Chapter 19 slide 1 Mutations and Recombination Dr.Aida Fadhel Biawi 2013 Chapter 19 slide 2 Mutations Defined 1.A mutation is a change in a DNA base-pair or a chromosome. a.Somatic mutations affect only the individual in which they arise. b.Germ-line mutations alter gametes, affecting the next generation. 2.Mutations are quantified in two different ways: a.Mutation rate is the probability of a particular kind of mutation as a function of time (e.g., number per gene per generation). b.Mutation frequency is number of times a particular mutation occurs in proportion to the number of cells or individuals in a population (e.g., number per 100,000 organisms). Chapter 19 slide 3 Types of Mutations Point Mutations Base Pair Substitutions Silent Missense new protein (Amino Acid Substitutions) Nonsense stop codon Base Pair Insertions and deletions Triplet Repeats Frameshift Mutations Chapter 19 slide 4 Types of Point Mutations Nonsense Mutation and Nonsense Suppressor Mutation There are two general categories of point mutations: base-pair substitutions and base-pair deletions or insertions. 1.A base-pair substitution replaces 1 base-pair with another. There are two types : a. Transitions convert a purine-pyrimidine pair to the other purine- pyrimidine pair (e.g., AT to GC or TA to CG). b. Transversions convert a purine-pyrimidine pair to a pyrimidine-purine pair (e.g., AT to TA, or AT to CG). 2.Base-pair substitutions are also defined by their effect on the protein sequence. Effects vary from none to severe. a. Nonsense mutations change a codon to a stop (nonsense) codon, resulting in premature termination of translation, and a truncated (often nonfunctional) protein. Chapter 19 slide 5 Possible effects of changing a single nucleotide base in the coding region of an mRNA chain. Sense mutations or Chapter 19 slide 6 [Sense mutations do not alter genetic code] !! Chapter 19 slide 7 Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings. Fig. 19.3a-d Types of base-pair substitution mutations Chapter 19 slide 8 Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings. Fig. 19.3e-g Types of base-pair substitution mutations Chapter 19 slide 9 Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings. Fig A nonsense mutation and its effect on translation Chapter 19 slide 10 b.Missense mutations have a base-pair change resulting in a different mRNA codon, and therefore a different amino acid in the protein. c.Phenotypic effects may or may not occur, depending on the specific amino acid change. i. Neutral mutations change a codon, but the resulting amino acid substitution produces no detectable change in the function of the protein (e.g., AAA to AGA substitutes arginine for lysine. The amino acids have similar properties, so the proteins function may not be altered). ii. Silent mutations occur when the mutant codon encodes the same amino acid as the wild-type gene, so that no change occurs in the protein produced (e.g., AAA and AAG both encode lysine, so this transition would be silent). 3.Deletions and insertions can change the reading frame of the mRNA downstream of the mutation, resulting in a frameshift mutation. a.When the reading frame is shifted, incorrect amino acids are usually incorporated. b.Frameshifts may bring stop codons into the reading frame, creating a shortened protein. c.Frameshifts may also result in read-through of stop codons, resulting in a longer protein. d.Frameshift mutations result from insertions or deletions when the number of affected base pairs is not divisible by three. Chapter 19 slide 11 Frame-shift mutations as a result of addition or deletion of a base can cause an alteration in the reading frame of mRNA. Chapter 19 slide 12 e-Triplet Repeats mutation: In Cystic fibrosis: there is a three nucleotide deletion from the coding sequence. This will causes a deletion of Phenylalanine. Chapter 19 slide 13 Mutation can be classified according to their site in the gene Chapter 19 slide 14 Mutation in the outside The coding region Mutation in the inside The coding region Chapter 19 slide 15 Chapter 19 slide 16 Reverse Mutations and Suppressor Mutations (omitted) 1.Point mutations are divided into two classes based on their effect on phenotype: a.Forward mutations change the genotype from wild type to mutant. b.Reverse mutations (reversions or back mutations) change the genotype from mutant to wild-type or partially wild-type. Chapter 19 slide 17 Spontaneous and Induced Mutations 1.Most mutations are spontaneous, rather than induced by a mutagen. Chapter 19 slide 18 Spontaneous Mutations 1.All types of point mutations can occur spontaneously. 2.The spontaneous mutation rate in eukaryotes is between to per gene per generation, and in bacteria and phages to gene/generation. -Many spontaneous errors are corrected by the cellular repair systems, and so do not become fixed in DNA. -Spontaneous mutations result from DNA replication errors. Chapter 19 slide 19 DNA Replication Errors 1.DNA replication errors can be either point mutations, or small insertions or deletions. 2.Base-pair substitution mutations can result from wobble pairing. A normal form of the base-pairs with an incorrect partner due to different spatial positioning of the atoms involved in H-bonding.An example is a GC-to-AT transition. a.During DNA replication, G could wobble pair with T, producing a GT pair. b.In the next round of replication, G and A are likely to pair normally, producing one progeny DNA with a GC pair, and another with an AT pair. c.GT pairs are targets for correction by proofreading during replication, and by other repair systems. Only mismatches uncorrected before the next round of replication lead to mutations. Chapter 19 slide 20 Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings. Fig Normal and wobble base pairing in DNA Chapter 19 slide 21 Fig Production of a mutation as a result of a mismatch caused by wobble base pairing Chapter 19 slide Additions and deletions can occur spontaneously during replication : a. DNA loops out from the template strand, generally in a run of the same base. b. DNA polymerase skips the looped out bases, creating a deletion mutation. c. If DNA polymerase adds untemplated base(s), new DNA looping occurs, resulting in additional mutation. d. Insertions and deletions in structural genes generate frameshift mutations (especially if they are not multiples of three). Chapter 19 slide 23 Fig Spontaneous generation of addition and deletion mutants by DNA looping-out errors during replication Chapter 19 slide Spontaneous chemical changes include depurination and deamination of particular bases, creating lesions in the DNA. a. Depurination removes the purine (A or G) from DNA by breaking the bond with its deoxyribose in the backbone. i. Depurination is common. ii. If not repaired before the next round of replication, it will result in a random base at that site. b. Deamination removes an amino group from a base (e.g., cytosine to uracil).i. Uracil is an abnormal base in DNA, and it will usually be repaired. ii. If uracil is not replaced, it will pair with an A during replication iii. Both prokaryotic and eukaryotic DNA have small amounts of 5- methylcytosine (5 m C) in place of the normal C. (1) Deamination of 5 m C produces T. (2) T is a normal nucleotide in DNA, so it is not detected by repair mechanisms. (3) Locations of 5 m C in the chromosome are often detected as mutational hot spots. Chapter 19 slide 25 Fig Deamination of cytosine to uracil (a); deamination of 5-methylcytosine to thymine Chapter 19 slide 26 Induced Mutations 1.Exposure to physical mutagens plays a role in genetic research, where they are used to increase mutation frequencies to provide mutant organisms for study. 2.Radiation (e.g., X rays and UV) induces mutations. a.X rays are an example of ionizing radiation, which penetrates tissue and collides with molecules, knocking electrons out of orbits and creating ions. i. Ions can break covalent bonds, including those in the DNA sugar- phosphate backbone. ii. Ionizing radiation is the leading cause of human gross chromosomal mutations. iii. Ionizing radiation kills cells at high doses, and lower doses produce point mutations. iv. Ionizing radiation has a cumulative effect. A particular dose of radiation results in the same number of mutations whether it is received over a short or a long period of time. Chapter 19 slide 27 b.Ultraviolet (UV) causes photochemical changes in the DNA. i. UV is not energetic enough to induce ionization. ii. UV has lower-energy wavelengths than X rays, and so has limited penetrating power. iii. However, UV in the 254260 nm range is strongly absorbed by purines and pyrimidines, forming abnormal chemical bonds. (1)A common effect is dimer formation between adjacent pyrimidines, commonly thymines (designated T^T). (2)C^C, C^T and T^C dimers also occur, but at lower frequency. Any pyrimidine dimer can cause problems during DNA replication. (3)Most pyrimidine dimers are repaired, because they produce a bulge in the DNA helix. If enough are unrepaired, cell death may result. Chapter 19 slide 28 Fig Production of thymine dimers by ultraviolet light irradiation Chapter 19 slide 29 Chemical Mutagens Mutagenic Effects of 5BU 1. Chemical mutagens may be naturally occurring, or synthetic. They form different groups based on their mechanism of action: a. Base analogs i. Analogs are similar to normal nitrogen bases, and so are incorporated into DNA readily. ii. Once in the DNA, a shift in the analogs form will cause incorrect base pairing during replication, leading to mutation. iii. 5-bromouracil acts as an analog of thymine T Chapter 19 slide 30 Fig c Mutagenic effects of the base analog 5-bromouracil (5BU) Chapter 19 slide 31 b. Base-modifying agents can induce mutations at any stage of the cell cycle. They work by modifying the chemical structure and properties of the bases. Three types are (Figure 19.13): i. Deaminating agents remove amino groups. An example is nitrous acid (HNO 2 ), which deaminates G, C and A. (1) HNO 2 deaminates guanine to produce xanthine, which has the same base pairing as G. No mutation results. (2) HNO 2 deaminates cytosine to produce uracil, (3) HNO 2 deaminates adenine to produce hypoxanthine, which pairs with cytosine ii. Hydroxylating agents include hydroxylamine (NH 2 OH). (1) NH 2 OH specifically modifies C with a hydroxyl group (OH), so that it pairs only with A instead of with G. iii. Allkylating agents. Usually alkylation occurs at the 6-oxygen of G, producing O 6 -allkcylguanine. (1) An example is methylmethane sulfonate (MMS), which methylates G to produce O 6 -alkyl G. Chapter 19 slide 32 Fig a Action of three base-modifying agents Chapter 19 slide 33 Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings. Fig b, c Action of three base-modifying agents Chapter 19 slide 34 c. Intercalating agents insert themselves between adjacent bases in dsDNA. They are generally thin, plate-like hydrophobic molecules. i. At replication, a template that contains an intercalated agent will cause insertion of a random extra base. ii. The base-pair addition is complete after another round of replication, during which the intercalating agent is lost. iii. If an intercalating agent inserts into new DNA in place of a normal base, the next round of replication will result in a deletion mutation. Chapter 19 slide 35 Fig Intercalating mutations Chapter 19 slide 36 Chemical Mutagens in the Environment 1.A wide variety of chemicals exist in our environment, and many can have mutagenic effects. a.Mutagens typically produce base-pair substitutions or insertions or deletions. b.Most cancer development results from accumulated mutations in a number of genes (oncogenes, tumor suppressor). Like tobacco. Chapter 19 slide 37 Auxotrophic Mutations 1.Auxotrophic mutants are easily detected for microorganisms that normally can grow on minimal medium, using methods that have been developed for selection and screening. 2.An example is replica plating. Cells are first grown on supplemented medium, and then the colonies transferred to minimal medium, as well as to a control plate of supplemented medium. Colonies that grow on supplemented, but not minimal, media are selected for further study. Chapter 19 slide 38 Fig Replica-plating technique to screen for mutant strains of a colony-forming microorganism Chapter 19 slide 39 - Heat sensitivity is a common conditional mutation, in which a normal protein is produced at permissive temperature, and a nonfunctional protein results at the nonpermissive temperature. Screening is generally by replica plating and incubation at different temperatures. Heat sensitivity Mutations Chapter 19 slide 40 Resistance Mutations 1.Microorganisms like E. coli and yeast are easily screened for resistance to viruses, chemicals or drugs, because resistant cells will grow when wild-type cells will not. 2Targeted mutations require screening to detect individuals with the desired mutation. Techniques are similar to screens for human disease and DNA typing, and use PCR, restriction enzyme analysis and DNA probing. Chapter 19 slide 41 Recombination Recombination is the process by which genes are rearranged on a chromosome or plasmid (extrachromosomal DNA). Occurs at the level of the chromosome or plasmid. the location of the gene is changed. The base sequence of a gene is unchanged. Chapter 19 slide 42 Mechanism of Recombination 1.Cross-over 2.Transformation 3.Conjugation 4.Transduction Chapter 19 slide 43 Cross-Over Chromosomal crossover (or crossing over) is the exchange of genetic material between homologous chromosomes that results in recombinant chromosomes. It is one of the final phases of genetic recombination, which occurs during prophase I of meiosis. homologous chromosomesgenetic recombinationprophase Imeiosis Chapter 19 slide 44 Chapter 19 slide 45 Transformation 2.Transformation -- process in which a sequence of genes are transferred from a donor bacterial cell to a closely related recipient bacterial cell in the form of DNA in solution. A closely related cell belongs to the same genus or specie as the donor cell. Chapter 19 slide 46 This is a very basic technique that is used on a daily basis in a molecular biological laboratory. The purpose of this technique is to introduce a foreign plasmid into a bacteria and to use that bacteria to amplify the plasmid in order to make large quantities of it. Chapter 19 slide 47 Chapter 19 slide 48 Conjugation 3.Conjugation -- Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. Chapter 19 slide 49 Chapter 19 slide 50 Transduction is the process by which DNA is transferred from one bacterium to another by a virus.DNAbacteriumvirus Chapter 19 slide 51 Thank You