Lecture 4 BBT317 Mutation and Repair Part IV.pptx

BBT317 Molecular Genetics Lecture # 4  Mutation and Repair: Part IV Pierce Genetics: A Conceptual Approach (4e or 5e) Chapter 18 Snustad Principles of Genetics (6e) Chapter 13 Klug Essentials of Genetics (10e) Chapter 14 Watson Molecular Biology of the Gene (7e) Chapter 10 Griffith...

BBT317 Molecular Genetics Lecture # 4  Mutation and Repair: Part IV Pierce Genetics: A Conceptual Approach (4e or 5e) Chapter 18 Snustad Principles of Genetics (6e) Chapter 13 Klug Essentials of Genetics (10e) Chapter 14 Watson Molecular Biology of the Gene (7e) Chapter 10 Griffiths Introduction to Genetic Analysis (11e) Chapter 16  Spontaneous Mutations The Molecular Basis of Spontaneous Mutations: The Fluctuation Test (Griffiths) Gene mutations can arise spontaneously or they can be induced. Spontaneous mutations are naturally occurring mutations and arise in all cells. Induced mutations arise through the action of certain agents called mutagens that increase the rate at which mutations occur. Q1. Explain the Luria-Delbrück fluctuation test.  Spontaneous Mutations The Molecular Basis of Spontaneous Mutations: The Fluctuation Test (Griffiths) Q1. Explain the Luria-Delbrück fluctuation test.  Spontaneous Mutations The Molecular Basis of Spontaneous Mutations: The Fluctuation Test (Griffiths) This result led to the reigning “paradigm” of mutation; that is, whether in viruses, bacteria, or eukaryotes, mutations can occur in any cell at any time and their occurrence is random. For this and other work, Luria and Delbrück were awarded the Nobel Prize in Physiology or Medicine in 1969. This elegant analysis suggests that the resistant cells are selected by the environmental agent (here, phage) rather than produced by it. Q1. Explain the Luria-Delbrück fluctuation test.  Spontaneous Mutations The Molecular Basis of Spontaneous Mutations: The Fluctuation Test (Griffiths) Q1. Explain the Luria-Delbrück fluctuation test.  Spontaneous Mutations The Molecular Basis of Spontaneous Mutations: Replica Plating (Griffiths) Can the existence of mutants in a population before selection be demonstrated directly? This demonstration was made possible by the use of a technique called replica plating, developed largely by Esther and Joshua Lederberg in 1952. A population of bacteria was plated on nonselective medium—that is, medium containing no phage—and from each cell a colony grew. This plate was called the master plate. A sterile piece of velvet was pressed down lightly on the surface of the master plate, and the velvet picked up cells wherever there was a colony (Figure 16-6). In this way, the velvet picked up a colony “imprint” from the whole plate. The velvet was then touched to replica plates containing selective medium (that is, containing T1 phage). On touching velvet to plates, cells clinging to the velvet are inoculated onto the replica plates in the same relative positions as those of the colonies on the original master plate. As expected, rare resistant mutant colonies were found on the replica plates, but the multiple replica plates showed identical patterns of resistant colonies (Figure 16-7). If the mutations had occurred after exposure to the selective agents, the patterns for each plate would have been as random as the mutations themselves. The mutation events must have occurred before exposure to the selective agent. Again, these results confirm that mutation is occurring randomly all the time, rather than in response to a selective agent. Q1. What is replica plating?  Spontaneous Mutations The Molecular Basis of Spontaneous Mutations: Replica Plating Q1. What is replica plating?  Spontaneous Mutations The Molecular Basis of Spontaneous Mutations: Replica Plating Q1. What is replica plating?  Spontaneous- vs Adaptive Mutations Both Spontaneous- and Adaptive mutations occur The classic experiments of Luria and Delbriick (1943) and Lederberg and Lederberg (1952) demonstrated that mutations in bacterial cells occur prior to exposure to a selective agent. Such 'spontaneous' mutations presumably reflect random errors made during DNA synthesis and hence occur without regard to their possible utility. Although these experiments demonstrated the occurrence of spontaneous mutation events, they utilized a lethal selection scheme that precluded the detection of mutational events that might be occurring after exposure to the selective agent. https://www.ias.ac.in/article/fulltext/jgen/078/01/0051-0055 Adaptive mutation is a process that during nonlethal selection produces mutations that relieve the selective pressure, whether or not other, non-selected mutations are also produced. It remains to be seen whether this occurs via an evolved mechanism, or because the cells are simply unable to maintain the integrity of their DNA repair systems. For example, mutations arise in non-dividing, nutritionally deprived cells of Escherichia coli, apparently in response to selective pressure. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2929355/#:~:text=Examples%20of %20adaptive%20mutation%20or,mutation%20rates%20under%20adverse %20conditions. Q1. Is there any example of adaptive mutation?  Induced Mutations Chemically Induced Mutations Although many mutations arise spontaneously, a number of environmental agents are capable of damaging DNA, including certain chemicals and radiation. Any environmental agent that significantly increases the rate of mutation above the spontaneous rate is called a mutagen. Mutations can be induced by chemicals that can be classified in the following ways: 1. Base analogs 2. Alkylating agents 3. Deamination by nitrous acid (HNO2) 4. Hydroxylation by hydroxylamine 5. Oxidation reactions by reactive oxygen species (ROS) 6. Intercalating agents 7. Adduct-forming agents Q1. Discuss the key characteristics of different types of mutations. Chemically Induced Mutations Base analog-induced mutations Q1. Discuss how 5-bromouracil can induce mutation. Chemically Induced Mutations Base analog-induced mutations Q1. Discuss how 5-bromouracil can induce mutation.  Chemically Induced Mutations Alkylating agents like Ethylmethanesulfonate (EMS) can induce mutations Q1. Discuss how EMS can induce mutation.  Chemically Induced Mutations Deamination of Cytosine by Nitrous Acid can induce mutations Q1. Discuss how nitrous acid can induce mutation.  Chemically Induced Mutations Hydroxylation of Cytosine by Hydroxylamine can induce mutations Q1. Discuss how hydroxylamine can induce mutation.  Chemically Induced Mutations Oxidation of Bases by Oxidizing agents like Reactive Oxygen Species can induce mutations Q1. Discuss how oxidation of guanine by reactive oxygen species (ROS) can induce mutation.  Chemically Induced Mutations DNA Intercalating Agents can induce mutations Q1. Discuss how DNA intercalating agents can induce mutation.  Chemically Induced Mutations DNA-Adduct Forming Agents like Heterocyclic Amines can induce mutations (Klug) Another group of chemicals that cause mutations are known as adduct-forming agents. A DNA adduct is a substance that covalently binds to DNA, altering its conformation and interfering with replication and repair. Two examples of adduct- forming substances are acetaldehyde (a component of cigarette smoke) and heterocyclic amines (HCAs). HCAs are cancer-causing chemicals that are created during the cooking of meats such as beef, chicken, and fish. HCAs are formed at high temperatures from amino acids and creatine. Many HCAs covalently bind to guanine bases. At least 17 different HCAs have been linked to the development of cancers, such as those of the stomach, colon, and breast. Acetaldehyde Examples of DNA adduct-forming heterocyclic amines https://www.semanticscholar.org/paper/DNA-adducts-of-heterocyclic-amine-food- mutagens%3A-Schut-Snyderwine/933c89ed5c05cd390b1501f70db287e1c92f1c21 Q1. What are DNA-adduct forming agents? No question in exam on the chemical structures of this slide.  Radiation Induced Mutations Mutations induced by Radiation (Klug, Snustad) All electromagnetic radiation consists of energetic waves that we define by their different wavelengths (Figure 14.7). The full range of wavelengths is referred to as the electromagnetic spectrum, and the energy of any radiation in the spectrum varies inversely with its wavelength. Waves in the range of visible light and longer are benign when they interact with most organic molecules. However, waves of shorter length than visible light, being inherently more energetic, have the potential to disrupt Q1. What types of organic molecules. radiation can affect organic molecules? The portion of the electromagnetic spectrum with wavelengths shorter and of higher energy than visible light is subdivided into ionizing radiation (X rays, gamma rays, and cosmic rays) and nonionizing radiation (ultraviolet light).  Radiation Induced Mutations Mutations induced by Ionizing Radiation (Snustad, Klug) The energy of radiation varies inversely with wavelength. Therefore, X rays, gamma rays, and cosmic rays are more energetic than UV radiation (Figure 14.7). Ionizing radiations are of high energy and are useful for medical diagnosis because they penetrate living tissues for substantial distances. In the process, these high-energy rays collide with atoms and cause the release of electrons, creating positively charged free radicals or ions. The ions, in turn, collide with other molecules and cause the release of additional electrons. The result is that a cone of ions is formed along the track of each high- energy ray as it passes through living tissues. As ionizing radiation penetrates cells, stable molecules and atoms are transformed into free radicals—chemical species containing one or more unpaired electrons. Free radicals can directly or indirectly affect the genetic material, altering purines and pyrimidines in DNA, breaking phosphodiester bonds, disrupting the integrity of chromosomes, and producing a variety of chromosomal aberrations, such as deletions, translocations, and chromosomal fragmentation. Although it is often assumed that radiation from artificial sources such as nuclear power plant waste and medical X rays are the most significant sources of radiation exposure for humans, scientific data indicate otherwise. Scientists estimate that less than 20 percent of human radiation exposure arises from human-made sources. The greatest radiation exposure comes from radon gas, cosmic rays, and natural soil radioactivity. More than half of human-made radiation exposure comes from medical X rays and radioactive pharmaceuticals. Q1. How do ionizing radiations induce mutations?  Radiation Induced Mutations Mutations induced by Ionizing Radiation (Griffiths) Q1. How do ionizing radiations induce mutations?  Radiation Induced Mutations Mutations induced by Nonionizing (UV) Radiation (Snustad, Watson) Ultraviolet (UV) radiation does not possess sufficient energy to induce ionizations. However, it is readily absorbed by many organic molecules such as the purines and pyrimidines in DNA, which then enter a more reactive or excited state. UV rays penetrate tissue only slightly. In multicellular organisms, only the epidermal layer of cells usually is exposed to the effects of UV. However, ultraviolet light is a potent mutagen for unicellular organisms. The maximum absorption of UV by DNA is at a wavelength of 254 nm. Maximum mutagenicity also occurs at 254 nm, suggesting that the UV-induced mutation process is mediated directly by the absorption of UV by purines and pyrimidines. In vitro studies show that the pyrimidines absorb strongly at 254 nm and, as a result, become very reactive. Two major products of UV absorption by pyrimidines (thymine and cytosine) are pyrimidine hydrates and pyrimidine dimers (Figure 13.12). Thymine dimers cause mutations in two ways: (1) Dimers perturb the structure of DNA double helices and interfere with accurate DNA replication. (2) Errors occur during the cellular processes that repair defects in DNA, such as UV-induced thymine dimers. In the case of a thymine adjacent to a cytosine, the resulting fusion is a thymine–cytosine adduct in which the thymine is linked via its carbon atom 6 to the carbon atom 4 of cytosine. Q1. How does UV radiation induce mutations?  Radiation Induced Mutations Mutations induced by Radiation (Snustad) Q1. How do ionizing radiations induce mutations?  Radiation Induced Mutations Mutations induced by Radiation (Griffiths) No question in exam from this slide.  Next Lecture: Mutation and Repair: Part V