Genetic Theory and Analysis

Danny E. Miller, MD, PhD is an Assistant Professor in the Department of Pediatrics, Division of Genetic Medicine and Laboratory Medicine & Pathology at the University of Washington in Seattle, WA, USA. He is the recipient of the 2017 Larry Sandler Memorial Award, the 2018 Lawrence E. Lamb Prize for Medical Research, and a 2022 National Institutes of Health Director’s Early Independence Award. Dr Miller is a leader in the field of long-read sequencing technology and the use of new technology to evaluate individuals with unsolved genetic disorders. Angela L. Miller is a Research Coordinator at the University of Washington in Seattle, WA, USA, with a background in journalism, visual communications, and molecular biology. She has published several peer-reviewed papers and has won multiple national awards for her work as a journal art director. R. Scott Hawley, PhD is an Investigator at the Stowers Institute for Medical Research, Kansas City, MO, USA. He is a member of the National Academy of Sciences and former President of the Genetics Society of America, with faculty positions at the University of Kansas Medical Center and the University of Missouri-Kansas City. During his distinguished career, Dr. Hawley has mentored hundreds of trainees, received numerous genetics awards, written six textbooks, and published extensively on meiosis.

• Preface xiIntroduction xiii1 Mutation 11.1 Types of Mutations 1Muller’s Classification of Mutants 2Nullomorphs 2Hypomorphs 4Hypermorphs 5Antimorphs 6Neomorphs 8Modern Mutant Terminology 10Loss-of-Function Mutants 10Dominant Mutants 10Gain-of-Function Mutants 11Separation-of-Function Mutants 11DNA-Level Terminology 11Base-Pair-Substitution Mutants 11Base-Pair Insertions or Deletions 12Chromosomal Aberrations 121.2 Dominance and Recessivity 13The Cellular Meaning of Dominance 13The Cellular Meaning of Recessivity 15Difficulties in Applying the Terms Dominant and Recessive to Sex-Linked Mutants 16The Genetic Utility of Dominant and Recessive Mutants 171.3 Summary 17References 172 Mutant Hunts 202.1 Why Look for New Mutants? 20Reason 1: To Identify Genes Required for a Specific Biological Process 21Reason 2: To Isolate more Mutations in a Specific Gene of Interest 31Reason 3: To Obtain Mutants for a Structure-Function Analysis 32Reason 4: To Isolate Mutations in a Gene So Far Identified only by Computational Approaches 322.2 Mutagenesis and Mutational Mechanisms 32Method 1: Ionizing Radiation 33Method 2: Chemical Mutagens 33Alkylating Agents 34Crosslinking Agents 35Method 3: Transposons 35Identifying Where Your Transposon Landed 37Why not Always Screen With TEs? 40Method 4: Targeted Gene Disruption 40RNA Interference 40CRISPR/Cas9 41TALENs 42So Which Mutagen Should You Use? 432.3 What Phenotype Should You Screen (or Select) for? 442.4 Actually Getting Started 45Your Starting Material 45Pilot Screen 45What to Keep? 45How many Mutants is Enough? 46Estimating the Number of Genes not Represented by Mutants in Your New Collection 462.5 Summary 48References 483 Complementation 513.1 The Essence of the Complementation Test 513.2 Rules for Using the Complementation Test 55The Complementation Test Can be Done Only When Both Mutants are Fully Recessive 55The Complementation Test Does Not Require that the Two Mutants Have Exactly the Same Phenotype 56The Phenotype of a Compound Heterozygote Can be More Extreme than that of Either Homozygote 563.3 How the Complementation Test Might Lie to You 57Two Mutations in the Same Gene Complement Each Other 57A Mutation in One Gene Silences Expression of a Nearby Gene 57Mutations in Regulatory Elements 593.4 Second-Site Noncomplementation (Nonallelic Noncomplementation) 59Type 1 SSNC (PoisonousInteractions): The Interaction is Allele Specific at Both Loci 60An Example of Type 1 SSNC Involving the Alpha- and Beta-Tubulin Genes in Yeast 60An Example of Type 1 SSNC Involving the Actin Genes in Yeast 62Type 2 SSNC (Sequestration): The Interaction is Allele Specific at One Locus 66An Example of Type 2 SSNC Involving the Tubulin Genes in Drosophila 66An Example of Type 2 SSNC in Drosophila that Does Not Involve the Tubulin Genes 69An Example of Type 2 SSNC in the Nematode Caenorhabditis elegans 71Type 3 SSNC (Combined Haploinsufficiency): The Interaction is Allele-Independent at Both Loci 72An Example of Type 3 SSNC Involving Two Motor Protein Genes in Flies 72Summary of SSNC in Model Organisms 72SSNC in Humans (Digenic Inheritance) 73Pushing the Limits: Third-Site Noncomplementation 743.5 An Extension of SSNC: Dominant Enhancers 74A Successful Screen for Dominant Enhancers 753.6 Summary 76References 774 Meiotic Recombination 814.1 An Introduction to Meiosis 81A Cytological Description of Meiosis 88A More Detailed Description of Meiotic Prophase 894.2 Crossing Over and Chiasmata 924.3 The Classical Analysis of Recombination 934.4 Measuring the Frequency of Recombination 96The Curious Relationship Between the Frequency of Recombination and Chiasma Frequency 97Map Lengths and Recombination Frequency 97The Mapping Function 99Tetrad Analysis 100Statistical Estimation of Recombination Frequencies 101Two-Point Linkage Analysis 101What Constitutes Statistically Significant Evidence for Linkage? 104An Example of LOD Score Analysis 105Multipoint Linkage Analysis 105Local Mapping via Haplotype Analysis 106The Endgame 108The Actual Distribution of Exchange Events 109The Centromere Effect 110The Effects of Heterozygosity for Aberration Breakpoints on Recombination 110Practicalities of Mapping 1104.5 The Mechanism of Recombination 111Gene Conversion 111Early Models of Recombination 112The Holliday Model 112The Meselson–Radding Model 114The Currently Accepted Mechanism of Recombination: The Double-Strand Break Repair Model 114Class I Versus Class II Recombination Events 1164.6 Summary 117References 1185 Identifying Homologous Genes 1265.1 Homology 126Orthologs 127Paralogs 127Xenologs 1285.2 Identifying Sequence Homology 128Nucleotide–Nucleotide BLAST (blastn) 129An Example Using blastn 129Translated Nucleotide–Protein BLAST (blastx) 131An Example Using blastx 131Protein–Protein BLAST (blastp) 132An Example Using blastp 132Translated BLASTx (tblastx) and Translated BLASTn (tblastn) 1335.3 How Similar is Similar? 1335.4 Summary 134References 1346 Suppression 1366.1 Intragenic Suppression 137Intragenic Suppression of Loss-of-Function Mutations 137Intragenic Suppression of a Frameshift Mutation by the Addition of a Second, Compensatory Frameshift Mutation 138Intragenic Suppression of Missense Mutations by the Addition of a Second and Compensatory Missense Mutation 140Intragenic Suppression of Antimorphic Mutations that Produce a Poisonous Protein 1416.2 Extragenic Suppression 1416.3 Transcriptional Suppression 141Suppression at the Level of Gene Expression 142A CRISPR Screen for Suppression of Inhibitor Resistance in Melanoma 142Suppression of Transposon-Insertion Mutants by Altering the Control of mRNA Processing 143Suppression of Nonsense Mutants by Messenger Stabilization 1436.4 Translational Suppression 144tRNA-Mediated Nonsense Suppression 144The Numerical and Functional Redundancy of tRNA Genes Allows Suppressor Mutations to be Viable 146tRNA-Mediated Frameshift Suppression 1466.5 Suppression by Post-Translational Modification 1476.6 Conformational Suppression: Suppression as a Result of Protein–Protein Interaction 147Searching for Suppressors that Act by Protein–Protein Interaction in Eukaryotes 148Actin and Fimbrin in Yeast 148Mediator Proteins and RNA Polymerase II in Yeast 150“Lock-and-key” Conformational Suppression 152Suppression of a Flagellar Motor Mutant in E. coli 152Suppression of a Mutant Transporter Gene in C. elegans 152Suppression of a Telomerase Mutant in Humans 1536.7 Bypass Suppression: Suppression Without Physical Interaction 154“Push me, Pull You” Bypass Suppression 155Multicopy Bypass Suppression 1566.8 Suppression of Dominant Mutations 1576.9 Designing Your Own Screen for Suppressor Mutations 1576.10 Summary 158References 1587 Epistasis Analysis 1637.1 Ordering Gene Function in Pathways 163Biosynthetic Pathways 164Nonbiosynthetic Pathways 1657.2 Dissection of Regulatory Hierarchies 167Epistasis Analysis Using Mutants with Opposite Effects on the Phenotype 167Hierarchies for Sex Determination in Drosophila 169Epistasis Analysis Using Mutants with the Same or Similar Effects on the Final Phenotype 170Using Opposite-Acting Conditional Mutants to Order Gene Function by Reciprocal Shift Experiments 170Using a Drug or Agent that Stops the Pathway at a Given Point 170Exploiting Subtle Phenotypic Differences Exhibited by Mutants that Affect the Same Signal State 1727.3 How Might an Epistasis Experiment Mislead You? 1727.4 Summary 173References 1738 Mosaic Analysis 1758.1 Tissue Transplantation 176Early Tissue Transplantation in Drosophila 176Tissue Transplantation in Zebrafish 1778.2 Mitotic Chromosome Loss 178Loss of the Unstable Ring-X Chromosome 179Other Mechanisms of Mitotic Chromosome Loss 179Mosaics Derived from Sex Chromosome Loss in Humans and Mice (Turner Syndrome) 1808.3 Mitotic Recombination 181Gene Knockout Using the FLP/FRT or Cre-Lox Systems 1828.4 Tissue-Specific Gene Expression 184Gene Knockdown Using RNAi 184Tissue-Specific Gene Editing Using CRISPR/Cas9 1858.5 Summary 187References 1889 Meiotic Chromosome Segregation 1919.1 Types and Consequences of Failed Segregation 1929.2 The Origin of Spontaneous Nondisjunction 193MI Exceptions 194MII Exceptions 1949.3 The Centromere 195The Isolation and Analysis of the Saccharomyces cerevisiae Centromere 195The Isolation and Analysis of the Drosophila Centromere 198The Concept of the Epigenetic Centromere in Drosophila and Humans 200Holocentric Chromosomes 2019.4 Chromosome Segregation Mechanisms 202Chiasmate Chromosome Segregation 202Segregation Without Chiasmata (Achiasmate Chromosome Segregation) 203Achiasmate Segregation in Drosophila Males 203Achiasmate Segregation in D. melanogaster Females 204Achiasmate Segregation in S. cerevisiae 205Achiasmate Segregation in S. pombe 207Achiasmate Segregation in Silkworm Females 2079.5 Meiotic Drive 207Meiotic Drive Via Spore Killing 207An Example in Schizosaccharomyces pombe 207An Example in D. melanogaster 208Meiotic Drive Via Directed Segregation 2089.6 Summary 210References 210Appendix A: Model Organisms 215Appendix B: Genetic Fine-Structure Analysis 228Appendix C: Tetrad Analysis 250Glossary 262Index 275