Genetic Theory and Analysis Finding Meaning in a Genome

by Miller, Danny E.; Miller, Angela L.; Hawley, R. Scott

• ISBN13: 9781118086926
• ISBN10: 1118086929
• Edition: 2nd
• Format: Paperback
• Copyright: 2023-08-29
• Publisher: Wiley

Summary

GENETIC THEORY AND ANALYSIS

Understand and apply what drives change of characteristic genetic traits and heredity

Genetics is the study of how traits are passed from parents to their offspring and how the variation in those traits affects the development and health of the organism. Investigating how these traits affect the organism involves a diverse set of approaches and tools, including genetic screens, DNA and RNA sequencing, mapping, and methods to understand the structure and function of proteins. Thus, there is a need for a textbook that provides a broad overview of these methods.

Genetic Theory and Analysis meets this need by describing key approaches and methods in genetic analysis through a historical lens. Focusing on the five basic principles underlying the field—mutation, complementation, recombination, segregation, and regulation—it identifies the full suite of tests and methodologies available to the geneticist in an age of flourishing genetic and genomic research. This second edition of the text has been updated to reflect recent advances and increase accessibility to advanced undergraduate students.

Genetic Theory and Analysis, 2nd edition readers will also find:

• Detailed treatment of subjects including mutagenesis, meiosis, complementation, suppression, and more
• Updated discussion of epistasis, mosaic analysis, RNAi, genome sequencing, and more
• Appendices discussing model organisms, genetic fine-structure analysis, and tetrad analysis

Genetic Theory and Analysis is ideal for both graduate students and advanced undergraduates undertaking courses in genetics, genetic engineering, and computational biology.

Author Biography

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.

Table of Contents

Preface

 

Introduction

 

Chapter 1: Mutation

This chapter describes different types of mutations and the various terminology used to describe mutations.

1.1  Types of Mutations

• Muller’s classification of mutants

–   Nullomorphs

–   Hypomorphs          

–   Hypermorphs

–   Antimorphs

–   Neomorphs

• Modern mutant terminology

–   Loss-of-function mutants

–   Dominant mutants

–   Gain-of-function mutants

–   Separation-of-function mutants

• DNA-level terminology

–   Base-pair-substitution mutants

–   Base-pair insertions or deletions

–   Chromosomal aberrations

1.2 Dominance and recessivity

• The cellular meaning of dominance
• The cellular meaning of recessivity
• Difficulties in applying the terms dominant and recessive to sex-linked mutants
• The genetic utility of dominant and recessive mutants

Summary

Boxes

• Box 1.1 DNA-level terminology
• Box 1.2 Detecting gene expression by RNA-seq
• Box 1.3 De novo mutation

References

 

Chapter 2: Mutant Hunts

This chapter describes why identifying new genetic mutants is useful, ways to create mutants, and how to screen for mutant phenotypes.

2.1 Why look for new mutants?

• Reason 1: To identify genes required for a specific biological process
• Reason 2: To isolate more mutations in a specific gene of interest
• Reason 3: To obtain mutants for a structure-function analysis
• Reason 4: To isolate mutations in a gene so far identified only by computational approaches

2.2 Mutagenesis and mutational mechanisms

• Method 1: Ionizing radiation
• Method 2: Chemical mutagens

–   Alkylating agents

–   Crosslinking agents

• Method 3: Transposons

–   Identifying where your transposon landed

–   Why not always screen with TEs?

• Method 4: Targeted gene disruption

–   RNA interference

–   CRISPR/Cas9

–   TALENs

• So which mutagen should you use?

2.3 What phenotype should you screen (or select) for?

2.4 Actually getting started

• Your starting material
• Pilot screen
• What to keep?
• How many mutants is enough?

–   Estimating the number of genes not represented by mutants in your new collection

Summary

Boxes

• Box 2.1 A screen for embryonic lethal mutations in Drosophila
• Box 2.2 A screen for sex-linked lethal mutations in Drosophila

–   Objective

–   Basic stocks

–   The screen itself

–   A complication

• Box 2.3 The balancer chromosome
• Box 2.4 De novo genome and transcriptome assembly
• Box 2.5 Identifying new transposon insertion sites

References

 

Chapter 3: Complementation

This chapter describes methods for determining whether mutants isolated in a genetic screen are novel.

3.1 The essence of the complementation test

3.2 Rules for using the complementation test

• The complementation test can be done only when both mutants are fully recessive
• The complementation test does not require that the two mutants have exactly the same phenotype
• There are cases where the phenotype of a compound heterozygote is more extreme than is that of either homozygote

3.3 How might the complementation test lie to you?

• Two mutations in the same gene complement each other
• A mutation in one gene silences expression of a nearby gene
• Mutations in regulatory elements

3.4 Second-site noncomplementation (nonallelic noncomplementation)

• Type 1 SSNC (poisonous interactions): the interaction is allele specific at both loci

–   An example of type 1 SSNC involving the alpha- and beta-tubulin genes in yeast

–   An example of type 1 SSNC involving the actin genes in yeast

• Type 2 SSNC (sequestration): the interaction is allele specific at one locus

–   An example of type 2 SSNC involving the tubulin genes in Drosophila

–   An example of type 2 SSNC in Drosophila that does not involve the tubulin genes

–   An example of type 2 SSNC in the nematode Caenorhabditis elegans

• Type 3 SSNC (combined haploinsufficiency): the interaction is allele independent at both loci

–   An example of type 3 SSNC involving two motor protein genes in flies

• Summary of SSNC in model organisms
• SSNC in humans (digenic inheritance)
• Pushing the limits: third-site noncomplementation

3.5 An extension of SSNC: dominant enhancers

• A successful screen for dominant enhancers

Summary

Boxes

• Box 3.1 A more rigorous definition of the complementation test
• Box 3.2 An example of using the complementation test in yeast
• Box 3.3 Transformation rescue is a variant of the complementation test
• Box 3.4 A method for determining whether a dominant mutation is an allele of a given gene
• Box 3.5 Pairing-dependent complementation: transvection
• Box 3.6 Synthetic lethality and genetic buffering

References

 

Chapter 4: Recombination

This chapter provides a description of meiotic recombination and how it is used to map the genomic regions affected by novel mutations.

4.1 An introduction to meiosis

• A cytological description of meiosis
• A more detailed description of meiotic prophase

4.2 Crossing over and chiasmata

4.3 The classical analysis of recombination

4.4 Measuring the frequency of recombination

• The curious relationship between the frequency of recombination and chiasma frequency
• Map lengths and recombination frequency

–   The mapping function

• Tetrad analysis
• Statistical estimation of recombination frequencies

–   Two-point linkage analysis

–   What constitutes statistically significant evidence for linkage?

–   An example of LOD score analysis

–   Multipoint linkage analysis

–   Local mapping via haplotype analysis

–   The endgame

• The actual distribution of exchange events
• The centromere effect
• The effects of heterozygosity for aberration breakpoints on recombination
• Practicalities of mapping

4.5 The mechanism of recombination

• Gene conversion
• Early models of recombination

–   The Holliday model

–   The Meselson-Radding model

• The currently accepted mechanism of recombination: the double-strand break repair model
• Class I versus class II recombination events

Summary

Boxes

• Box 4.1 The molecular biology of synapsis
• Box 4.2 Do specific chromosomal sites mediate pairing?

–   The role of telomeres in early pairing

–   The role of centric heterochromatin in chromosome pairing

–   Specific pairing sites in C. elegans

–   Specific euchromatic pairing sites in Drosophila

• Box 4.3 Crossing over in compound-X chromosomes
• Box 4.4 Does any sister-chromatid exchange occur during meiosis?

–   Genetic studies in yeast

–   Genetic studies in Drosophila

–   A direct molecular assessment in yeast

References

 

Chapter 5: Finding Homologous Genes

This chapter describes methods for determining whether a gene of interest identified in one organism has been described in another organism.

5.1 Homology

• Orthologs
• Paralogs
• Xenologs

5.2 Identifying sequence homology

• Nucleotide–nucleotide BLAST (blastn)

–   An example using blastn

• Translated nucleotide–protein BLAST (blastx)

–   An example using blastx

• Protein–protein BLAST (blastp)

–   An example using blastp

• Translated BLASTx (tblastx) and translated BLASTn (tblastn)

5.3 How similar is similar?

Summary

References

 

Chapter 6: Suppression

This chapter discusses how a mutant might suppress the phenotype of another mutant and how to screen for such suppressor mutants.

6.1 Intragenic suppression

• Intragenic suppression of loss-of-function mutations

–   Intragenic suppression of a frameshift mutation by the addition of a second, compensatory frameshift mutation

–   Intragenic suppression of missense mutations by the addition of a second and compensatory missense mutation

–   Intragenic suppression of antimorphic mutations that produce a poisonous protein

6.2 Extragenic suppression

6.3 Transcriptional suppression

• Suppression at the level of gene expression
• A CRISPR screen for suppression of inhibitor resistance in melanoma
• Suppression of transposon-insertion mutants by altering the control of mRNA processing
• Suppression of nonsense mutants by messenger stabilization

6.4 Translational suppression

• tRNA-mediated nonsense suppression

–   The numerical and functional redundancy of tRNA genes allows suppressor mutations to be viable

• tRNA-mediated frameshift suppression

6.5 Suppression by post-translational modification

6.6 Conformational suppression: suppression as a result of protein–protein interaction

• Searching for suppressors that act by protein-protein interaction in eukaryotes

–   Actin and fimbrin in yeast

–   Mediator proteins and RNA polymerase II in yeast

• “Lock-and-key” conformational suppression

–   Suppression of a flagellar motor mutant in E. coli

–   Suppression of a mutant transporter gene in C. elegans

–   Suppression of a telomerase mutant in humans

6.7 Bypass suppression: suppression without physical interaction

• “Push me, pull you” bypass suppression
• Multicopy bypass suppression

6.8 Suppression of dominant mutations

6.9 Designing your own screen for suppressor mutations

Summary

Boxes

• Box 6.1 Bypass suppression of a telomere defect in the yeast S. pombe

References

 

Chapter 7: Epistasis Analysis

This chapter describes methods for determining whether different genes function in the same biological pathway and, if so, the order in which they function.

7.1 Ordering gene function in pathways

• Biosynthetic pathways
• Nonbiosynthetic pathways

7.2 Dissection of regulatory hierarchies

• Epistasis analysis using mutants with opposite effects on the phenotype

–   Hierarchies for sex determination in Drosophila

• Epistasis analysis using mutants with the same or similar effects on the final phenotype

–   Using opposite-acting conditional mutants to order gene function by reciprocal shift experiments

–   Using a drug or agent that stops the pathway at a given point

–   Exploiting subtle phenotypic differences exhibited by mutants that affect the same signal state

7.3 How might an epistasis experiment mislead you?

Summary

References

 

Chapter 8: Mosaic Analysis

This chapter describes methods for determining in which tissue(s) or at what stage(s) of development a given gene functions.

8.1 Tissue transplantation

• Early tissue transplantation in Drosophila
• Tissue transplantation in zebrafish

8.2 Mitotic chromosome loss

• Loss of the unstable ring X chromosome
• Other mechanisms of mitotic chromosome loss
• Mosaics derived from sex chromosome loss in humans and mice (Turner syndrome)

8.3 Mitotic recombination

• Gene knockout using the FLP/FRT or Cre-Lox systems

8.4 Tissue-specific gene expression   

• Gene knockdown using RNAi
• Tissue-specific gene editing using CRISPR/Cas9

Summary

Boxes

• Box 8.1 The ethics of targeted gene editing in humans

References

 

Chapter 9: Meiotic Chromosome Segregation

This chapter describes the mechanisms that ensure meiotic chromosome segregation, which is the physical basis of Mendelian inheritance.

9.1 Types and consequences of failed segregation

9.2 The origin of spontaneous nondisjunction

• MI exceptions
• MII exceptions

9.3 The centromere

• The isolation and analysis of the S. cerevisiae centromere
• The isolation and analysis of the Drosophila centromere
• The concept of the epigenetic centromere in Drosophila and humans
• Holocentric chromosomes

9.4 Chromosome segregation mechanisms

• Chiasmate chromosome segregation
• Segregation without chiasmata (achiasmate chromosome segregation)

–   Achiasmate segregation in Drosophila males

–   Achiasmate segregation in Drosophila females

–   Achiasmate segregation in S. cerevisiae

–   Achiasmate segregation in S. pombe

–   Achiasmate segregation in silkworm females

9.5 Meiotic drive

• Meiotic drive via spore killing

–   An example in Schizosaccharomyces pombe

–   An example in Drosophila melanogaster

• Meiotic drive via directed segregation

Summary

Boxes

• Box 9.1 Identifying genes that encode centromere-binding proteins in yeast
• Box 9.2 Achiasmate heterologous segregation in Drosophila females

Figures

References

 

Appendix A: Model Organisms

This appendix presents useful information for performing genetic analyses in the various model organisms mentioned throughout this book.

A.1 Budding yeast: Saccharomyces cerevisiae

• Basic culture techniques
• Nomenclature
• Chromosome biology
• Useful guides and manuals

A.2 Plants: Arabidopsis thaliana

• Basic culture techniques
• Nomenclature
• Chromosome biology
• Useful guides and manuals

A.3 Worms: Caenorhabditis elegans

• Basic culture techniques
• Nomenclature
• Chromosome biology
• Useful guides and manuals

A.4 Fruit flies: Drosophila melanogaster

• Basic culture techniques
• Nomenclature
• Chromosome biology
• Useful guides and manuals

A.5 Zebrafish: Danio rerio

• Basic culture techniques
• Nomenclature
• Chromosome biology
• Useful guides and manuals

A.6 Mice: Mus musculus

• Basic culture techniques
• Nomenclature
• Chromosome biology
• Useful guides and manuals

A.7 Phage lambda

• Nomenclature
• Useful guides and manuals

 

Appendix B: Genetic Fine-Structure Analysis

This appendix describes the classical approach to mapping the exact location of a mutation within a gene.

B.1 Intragenic mapping (then)

• The first efforts toward finding structure within a gene
• The unit of recombination and mutation is the base pair

B.2 Intragenic complementation meets intragenic recombination: the basis of fine-structure analysis

• The formal analysis of intragenic complementation

B.3 Fine-structure analysis of a eukaryotic gene encoding a multifunctional protein   

• Genetic and functional dissection of the HIS4 gene in yeast
• Genetic and functional dissection of the rudimentary gene in Drosophila

B.4 Fine-structure analysis of genes with complex regulatory elements in eukaryotes

• Genetic and functional dissection of the cut gene in Drosophila

B.5 Pairing-dependent intragenic complementation

• Genetic and functional dissection of the yellow gene in Drosophila
• The influence of the zeste gene on pairing-dependent complementation at the white locus in Drosophila
• Genetic and functional dissection of the bithorax complex in Drosophila

Summary

References

 

Appendix C: Tetrad Analysis

This appendix describes approaches for measuring map length and the frequency of recombination.

C.1 Tetrad analysis in linear asci

C.2 Unordered tetrad analysis

C.3 Half-tetrad analysis

C.4 Algebraic tetrad analysis

• A simple example of algebraic tetrad analysis
• A more complicated example of algebraic tetrad analysis

Boxes

• Box C.1 Using tetrad analysis to determine linkage
• Box C.2 Mapping centromeres in fungi with unordered tetrads

References

 

Glossary

 

Index