Chapter 7: Anatomy and Function of a Gene: Dissection Through Mutation Chapter of the textbook: Genetics: From Genes to Genomes, 4th edition (2011), Hartwell H et al CHAPTER OUTLINE: 7.1 Mutations: Primary Tools of Genetic Analysis 7.2 What Mutations Tell Us About Gene Structure 7.3 What Mutations Tell Us About Gene Function 7.4 A Comprehensive Example: Mutations That Affect Vision VNU-University of Science - DNThai 7.1 Mutations: Primary tools of genetic analysis • Mutations are heritable changes in DNA base sequences • Forward mutation – changes wild-type allele to a different allele – e.g A+  a or b+  B • Reverse mutation (reversion) – changes a mutant allele back to wild type – e.g a  A+ or B  b+ • Forward mutation rate is usually greater than reversion rate VNU-University of Science - DNThai Classification of mutations by effect on DNA molecule • Substitution – replacement of a base by another base – Transition – purine replaced by another purine, or pyrimidine replaced by another pyrimidine – Transversion – purine replaced by a pyrimidine, or pyrimidine replaced by a purine • Deletion – block of or more bp lost from DNA • Insertion – block of or more bp added to DNA • Inversion – 180° rotation of a segment of DNA • Reciprocal translocation – parts of two nonhomologous chromosomes change places VNU-University of Science - DNThai Mutations classified by their effect on DNA Fig 7.2 a-c VNU-University of Science - DNThai Mutations classified by their effect on DNA (2) Fig 7.2d VNU-University of Science - DNThai Mutations classified by their effect on DNA (3) Fig 7.2e VNU-University of Science - DNThai Rates of spontaneous mutation • Mutant mouse coat colors: albino VNU-University of Science - DNThai brown Rates of spontaneous mutation • Rates of recessive forward mutations at five coat color genes in mice – 11 mutations per gene every 106 gametes • Mutation rates in other organisms – to 12 mutations per gene every 106 gametes Fig 7.3b VNU-University of Science - DNThai General observations of mutation rates • Mutations affecting phenotype occur very rarely • Different genes mutate at different rates • Rate of forward mutation is almost always higher than rate of reverse mutation • Average mutation rate in gamete-producing eukaryotes is higher than that of prokaryotes – Many cell divisions take place between zygote formation and meiosis in germ cells (More chance to accumulate mutations) • Can diploid organisms tolerate more mutations than haploid organisms? VNU-University of Science - DNThai Experimental evidence that mutations in bacteria occur spontaneously • S Luria and M Delbrück (1943) − fluctuation test • Examined origin of bacterial resistance to phage infection • Infected wild-type bacteria with phage • Majority of cells die, but a few cells can grow and divide – Had the cells altered biochemically? – Did the cells carry heritable mutations for resistance? – Did the mutations arise by chance or did they arise in response to the phage? VNU-University of Science - DNThai 10 7.2 What Mutations Tell Us About Gene Structure • Complementation testing – Reveals whether two mutations are in a single gene or in different genes – "Complementation group" is synonymous with a gene • Fine structure mapping – Seymour Benzer used phage T4 mutants – Experimental evidence that a gene is a linear sequence of nucleotide pairs – Some regions of chromosomes have "hot spots" for mutations VNU-University of Science - DNThai 42 Drosophila eye color mutations produce a variety of phenotypes •Flies carrying different X-linked eye color mutations From the left: ruby, white, and apricot; a wild-type eye is at the far right •Do these phenotypes result from allelic mutations or from mutations in different genes? Fig 7.17 VNU-University of Science - DNThai 43 Complementation testing of Drosophila eye color mutations • (a) A heterozygote has one mutation (m 1) on one chromosome and a different mutation (m 2) on its homolog If the mutations are in different genes, the heterozygote will be wild type; the mutations complement each other (left) If both mutations affect the same gene, the phenotype will be mutant; the mutations not complement each other (right) Complementation testing makes sense only when both mutations are recessive to wild type VNU-University of Science - DNThai Fig 7.18a 44 A complementation table for X-linked eye color mutations in Drosophila • (b) This complementation table reveals five complementation groups (fi ve different genes) for eye color A “+” indicates mutant combinations with wild type eye color; these mutations complement and are thus in different genes Several mutations fail to complement (–) and are thus alleles of one gene, white VNU-University of Science - DNThai Fig 7.18b 45 A complementation table for X-linked eye color mutations in Drosophila (2) • (c) Recombination mapping shows that mutations in different genes are often far apart, while different mutations in the same gene are very close together VNU-University of Science - DNThai Fig 7.18 c 46 A gene is a linear sequence of nucleotide pairs • Seymore Benzer mid 1950s – 1960s – If a gene is a linear set of nucleotides, recombination between homologous chromosomes carrying different mutations within the same gene should generate wild-type – T4 phage as an experimental system: • Can examine a large number of progeny to detect rare mutation events • Easy to produce large numbers of progeny to detect rare events • Could allow only recombinant phage to proliferate while parental phages die VNU-University of Science - DNThai 47 How recombination within a gene could generate a wild-type allele Fig 7.19 VNU-University of Science - DNThai 48 Working with bacteriophage T4 (a.1) (a.2) (a.3) Fig 7.20a VNU-University of Science - DNThai 49 Counting bacteriophages by serial dilution (a.4) Fig 7.20a VNU-University of Science - DNThai 50 Phenotypic properties of rII – mutants of bacteriophage T4 (cont) • rll – mutants, when plated on E coli B cells, produce plaques that are larger and more distinct (with sharper edges) than plaques formed by rll + wild-type phage • rll – mutants are particularly useful for looking at rare recombination events because they have an altered host range In trast to rll + wild-type phages, rll – mutants cannot form plaques in lawns of E coli strain K(l) host bacteria VNU-University of Science - DNThai Fig 7.20b 51 Benzer's experimental approach to fine structure mapping of the rII locus Generated 1612 spontaneous point mutations and several deletions in rII locus • Identified two complementation groups, rIIA and rIIB (Fig 7.20c) • Mapped locations of deletions relative to each other using recombination (Fig 7.21a) • Mapped locations of point mutations relative to the deletions (Fig 7.21a) • Tested for recombination between all point mutations within the same complementation group (Fig 7.20d) VNU-University of Science - DNThai 52 A customized complementation test between rII – mutants of bacteriophage T4 (c.1) Complementation test (trans configuration) VNU-University of Science - DNThai (c.2) Control (cis configuration) Fig 7.20c 53 Detecting recombination between two allelic mutations (d.1) Recombination test (d.2) Control Fig 7.20d VNU-University of Science - DNThai 54 Using deletions for rapid mapping Fig 7.21a VNU-University of Science - DNThai 55 Fine structure of the phage T4 rII region Mutation hotspots suggest that some nucleotides are more susceptible to mutations than others VNU-University of Science - DNThai 56 ... DNThai General observations of mutation rates • Mutations affecting phenotype occur very rarely • Different genes mutate at different rates • Rate of forward mutation is almost always higher than... VNU-University of Science - DNThai Rates of spontaneous mutation • Mutant mouse coat colors: albino VNU-University of Science - DNThai brown Rates of spontaneous mutation • Rates of recessive forward mutations...7.1 Mutations: Primary tools of genetic analysis • Mutations are heritable changes in DNA base sequences • Forward mutation – changes wild-type allele to a different allele – e.g A+  a or b+