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

 

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