Chapter 25, Problem 20DAP

$\left(\text{a}\right)$

Summary Introduction

To determine: The presence of mutants even when no $\text{R7000}$ drug was present.

Introduction:

Drug $\text{R7000}\left(\text{7 -methoxy-2-nitronaphtho[2, 1-b ]furan}\right)$ is carcinogenic and induces mutations in the genome of bacteria like E.coli $E.coli$. It reacts with enzyme nitroreductase and forms adducts with $\text{DNA}$.

Explanation of Solution

Explanation:

Experiments were carried out in two strains of bacteria with wild type ${\text{Uvr}}^{+}$ strain and mutant ${\text{Uvr}}^{-}$. These bacteria are grown in the presence of protein inhibitor rifampicin. Rifampicin inhibits the action of $\text{RNA}$ polymerase which are made resistant by exposing these strains to ultra-violet radiation.

These strains are then grown in culture media with observance of few colonies in mutant ${\text{Uvr}}^{-}$ strain as compared to ${\text{Uvr}}^{+}$ strain. This occurs because of the presence of mutations in the recombination excision repair mechanism system in ${\text{Uvr}}^{-}$ strain even when the drug was not added but exposure to ultra-violet radiations is still there during treatment.

Conclusion

Conclusion:

${\text{Uvr}}^{-}$ strain grows in culture media when drug is not added as it is a mutant strain containing deficient nucleotide excision repair system still some background colonies are leading to growth of some colonies of mutant strain.

$\left(\text{b}\right)$

Summary Introduction

To explain: That treatment with drug $\text{R7000}\left(\text{7 -methoxy-2-nitronaphtho[2, 1-b ]furan}\right)$ is lethal for the cell.

Introduction:

Genotoxic activity of drug is its chemical property that induces mutations in the genome.

Explanation of Solution

Explanation:

Treatment with drug $\text{R7000}\left(\text{7 -methoxy-2-nitronaphtho[2, 1-b ]furan}\right)$ is lethal as it alkylates the nucleotides in the genome leading to formation of mutant stains in bacteria $E.coli$.

Conclusion

Conclusion:

The addition of this drug alkylates nucleotides leading to the formation of mutations in the genome.

$\left(\text{c}\right)$

Summary Introduction

To determine: The differences in the mutagenesis curve and survival curves in the two bacterial strains ${\text{Uvr}}^{+}$ and ${\text{Uvr}}^{-}$ strain.

Introduction:

Mutagenesis curve growth rate analyzes the rate of growth of bacterial strain after increasing the concentration of mutation causing drug in bacteria. Survival curve analyzes the percent of bacteria which survive after adding the mutation causing drug.

Explanation of Solution

Explanation:

The percent survival of ${\text{Uvr}}^{+}$ strain decreases from $100\%$ to $10\%$ after increasing the concentration of drug and survival rate of ${\text{Uvr}}^{-}$ strain decreases from $100\%$ to $1\%$ after increasing the concentration of drug inside bacteria. ${\text{Uvr}}^{-}$ strain is more sensitive to mutations as compared to ${\text{Uvr}}^{+}$ strain. The repair system of mutant strain is defective as compared to wild type strains.

Conclusion

Conclusion:

There is more decrease in the survival rate of ${\text{Uvr}}^{-}$ strain as compared to the survival rate of ${\text{Uvr}}^{+}$ strain of bacteria due to defect in the nucleotide repair system in ${\text{Uvr}}^{-}$ strain.

$\left(\text{d}\right)$

Summary Introduction

To explain: The amount of drops in $\text{H}$ over time in ${\text{Uvr}}^{+}$ strain as compared to rise over time in ${\text{Uvr}}^{-}$ strain.

Introduction:

Radioactive labeling with thymidine incorporates these labeled atoms inside the formation of adducts of drug- $\text{DNA}$ complex leading to the measurements of radioactive counts.

Explanation of Solution

Explanation:

${\text{Uvr}}^{+}$ strain is a wild type strain which has an active nucleotide excision repair system as compared to ${\text{Uvr}}^{-}$ strain which has a deficient nucleotide repair system. The addition of radiolabeled $\text{[}\text{H}\text{]R7000}$ in ${\text{Uvr}}^{+}$ strain causes decrease in the amount of attached drug in ${\text{Uvr}}^{+}$ strains due to presence of repair system functioning. It decreases drops over time as compared to ${\text{Uvr}}^{-}$ strain where the counts increase due to deficient functioning of excision repair system.

Conclusion

Conclusion:

The counts of incorporation of radioactive drug decreases in wild type strain and increases in mutant strain of $Ecoli$.

$\left(\text{e}\right)$

Summary Introduction

To determine: The types of mutations which show significant increases due to treatment with $\text{R7000}\left(\text{7 -methoxy-2-nitronaphtho[2, 1-b ]furan}\right)$.

Introduction:

Mutations are changes in nucleotides present at specific positions in the genome. These mutations convert purines to pyrimidines and pyrimidines to purines in the genome.

Explanation of Solution

Explanation:

The strains used in this experimental study include the following with the nomenclature provided as follows: $\text{CC101,CC102,CC103,CC104,CC105,CC106}$. The conversions occur in all the strains depending upon the amount of drug added and characteristics properties of these strains. The frequency of occurrence of mutations is different in different strains of bacteria.

Conclusion

Conclusion:

There is occurrence of mutations in all strains of bacteria but the frequency is different depending upon the characteristics of strain and amount of drug added to the strain of bacteria.

$\left(\text{f}\right)$

Summary Introduction

To determine: The different mutations occurring due to the formation of $\text{R7000}$ to a $\text{GC}$ base pair.

Introduction:

Mutations occur in the genome of bacteria due to conversion of nucleotides after the formation of adducts of $\text{DNA-drug}$.

Explanation of Solution

Explanation:

The adduct formation takes place at guanine with formation of derivates with drug. This causes the change in base pairing from $\text{GC}$ to $\text{AT}$ and from $\text{AT}$ to $\text{T}\text{}\text{A}$. The adducts attach to $\text{A or T}$.

Conclusion

Conclusion:

The adducts are formed by the addition of drug which changes the base pairing from $\text{GC}$ to $\text{AT}$ or $\text{T}\text{}\text{A}$.

$\left(\text{g}\right)$

Summary Introduction

To determine: The base pairs that form $\text{R7000-GC}$ adduct inside the cell.

Introduction:

$\text{R7000-GC}$ adducts are formed after the addition of drugs inside the bacterial cells. This occurs due to methylation of nucleotides in the genome of bacteria.

Explanation of Solution

Explanation:

Refer to Figure $\text{25-27b}$ “Examples of how $\text{DNA}$ damage results in mutations”. The effect of formation of adducts of $\text{R7000-GC}$ and the types of base pairs that are formed include formation of guanine, cytosine base pairs with conversion to $\text{G-A}$ products which are finally converted to $\text{T-A}$ products. If formation of $\text{G-T}$ products takes place it gets converted to $\text{A-T}$ products.

Conclusion

Conclusion:

The base pairs that form $\text{R7000-GC}$ adducts inside the cell include the conversion of $\text{G-C}$ to $\text{A-T}$ base pairs inside the genome.

$\left(\text{h}\right)$

Summary Introduction

To determine: Whether all the mutation types are repaired with same fidelity.

Introduction:

Fidelity is the precision with which the nucleotides are repaired in the genome. It depends upon the enzyme complexes which take an active part in excising and replacing the error prone nucleotides in the genome.

Explanation of Solution

Explanation:

The different mutation types are not repaired with the same fidelity as given in the table below:

$\text{A-T}$ to $\text{C-G}$	Higher repair, less number of colonies.
$\text{G-C}$ to $\text{A-T}$	Not much difference
$\text{G-C}$ to $\text{C-G}$	Less repair.
$\text{G-C}$ to $\text{T-A}$	High repair in both strains.
$\text{A-T}$ to $\text{T-A}$	High repair in both strains
$\text{A-T}$ to $\text{G-C}$	Low repair.

Conclusion

Conclusion:

The formation of different base-pairs with transitions occurs in both the wild type and mutant strains with formation of $\text{DNA-adduct}$ complexes. The fidelity of repair is different in different strains based on whether they are wild type for gene $\text{Uvr}$ or mutant for gene $\text{Uvr}$.