will expose their all their plates at one of these time points. CAUTION: Do not expose your skin or eyes to the UV lamp. Put on opaque gloves if you must put you hands under the UV lamp while it is on. Do not look directly at the UV lamp when it is on. Room lights will be turned off during exposure. As soon as your plates have been exposed, wrap them in Al foil to exclude light. Then stash them in the 37 C incubator and you're done for the day. The polystyrene plastic Petri dishes are opaque to UV. You must remove the lids of the plates while they are in Ultraviolator. At the next lab we will re-streak mutants to isolate a number of SC 108 revertants as pure cultures, and these will be subsequently tested for reversion of the other gene. i.e. If you isolate an Arg+ revertant of SC 108, you will then subsequently test it to determine whether or not it is also a Lac+ revertant (see item 4 in the Experimental Strategy section.) 4

5 ASSIGNMENT 1. Test your sophistication in the application of the genetic code table by working out the following: For any amber codon (5' UAG 3'), a single base pair substitution can covert the UAG to any one of 9 other codons. Work out the effect on the protein product that would result from each of the 9 substitutions. 2. Write a discussion of the class results divided into 4 sections. Each section should directly address, in turn, the 4 points listed in the Experimental Strategy section. i.e. Do are data show increasing cell killing with increasing UV dose? etc. 5 of 5