Download Genetics - Semantic Scholar

Genetics
Toby Dylan Hocking
based on lectures by
Sharon Amacher
Tom Kline
Fyodor Urnov
Molecular and Cell Biology 140
UC Berkeley
Spring Semester 2005
May 16, 2005
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Contents
1 Classical and Molecular Genetics
1.1 Mitosis and Meiosis . . . . . . . . . . . . . . . . . . . . . . .
1.1.1 Stages of the Cell Cycle . . . . . . . . . . . . . . . .
1.1.2 Definitions . . . . . . . . . . . . . . . . . . . . . . . .
1.1.3 Phases of Mitosis . . . . . . . . . . . . . . . . . . . .
1.1.4 Phases of Meiosis . . . . . . . . . . . . . . . . . . . .
1.1.5 Comparing Mitosis and Meiosis . . . . . . . . . . . .
1.2 Background of Mendel . . . . . . . . . . . . . . . . . . . . .
1.3 Elementary Genetic Analysis . . . . . . . . . . . . . . . . . .
1.3.1 Monohybrid Cross . . . . . . . . . . . . . . . . . . .
1.3.2 Dihybrid Cross . . . . . . . . . . . . . . . . . . . . .
1.3.3 Test Cross . . . . . . . . . . . . . . . . . . . . . . . .
1.4 Complications to Basic Genetics . . . . . . . . . . . . . . . .
1.4.1 Incomplete Dominance . . . . . . . . . . . . . . . . .
1.4.2 Codominance . . . . . . . . . . . . . . . . . . . . . .
1.4.3 Recessive Lethality . . . . . . . . . . . . . . . . . . .
1.5 Sex Determination Proves Chromosomal Inheritance . . . . .
1.5.1 Sex Detemination Summary . . . . . . . . . . . . . .
1.5.2 Nomenclature in Drosophila . . . . . . . . . . . . . .
1.5.3 Establishing Sex Linkage . . . . . . . . . . . . . . . .
1.5.4 Primary Nondisjunction . . . . . . . . . . . . . . . .
1.5.5 2◦ Nondisjunction . . . . . . . . . . . . . . . . . . . .
1.5.6 Barred Phenotype Crosses Reveal Meiosis I as Point of
Nondisjunction . . . . . . . . . . . . . . . . . . . . .
1.6 Pedigree Analysis . . . . . . . . . . . . . . . . . . . . . . . .
1.6.1 Autosomal Dominant . . . . . . . . . . . . . . . . . .
1.6.2 Autosomal Recessive . . . . . . . . . . . . . . . . . .
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CONTENTS
1.7
1.8
1.9
1.10
1.11
1.12
1.13
1.14
1.15
1.6.3 X-Linked Recessive . . . . . . . . . . .
1.6.4 X-Linked Dominant . . . . . . . . . . .
Linkage . . . . . . . . . . . . . . . . . . . . .
1.7.1 X-Linked Mutant Cross . . . . . . . .
1.7.2 Autosomal Mutant Cross . . . . . . . .
1.7.3 χ2 Test of Linkage . . . . . . . . . . .
1.7.4 Summary of Linkage . . . . . . . . . .
Genetic Mapping . . . . . . . . . . . . . . . .
1.8.1 Mapping 5 Genes With 2-Point Crosses
1.8.2 Mapping 3 Genes With 3-Point Crosses
1.8.3 Interference . . . . . . . . . . . . . . .
Tetrad Analysis . . . . . . . . . . . . . . . . .
1.9.1 Fungi As A Model . . . . . . . . . . .
1.9.2 Meiosis in S. cerevisiae . . . . . . . . .
1.9.3 Genetics of S. cerevisiae . . . . . . . .
1.9.4 Recombination Frequency . . . . . . .
1.9.5 Neurospora crassa . . . . . . . . . . .
Recombination Mechanisms . . . . . . . . . .
1.10.1 Physical Exchange . . . . . . . . . . .
1.10.2 Breaking and Rejoining . . . . . . . . .
1.10.3 Gene Conversion . . . . . . . . . . . .
1.10.4 Models of Recombination . . . . . . . .
Pathway Dissections . . . . . . . . . . . . . .
1.11.1 One Gene-One Protein . . . . . . . . .
1.11.2 Arg Mutants . . . . . . . . . . . . . .
Complementation Test . . . . . . . . . . . . .
Zebrafish . . . . . . . . . . . . . . . . . . . . .
1.13.1 Modeling Development . . . . . . . . .
1.13.2 Development . . . . . . . . . . . . . .
1.13.3 Alternate Complementation Test . . .
1.13.4 Haploid Embryos . . . . . . . . . . . .
1.13.5 Expression Screen . . . . . . . . . . . .
1.13.6 Half Tetrads . . . . . . . . . . . . . . .
Chromosomal Rearrangements . . . . . . . . .
1.14.1 Origins of Mutations . . . . . . . . . .
1.14.2 Types . . . . . . . . . . . . . . . . . .
Deletions . . . . . . . . . . . . . . . . . . . . .
1.15.1 Overview . . . . . . . . . . . . . . . .
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CONTENTS
1.16
1.17
1.18
1.19
1.20
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1.15.2 Allele Screens . . . . . . . . .
1.15.3 Deletion Characteristics . . .
Duplications . . . . . . . . . . . . . .
Inversions . . . . . . . . . . . . . . .
1.17.1 Pericentric Inversion . . . . .
1.17.2 Paracentric Inversion . . . . .
1.17.3 Diagnostics for Inversions . .
1.17.4 Utility of Inversions . . . . . .
Translocations . . . . . . . . . . . . .
1.18.1 Overview . . . . . . . . . . .
1.18.2 Diagnostics of Translocations
1.18.3 Robertsonian Translocation .
Ploidy . . . . . . . . . . . . . . . . .
1.19.1 Terms . . . . . . . . . . . . .
1.19.2 Monoploidy . . . . . . . . . .
1.19.3 Polyploidy . . . . . . . . . . .
1.19.4 Autotetraploidy . . . . . . . .
1.19.5 Allotetraploidy . . . . . . . .
1.19.6 Aneuploidy . . . . . . . . . .
Organelle Genetics . . . . . . . . . .
1.20.1 Mitochondrial DNA . . . . . .
1.20.2 Chloroplast DNA . . . . . . .
1.20.3 Bacterial Similarities . . . . .
1.20.4 Four O’ Clocks . . . . . . . .
1.20.5 Xenopus . . . . . . . . . . . .
1.20.6 Cultivating cpDNA . . . . . .
1.20.7 LHON . . . . . . . . . . . . .
1.20.8 Chlamydamonas . . . . . . . .
1.20.9 Yeast . . . . . . . . . . . . . .
1.20.10 Diagnostics . . . . . . . . . .
2 Genetics in Society
2.1 The Human Genome . . . . . . .
2.1.1 Size . . . . . . . . . . . .
2.1.2 Repetition . . . . . . . . .
2.1.3 Contructing Genome Maps
2.1.4 Chromosomal Maps . . . .
2.1.5 Linkage Maps . . . . . . .
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CONTENTS
2.2
2.3
2.4
2.5
2.6
2.1.6 Radiation Hybrid Maps . . . . . .
2.1.7 DNA Fingerprinting . . . . . . . .
2.1.8 The Genome Projects . . . . . . . .
2.1.9 Other Projects . . . . . . . . . . .
Human Disease Genes . . . . . . . . . . .
2.2.1 Classes . . . . . . . . . . . . . . . .
2.2.2 Alkaptonura . . . . . . . . . . . . .
2.2.3 Sickle Cell Anemia . . . . . . . . .
2.2.4 Mouse Models . . . . . . . . . . . .
2.2.5 Human Crosses? . . . . . . . . . .
2.2.6 Carrier Screening . . . . . . . . . .
2.2.7 X-Linked SCIDs . . . . . . . . . . .
Cancer . . . . . . . . . . . . . . . . . . . .
2.3.1 Retinoblastoma . . . . . . . . . . .
2.3.2 Alterations in Cancer Cells . . . . .
2.3.3 Heterogeneity of Cancer . . . . . .
2.3.4 Genetic Cancer Causation . . . . .
2.3.5 Cell Division Cycle . . . . . . . . .
2.3.6 Oncogenes . . . . . . . . . . . . . .
2.3.7 Tumor Suppressors . . . . . . . . .
Genetics of Human Diversity . . . . . . . .
2.4.1 Introduction . . . . . . . . . . . . .
2.4.2 Examples . . . . . . . . . . . . . .
2.4.3 Race-Based Medicine? . . . . . . .
Inheritance of Quantitative Traits . . . . .
2.5.1 Pseudoscience . . . . . . . . . . . .
2.5.2 Quantitative Traits . . . . . . . . .
Bacterial Genetics . . . . . . . . . . . . . .
2.6.1 Classical Genetics . . . . . . . . . .
2.6.2 Rise of Molecular Genetics . . . . .
2.6.3 Bacterial Virus Resistance . . . . .
2.6.4 One Gene-One Enzyme Hypothesis
2.6.5 The Operon . . . . . . . . . . . . .
2.6.6 Cis-Trans Test . . . . . . . . . . .
2.6.7 Determining the Suppressor . . . .
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CONTENTS
3 Analysis Techniques
3.1 Themes in Genetics . . . . . . . . . . . .
3.1.1 Introduction . . . . . . . . . . . .
3.1.2 Distinctions . . . . . . . . . . . .
3.1.3 Relevance of the Mendelian Test .
3.2 Viral Genetics . . . . . . . . . . . . . . .
3.2.1 Introduction . . . . . . . . . . . .
3.2.2 T4 Plaque Morphology . . . . . .
3.2.3 Viral Complementation . . . . . .
3.2.4 Reverting the Dominance . . . .
3.3 Importance of Mutations . . . . . . . . .
3.3.1 Benzer’s Deductions . . . . . . .
3.3.2 Deletion Mapping . . . . . . . . .
3.3.3 Mutagenesis . . . . . . . . . . . .
3.4 Mutation Classes . . . . . . . . . . . . .
3.4.1 Kline’s Sex Lethal . . . . . . . .
3.4.2 Amorphic . . . . . . . . . . . . .
3.4.3 Hypomorphic . . . . . . . . . . .
3.4.4 Hypermorphic . . . . . . . . . . .
3.4.5 Antimorphic . . . . . . . . . . . .
3.4.6 Neomorphic . . . . . . . . . . . .
3.4.7 Summary . . . . . . . . . . . . .
3.5 Conditional Mutations . . . . . . . . . .
3.5.1 Introduction . . . . . . . . . . . .
3.5.2 Gene and Allele Specific . . . . .
3.5.3 Gene Specific, Allele Nonspecific .
3.5.4 Gene Nonspecific, Allele Specific .
3.5.5 Temperature Sensitive . . . . . .
3.6 Mutagenesis . . . . . . . . . . . . . . . .
3.6.1 Introduction . . . . . . . . . . . .
3.6.2 Radiation . . . . . . . . . . . . .
3.6.3 Mobile Genetic Elements . . . . .
3.6.4 Balancer Chromosomes . . . . . .
3.7 Genetic Mosaics . . . . . . . . . . . . . .
3.7.1 Introduction . . . . . . . . . . . .
3.7.2 Genetic Screens . . . . . . . . . .
3.7.3 Mitotic Recombination . . . . . .
3.7.4 Maternal Effect Lethality . . . .
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75
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8
CONTENTS
3.8
Transient Phenocopies . . . . . . . . . .
3.8.1 Motivation . . . . . . . . . . . . .
3.8.2 The Discovery . . . . . . . . . . .
3.8.3 Mechanism . . . . . . . . . . . .
3.8.4 Limitations . . . . . . . . . . . .
3.8.5 Speculation . . . . . . . . . . . .
3.9 Sex Determination . . . . . . . . . . . .
3.9.1 Introduction . . . . . . . . . . . .
3.9.2 Evolution of Sex . . . . . . . . .
3.9.3 The Effects of Sex . . . . . . . .
3.9.4 Environmental Sex Determination
3.9.5 Genotypic Sex Determination . .
3.9.6 Mechanisms of GSD . . . . . . .
3.10 Dosage Compensation . . . . . . . . . .
3.10.1 The Lion Hypothesis . . . . . . .
3.10.2 Defining the X Chromosome . . .
3.10.3 Noncoding RNA . . . . . . . . .
3.10.4 Exceptions . . . . . . . . . . . . .
A Glossary
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94
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107
Chapter 1
Classical and Molecular
Genetics
1.1
Mitosis and Meiosis
A good understanding of meiosis can be used to derive any genetics problem.
1.1.1
Stages of the Cell Cycle
The cell cycle is divided up into these stages:
Mitosis (M) 

Gap 1 (G1)
Interphase DNA Synthesis (S)


Gap 2 (G2)
1.1.2
Definitions
Mitosis is the process of chromosome separation in somatic cells that produces two identical daughter cells.
Meiosis is the process of segregating alleles into gametes.
Chromatin is loosely defined as a tangled DNA/protein complex.
A Chromosome is a linear array of genes and noncoding regions.
Homologous Chromosomes match in size, shape, and order of genes.
Autosomes are chromosomes that do not influence sex determination.
9
10
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
Sex Chromosomes are chromosomes involved in sex determination.
Chromatids are the two segments of DNA joined by a centromere near
the center of the replicated chromosome.
Sister Chromatids are identical DNA segments connected at a centromere that exist only after DNA replication in S phase and before cell
division in mitosis.
1.1.3
Phases of Mitosis
Mitosis is divided into these phases:
Phase
Prophase
Features
Chromosome condensation
Nuclear envelope breakdown
Metaphase Chromosomes align, unordered, on metaphase plate
Anaphase Chromosomes are pulled to opposite poles
Telophase Dual nuclear envelope reformation
1.1.4
Phases of Meiosis
Meiosis is divided into these phases:
Phase Features
Prophase I Chromosome condensation
Nuclear envelope breakdown
Protein-mediated synapsis, crossing-over
Metaphase I Synapsed chromosomes (Tetrads) align on metaphase plate
Anaphase I Homologous replicated chromosomes are pulled to opposite poles
Telophase I Dual nuclear envelope reformation
Interkinesis Two new daughter cells form from division
No new replication
Prophase II Chromosome condensation
Nuclear envelope breakdown
Metaphase II Chromosomes align on metaphase plate
Anaphase II Sister chromatids are pulled to opposite poles
Telophase II Dual nuclear envelope reformation
1.2. BACKGROUND OF MENDEL
11
Note that, unlike mitosis, the products of meiosis are four haploid germ
cells with new varieties of chromosomes because:
1. Random Metaphase I alignment results in independent assortment
2. Crossing-over results in new combinations of alleles on a chromosome
1.1.5
Comparing Mitosis and Meiosis
Mitosis
Cell Type Somatic
Divisions
1
Homologous Chromosome Pairing
Rarely
Genetic Recombination
Rarely
Sister Chromatid Separation Anaphase
2
Daughter Cells Produced
1.2
Meiosis
Germ
2
Always
Always
Anaphase II
4
Background of Mendel
Gregor Mendel was an Austrian monk who established the basic laws of inheritance through radical breeding experiments with pea plants in the 1860s.
At the time of his publication, there were two other prevailing theories of
inheritance:
1. Blending inheritance
2. Uniparental “homunculus” inheritance
Modern recognition for Mendel’s scientific success stems from his good
experimental setup:
1. Pea plants were an ideal model system since they have short generation
times and are capable of self-ferilization
2. Traits monitored were dichotomous and easily scorable
3. Pure-breeding lines were established so as to be confident in breeding
results
4. Controlled matings did not allow any possibility of undocumented fertilization
12
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
5. Quantitative counts of bred plants resulted in clear ratios
1.3
Elementary Genetic Analysis
Observing the pea plants’ Phenotypes, or observable inherited characteristics, led to the deduction of their Genotypes, or inherited genetic material.
Genes are the modern name for the discrete units that Mendel observed to
be inherited. Many individual varieties, or Alleles of each gene exist.
For genotypes, Dominant alleles are denoted by the upper case of the
first letter of the dominant phenotype. Recessive alleles are denoted by the
lower case of the first letter of the dominant phenotype.
The first Parental generation’s (P) offspring are referred to as the first
Filial generation (F1 ). These individuals’ offspring are referred to as the
second Filial generation (F2 ). “Filial” is a word defined as “of or suitable to
a son or daughter.”
1.3.1
Monohybrid Cross
Pure-breeding yellow plants were crossed with pure-breeding green plants.
P
Yellow
×
Green
:
F1
Yellow
× Yellow
:
Yellow
Yellow 3
Green 1
F1
F2
These deductions were made from the above results:
1. Two types of yellow plants exist (pure and hybrids)
2. Yellow is dominant over green in inheritance
3. Law of Segregation: The two alleles present for each trait separate
during meiosis and unite randomly with an allele from another gamete
at fertilization
And these genotypes were deduced:
P
YY
×
yy
:
F1
Yy
× Yy
:
Yy
1 YY
2 Yy
1 yy
F1
F2
1.3. ELEMENTARY GENETIC ANALYSIS
1.3.2
13
Dihybrid Cross
Peas purebred yellow and round were crossed with peas purebred green and
wrinkled.
Yellow, Round × Green, Wrinkled
P
F1
Yellow, Round ×
Yellow, Round
:
:
9
3
3
1
Yellow, Round
Yellow, Round
Yellow, Wrinkled
Green, Round
Green, Wrinkled
F1
F2
The Law of Independent Assortment was deduced from the dihybrid
cross. It states that pairs of alleles separate at meiosis and join at fertilization
independent of other pairs of alleles.
These genotypes were also deduced, as displayed in a Punnett square:
P
RY
ry RYry
F1
RY
Ry
rY
ry
RY
RRYY
RRYy
RrYY
RrYy
Ry
RRYy
RRyy
RrYy
Rryy
rY
RRYY
RrYy
rrYY
rrYy
ry
RrYy
Rryy
rrYy
rryy
So, in case you didn’t notice, the F2 generation genotypes occur in a ratio
of
1:1:1:1:2:2:2:2:4
1.3.3
Test Cross
A Test Cross is performed to determine the genotype of an individual with
dominant phenotype, and is especially useful for organisms which can’t be
self-fertilized. The unknown individual (A-B-) is crossed with a purebred
recessive individual (aabb), and the genotype is deduced as follows:
1. If no recessive phenotype is seen in the offspring, then the unknown
must be Homozygous dominant
2. If a recessive to dominant phenotype ratio of 1 : 1 emerges in the
offspring, then the unknown must be Heterozygous
14
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
1.4
Complications to Basic Genetics
The following observations contradict the simple model proposed by Mendel.
1. Incomplete dominance
2. Codominance
3. > 1 trait influenced by one gene
4. Not all genotypes are equally viable
1.4.1
Incomplete Dominance
This phenomenon is marked by emergence of a novel phenotype in the F1
generation, which normally consists of dominant phenotype heterozygotes.
Incomplete dominance results in expression of both dominant and expressive
alleles, resulting in a blended phenotype.
For example, in snapdragons, the flower color is determined by an incompletely dominant red allele:
1.4.2
P
Red (RR)
× White (rr)
:
F1
Pink (Rr)
×
:
Pink (Rr)
Pink (Rr)
1 Red (RR)
2 Pink (Rr)
1 White (rr)
F1
F2
Codominance
Codominance occurs when more than two alleles are present for a gene and
more than one of them is dominant to another allele. This phenomenon is
manifest when the F1 generation displays both parental phenotypes.
For example, in humans, blood type is determined by codominant alleles:
P
I AI A
× IBIB
:
F1
I AI B
×
I AI B
:
I AI B
1 I AI A
2 I AI B
1 IBIB
F1
F2
1.5. SEX DETERMINATION PROVES CHROMOSOMAL INHERITANCE15
1.4.3
Recessive Lethality
Recessive lethality is marked by a departure from the usual F2 phenotype
ratios because homozygous recessive individuals are aborted in utero.
P
F1
× Yellow (AY A)
:
Yellow (AY A) × Yellow (AY A)
:
Grey (AA)
1
1
Grey (AA)
Yellow (AY A)
1 Aborted (AY AY )
2 Yellow (AY A)
1 Grey (AA)
F1
F2
From the first cross, it can be deduced that:
1. A single gene with two alleles determines the yellow phenotype
2. Yellow mice must carry the Agouti allele
3. Yellow must be dominant
From the second cross, it is apparent that:
1. The AY allele’s gene locus is Pleiotropic, i.e. the gene contributes to
more than one phenotype
2. The AY allele is dominant in color determination but recessive in determining the lethal phenotype (hence the term Recessive lethality)
1.5
1.5.1
Sex Determination Proves Chromosomal
Inheritance
Sex Detemination Summary
Autosomes
22 pairs
3 pairs
Organism
Human
Drosophila
Chicken
Some Insects
Female
XX
XX
WZ
XX
Male Sex Detemination
XY Presence of Y
XY Number of X chromosomes
ZZ
XO Number of X chromosomes
16
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
1.5.2
Nomenclature in Drosophila
1. Gene loci are abbreviated by the first letter of the mutant phenotype.
Capitalize if the mutant phenotype is dominant.
2. Denote the wild-type allele (defined as any allele with frequency > 1%)
with a superscript plus (+ )
1.5.3
Establishing Sex Linkage
A mutant male fly with white eyes arose in the F2 generation in breeding
experiments:
P
Red F
× White M
:
F1
Red F
×
:
Red M
All Red
2 Red F
1 Red M
1 White M
F1
F2
The genotypes were deduced as:
w+
X
w+
P
X
F1
Xw Xw
+
×
w
X Y
+
× Xw Y
:
:
1
1
1
1
1
1
+
Xw Xw
+
Xw Y
+
+
Xw Xw
+
Xw Xw
+
Xw Y
X wY
F1
F2
A reciprocal cross was also performed to get more information about the
gene:
P White F
P
X wX w
× Red M
×
+
Xw Y
:
:
1
1
1
1
Red F
White M
+
Xw Xw
X wY
F1
F1
The males who had only one recessive allele yet showed the white phenotype became known as Hemizygotes. The eye color trait’s linkage with
sex determination caused a gene with this characteristic to become referred
to as “X-linked.” Also, these experiments provided firm evidence for the
chromosomal theory of inheritance.
1.5. SEX DETERMINATION PROVES CHROMOSOMAL INHERITANCE17
1.5.4
Primary Nondisjunction
With the above reciprocal eye color cross, there were “exceptional” deviations
from the usually observed ratios with frequency of about 1 in 2000:
P White F
P
X wX w
× Red M
×
+
Xw Y
:
:
2000
2000
1
1
2000
2000
1
1
1
1
Red F
White M
White F
Red M
+
Xw Xw
X wY
+
X w X wX w
X wX wY
+
Xw
Y
F1
F1
This was explained by attributing the exceptional individuals to a rare
failure of the X chromosome to segregate properly in meiosis I called Nondisjunction. Instead of producing four normal eggs each with one X w , the
female fly was deduced to have produced two eggs with no sex chromosomes
and two eggs with X w X w .
The exceptional female with white eyes and a Y chromosome was fertile, while the red male with no Y chromosome was sterile. The other two
genotypes, trisomy X and only Y, were fatal.
Using this interpretation of the data, it was deduced that sex in Drosophila
was determined by the number of X chromosomes rather than presence of Y.
1.5.5
2◦ Nondisjunction
The exceptional female generated by the primary nondisjunction cross above
was crossed to a standard red white male, and a 2◦ nondisjunction occured
with frequency 4%:
F1
Xw
X wY
X wX w
Y
+
Xw
+
Xw Xw
+
X w X wY
+
X w X wX w
+
Xw Y
Y
X wY
X wY Y
X wX wY
YY
Once again, the trisomy X and lack of X genotypes were fatal.
18
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
1.5.6
Barred Phenotype Crosses Reveal Meiosis I as
Point of Nondisjunction
Another characteristic was examined to more precisely determine the point
of nondisjunction was the dominant mutant “barred” phenotype (B-). The
following cross of an exceptional, red, non-barred female with a normal, red,
barred male was performed:
Xw
+ B+
X wB+ Y × X w
+B
Y
The expectation of this cross (without nondisjunction) was that all females would be red/barred while all males would be non-barred, half red, half
white. This experiment made looking for exceptional females easy since they
would exhibit a non-barred phenotype if nondisjunction occured at Meiosis I.
Conversely, if nondisjunction happened at Meiosis II, one would expect to
see white or red non-barred females. Keep in mind that nondisjunction at
Meiosis I implies gametes with the same alleles as the parent cell, whereas
nondisjunction at Meiosis II implies twice as much of one allele from the
parent chromosome.
1.6
Pedigree Analysis
Because breeding experiments can’t be performed on humans, a solution to
exploring human genetics can be found in pedigree analysis.
However, this method of analysis suffers from four major drawbacks:
1. No controlled crosses
2. Imperfect family records
3. Rarely large number of offspring (hard to gauge ratios)
4. Mistaken paternity causes misinterpretation
1.6.1
Autosomal Dominant
Examples of autosomal dominant traits include:
1.6. PEDIGREE ANALYSIS
19
Achondroplasia Dwarfism caused by a mutation in FGF3R that causes
either constitutive activity or increased ligand affinity. This results in
a faster onset (before maturity) of bone chondrocyte differentiation and
consequently shorter individuals.
Piebald Spotting An autosomal dominant disease is manifest with strange
white skin coloration that usually occurs in the middle of the ventral
part of the body. It results from a mutation in the gene c-kit, a growth
factor receptor kinase. Since the receptor acts as a dimer, the mutation
causes at 75% reduction of receptor activity and a consequent halt in
the signaling pathway.
Brachydactyly Results in malformed fingers and is caused by a mutation
in the Indian hedgehog gene, a gene so named because it was originally
a fly mutation that caused the fly to look very bristly like a hedgehog. Like piebald spotting, it acts through Haploinsufficiency, the
condition of not being able to sustain normal phenotype with only one
functional allele.
Huntington’s Disease A neurological disorder that is marked by a late
onset and slurred speech
Characteristics of autosomal dominant traits include:
1. Every affected individual has an affected parent
2. Vertical — every generation is affected
3. Affected × Normal : 1/2 affected progeny
4. Early onset deleterious traits unlikely to be passed on
1.6.2
Autosomal Recessive
Examples of autosomal recessive traits include:
Albinism The complete lack of pigment in the skin
Phenylketonuria Enzyme deficiency
Sickle-Cell Anemia
20
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
Tay-Sachs Neurodegenerative disease
Cystic Fibrosis Deficiency in lung immune system that allows bacteria to
grow and inhibit gas exchange
Characteristics of autosomal recessive traits include:
1. Affected individuals have unaffected parents
2. Chance union of 2 unrelated heterozygotes is small so therefore related
crosses (incest) are of use in determining the nature of the trait’s inheritance
3. Carrier × Carrier : 3 Norm : 1 Affected
4. Horizontal — appears suddenly in one generation
1.6.3
X-Linked Recessive
Examples of X-linked recessive traits include:
Red-Green Colorblindness
Duchenne Muscular Dystrophy
Hemophilia A
Characteristics of X-linked recessive traits include:
1. More males affected
2. All sons of affected mother affected
3. All progeny of an affected male will be normal and all daughters will
be carriers
4. Often skips a generation
1.7. LINKAGE
1.6.4
21
X-Linked Dominant
Characteristics of X-linked dominant traits include:
1. More females than males affected
2. Affected fathers pass it on to all daughters but no sons
3. Affected mothers pass it on to half her progeny
4. Phenotype less severe in females than males
1.7
Linkage
One of the assumptions of Mendelian inheritance laws is that all genes assort independently. This is true of many characteristics, but genes that are
sufficiently close to one another on the same chromosome do not follow independent assortment, and are known as Linked.
1.7.1
X-Linked Mutant Cross
Dihybrid crosses were performed for the following X-linked mutant traits:
w+
w
y+
y
Red
White
Brown
Yellow
Here are the crosses, with only the male F2 progeny displayed:
+
+
+
× Xw yY
+
+y
× X wy Y
P
X wy X wy
F1
X wy X w
+
+
:
:
+
1 X wy X w y
+
1 X wy Y
+
4484 X wy Y
+
4413 X w y Y
76 X wy Y
+ +
53 X w y Y
F1
F2
Note the departure from expectation in the F2 generation. The F2 sons
that appeared less frequently were produced by an egg with a pair of X
chromosomes that had participated in a crossing-over event between the w
22
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
and y gene loci. These progeny are referred to as Recombinant, since their
genotypes are not among the parental genotypes.
The proportion of recombinant progeny in these crosses is, empirically:
76 + 53
= 0.01429
4484 + 4413 + 76 + 53
This number, a measure of recombination frequency, is usually viewed as
a percent (1.429%) which is equivalent to centiMorgans (1.429cM) which is
equivalent to map units (1.429m.u.).
To a certain degree, the physical distance between genes can be approximated as proportional to recombination frequency. However, if an approximation is used, it must be specific to the organism and chromosome on which
the genes are linked.
1.7.2
Autosomal Mutant Cross
Dihybrid crosses were performed for the following autosomal mutant traits:
b+
b
c+
c
Brown
Black
Straight
Curved
Here are the crosses. Note that the F1 cross is actually a test cross, as
denoted by F1 T:
P
F1 T
bc+ /bc+ F × b+ c/b+ c M
:
bc+ /b+ c F ×
:
bc/bc M
bc+ /b+ c
2934 bc+ /bc
2768 b+ c/bc
871 bc/bc
846 b+ c+ /bc
F1
F2
These results indicate that the b and c gene loci are separated by approximately 23.14cM.
1.7. LINKAGE
1.7.3
23
χ2 Test of Linkage
With the above data, there is not much doubt that the genes are linked.
However, with genes that are observed to be separated by > 40cM, one
begins to question whether the genes are linked or unlinked.
To differentiate between these two possibilities, the statistical method of
applying a χ2 test statistic to a multinomial model is used. First, define the
hypotheses:
H0 : Genes are unlinked
HA : Genes are linked
Let O1 , O2 , ..., On be the observed
Pn frequencies of progeny. Then the total
number of observations is T = i=1 Oi . Considering that 50% parental gametes and 50% recombinant gametes are expected under H0 , assign expected
proportions p1 , p2 , ..., pn to each observed category of progeny. For example,
consider the simple dihybrid cross above:
P
bc+ /bc+ F
× b+ c/b+ c M
:
F1 T
bc+ /b+ c F
×
:
bc/bc M
bc+ /b+ c
0.25 bc+ /bc
0.25 b+ c/bc
0.25 bc/bc
0.25 b+ c+ /bc
F1
F2
In this case, pi = 1/4 |4i=1 . Now calculate expected counts E1 , E2 , ..., En ,
where Ei = T pi .
Finally, we have enough information to calculate the χ2 test statistic.
Pick your favorite of these two equivalent test statistics:
P
Pearson’s X 2 = ni=1 (Oi − Ei )2 /Ei
P
Likelihood Ratio X 2 = 2 ni=1 Oi ln(Oi /Ei )
Since X 2 ∼ χ2n−1 , a p-value can be calculated by finding the area under
χ2n−1 to the right of X 2 .
The p-value can be interpreted as the probability of observing deviations
from the expected values under H0 as large or larger than what was observed.
Therefore, a high p-value (p > 0.1) is strong evidence for linkage, and a low
p-value (p < 0.01) is strong evidence that the genes are unlinked.
In our example above, we have:
24
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
i
1
2
3
4
Oi
2934
2768
871
846
pi
1/4
1/4
1/4
1/4
Ei
Xi2
1854.75 2691.16
1854.75 2216.48
1854.75 -1317.71
1854.75 -1328.19
P4
2
2
Thus, X 2 =
i=1 Xi = 2262.73. The area under χ3 to the right of
2262.71 is approximately 0, so this is strong evidence that gene loci b and c
are linked.
1.7.4
Summary of Linkage
1. Physical linkage is somewhat related to genetic linkage, but varies with
organism and chromosome
2. Genetic recombination between linked loci results from the physical
event of crossing-over
3. 0 ≤ Recombination Frequency ≤ 0.5 since no recombination is expected for adjacent genes and at maximum 1/2 of progeny will be of
recombinant, rather than parental, genotypes
1.8
1.8.1
Genetic Mapping
Mapping 5 Genes With 2-Point Crosses
Consider the following observed gene separations, in cM:
m
y
w
v
r
m
y
w
- 34.3 32.8
1.1
-
v
4
33
32.1
-
r
17.8
42.9
42.1
24.1
-
Considering just the three loci m, w, and y, it is easy to deduce the
following topology:
1.8. GENETIC MAPPING
1.1
y
|
25
32.8
w
m
}
{z
34.3
Note that the two smaller map distances do not sum to the larger map
distance. This is expected for 2-point crosses.
By considering v and r, we can deduce the complete topology of these
genes:
y 1.1 w
|
1.8.2
m 4 v
32.8
r
}
17.8
{z
42.9
Mapping 3 Genes With 3-Point Crosses
Consider the following trihybrid F1 crosses, also known as 3-point crosses:
P
Homo- vg b pr
× Homo- vg + b+ pr+
:
F1 T
Heterozygotes
×
:
Homo- vg b pr
All Heterozygotes
1779 vg b pr
1654 vg + b+ pr+
252 vg + b pr
241 vg b+ pr+
9 vg + b+ pr
13 vg b pr+
118 vg b+ pr
131 vg + b pr+
To construct a genetic linkage map, the key point to realize is that the
probability of two independent recombination events happening is much less
likely than the probability of only one. This implies that the gene which
resides in between the other two will most likely be the one that crosses-over
by itself the least number of times.
In this case, the pr recombinants are observed the least, so pr must reside
between vg and b. This suggests the map:
vg
pr
b
F1
F2
26
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
Since pr recombinants reflect progeny with chromosomes that have crossedover twice between b and vg, these numbers must be counted twice to determine the map distance between vg and b. Also, these numbers must be
counted once along with the other vg and b recombinants to determine, respectively, the distances |vg − pr| and |b − pr|.
Thus, the map distances in this example are:
|vg − pr| =
9 + 13 + 252 + 241
= 0.12271
4197
|b − pr| =
9 + 13 + 118 + 131
= 0.06457
4197
|b − vg| = 0.06457 + 0.12271 = 0.18728
These numbers imply the following gene map (in units of cM):
vg
12.27
1.8.3
pr
6.46
b
Interference
Is it necessarily true that
P (Two Single Crossover Events) = P (Single Crossover Event)2
?
= P (Double Crossover Event)
No! In fact, the phenomenon of single crossover events somehow inhibiting the occurence of double crossover events is referred to as Interference
and is derived from the Coefficient of coincidence, which is defined as:
Kc = nObserved Doubles /nExpected Doubles
The interference is defined as I ≡ 1 − Kc . I values close to zero indicate that single crossover events inhibit almost all double crossover events.
I values close to 1 indicate that no inhibition is present.
For example, consider the example above. The observed proportion
of double crossovers is 22/4197, while the observed proportion of single
crossovers are 0.123 and 0.064. Therefore, the expected proportion of doubles
is
(0.123)(0.064) = 0.007872
1.9. TETRAD ANALYSIS
27
Therefore,
Kc =
0.007872
= 0.67:I = 0.33
22/4197
1.9
Tetrad Analysis
1.9.1
Fungi As A Model
In many organisms the products of individual meiosis events can’t be examined, so genetic analysis relies on interpretation of large samples of data and
the law of large numbers.
However, with the two yeasts Saccraromyces cerevisiae (baker’s yeast)
and Neurospora (bread mold), it is possible to pick out spores, put them
in a line on selective media, and count how many survive in each Tetrad
to deduce the specific products of individual meioses. Consequently, these
yeasts are great genetic models.
1.9.2
Meiosis in S. cerevisiae
There are two mating types, called a and α, in S. cerevisiae. An organism can
exist as a haploid (10 chromosomes) form of one mating type or a diploid
(2 copies each of 10 homologous chromosomes) form with a fusion of the
two mating types. The product of meiosis in S. cerevisiae is an ascus of
four haploid spores, half a and half α. The life cycle of the organism is
diagrammed below.
Ascus a, a, α, α
↑
Meiosis
&
↑
Diploid a/α
.
Haploid a
-.
1.9.3
&
.
Haploid α
-.
Genetics of S. cerevisiae
Upper case gene names denote dominant alleles, while lower case gene names
denote recessive mutant alleles.
To illustrate the possible outcomes of a yeast cell division, consider a
diploid organism that resulted from the union of an a with genotype his4 TRP1
28
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
and an α with genotype HIS4 trp1. Assuming these genes are unlinked
and that chromosome replication proceeds normally, consider the following
meioses:
Parental Ditype (PD) During meiosis I, parental chromosomes stay together, forming two yeasts with genotype his4 TRP1 and two yeasts
with genotype HIS4 trp1
Non-Parental Ditype (NPD) During meiosis I, parental chromosomes separate, forming two yeasts with genotype HIS4 TRP1 and two yeasts
with genotype his4 trp1
Tetratype (T) During metaphase I, a crossing-over event causes a rearrangement between two replicated chromosomes that results in the
creation of a two heterozygous chromosomes. Meiosis I separates these
two heterozygous chromosomes, and meiosis II separates the two different alleles into separate spores. This results in the spore products
his4 trp1, HIS4 TRP1, his4 TRP1, HIS4 trp1.
When genes are unlinked, the number of Parental Ditypes should be
approximately equal to the number of Non-Parental Ditypes. However, when
genes are linked, the PD NPD. This is because the genes do not assort
independently of one another and, assuming no crossing-over, are forced to
stay together throughout the life of the chromosome.
1.9.4
Recombination Frequency
Consider the cross of an a arg3 ura2 haploid with an α ARG3 URA2. A
diploid arg3 ura2/ARG3 URA2 results from the cross and produces the following haploid progeny:
PD NPD T
127
3
70
In all cases, we will take the recombination frequency to be, intuitively:
F = nRecombinant Tetrads /nTotal Tetrads
To determine the recombination frequency assuming at most 1 crossover,
the estimate is given by:
F1 =
nNPD + nT /2
nTotal
1.9. TETRAD ANALYSIS
29
This makes sense because all NPD’s will result from crossing-over and 1/2
of the spores produced in a Tetratype result from a crossing-over event.
However, double crossovers happen. So, under the assumption that all
double crossovers happen with equal probability and that there are no more
than two crossing-over events, a better estimate of recombination frequency
is given by:
nT /2 + 3nNPD
F2 =
nTotal
To understand this estimate, we must examine the four possibilities for
double crossover events.
1. An event involving two strands will produce a PD
2. The two possible events involving three strands will produce a T
3. An event involving all four strands of the two chromosomes will produce
an NPD
Remember that each single crossover event yields a T, and that no crossing over yields a PD.
Using this information, and assuming these four events are equally likely,
we can use the fact that NPD’s only show up in double crossovers to infer
that:
nSingle Crossover = nT − n3 Strand Double Crossovers = nT − 2nNPD
nDouble Crossover = 4nNPD
Therefore, we arrive at our assertion that:
nT /2 + 3nNPD
1/2(nT − 2nNPD ) + 4nNPD
=
nTotal
nTotal
Generally, the recombination frequency considering an arbitrary number
of crossover events c should be calculated as
c
X
Fc = 1/nTotal Tetrads
(2i/4)ni ,
F2 =
i=1
where ni is the number of tetrads that have done i crossover events, and
2i is the number of chromosomes involved in the i crossover events. Thus,
2i/4 = i/2 is the number of crossover events per cell.
For example, when c = 2, we have:
F2 = 2(1)/4n1 + 2(2)/4n2 = n1 /2 + n2
30
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
1.9.5
Neurospora crassa
The advantage to using this genetic model is that it is possible to map the
centromere relative to other genes on the chromosome because of the organism’s ordered tetrads.
The two haploid mating types of Neurospora crassa are a and A, and
their fusion results in a diploid A/a being capable of meiosis. First, an ascus
forms to protect the fungus’ spores. Then, meiosis I causes homologous
chromosome separation and cell division into two haploid cells with replicated
chromosomes. Then, meiosis II causes separation of sister chromatids and
formation of four haploid cells. One last mitotic cell division results in the
production of 8 haploid spores, an Octad, which are spatially oriented in
the ascus. Therefore, in each ascus the octad is comprised of four pairs of
adjacent, identical spores that may be treated as a tetrad. The orientation
of these spore pairs lets you deduce the events that happened in the meioses.
To map the centromere for genes in these cells, the map distance is defined
as:
nMeiosis II Segregates /2
This makes sense, because half of the observed meiosis II segregates will
be affected by a single crossing-over event. Additionally, during a crossing
over event, all chromatin from the location of the event to the end of the
chromosome is transferred to the homologous chromosome. Therefore, only
recombination events that occur between the gene of interest and the centromere will result in observing the recombinant phenotype.
1.10
Recombination Mechanisms
1.10.1
Physical Exchange
In 1931, Creighton and McClintock performed experiments on recombination
in maize to confirm the physical basis of genetic exchange.
They located a region on chromosome 9 where they could track the inheritance of two linked genes, each proximal to a readily identifiable cytogenetic
marker.
They created two individuals:
1. Double dominant with no cytogenetic markers
1.10. RECOMBINATION MECHANISMS
31
2. Double recessive with a marker proximal to each allele
When the recombinant progeny’s karyotype was observed, the markers
appeared on different chromosomes. This indicates that crossing-over is a
manifestation of a physical mechanism of trading chromatin between homologous chromosomes.
1.10.2
Breaking and Rejoining
To establish that breaking and rejoining was a universal phenomenon unrelated to proximal genes, the following experiment was performed.
Viral DNA was constructed for three linked genes:
1. Triple dominant grown on heavy isotope media
2. Triple recessive grown on light isotope media
The two types of viral DNA were mixed and allowed to infect a some host
bacteria. To rule out the possibility that the DNA would be replicated, an
inhibitor was added. After a certain amount of time, the virus repackaged
its DNA into new virions, which were separated out on a column by weight.
Unsurprisingly, the continuum of heterogeneity of the result confirmed the
hypothesis that crossing-over could occur anywhere.
1.10.3
Gene Conversion
Gene conversion is the unidirectional transfer of genetic information, which
can sometimes be provoked by Heteroduplex.
When examining hybrid crosses in S. cerevisiae, a 2:2 ratio of progeny is
expected without crossing-over.
When examining crosses of Neurospora crassa, a 4:4 ratio of progeny is
expected. However, exceptional ratios of 6:2, 5:3, and 3:1:1:3 are observed
rarely.
To explain these odd results, consider the results of meiosis if two heterogenous strands of DNA exist or are somehow produced as a result of
crossing-over. In this case, one strand could have the mutant allele, while the
other strand carried the wild-type allele. Then, the final mitosis event would
duplicate each strand of the heterogenous DNA and produce two spores with
different genotypes.
32
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
If each of the two heteroduplex DNA molecules are repaired with different strands before mitosis, the 5:3 case results. If both heteroduplex DNA
molecules are repaired with the same strand before mitosis, the 6:2 case
results. If no repair occurs, the 3:1:1:3 case is observed.
This immediately suggests the DNA repair mechanisms to respond to
genome damage, first explained in MCB 110.
From the trihybrid haploid cross abc × ABC, an ABC/abc diploid yeast
results. When this yeast produces spores, there are four observed gene conversion types:
1. ABC, ABC, aBc, abc
2. ABC, ABc, aBC, abc
3. ABC, AbC, abc, abc
4. ABC, Abc, abC, abc
Note that the A:a and C:c ratios are both 2:2, while the B:b ratio is 3:1
or 1:3. In the boxed types, it is clear that both recombination and gene
conversion has occured. The other options only provide evidence of gene
conversion.
1.10.4
Models of Recombination
Any physical model of the recombination process needs to consider:
1. Physical breaking and rejoining
2. Equal replication products
3. Can occur anywhere
4. No new mutations
5. Gene conversion can explain rare tetrads
Meselson revised the 1964 standard Holliday model and postulated that
1 single strand nick in DNA initiates the recombination event.
The model consists of the following steps:
1. Nicking
1.11. PATHWAY DISSECTIONS
33
2. Whisker displacement
3. First strand invasion
4. Second strand invasion
5. Repair and ligation
6. Branch migration
7. The Holliday intermediate
8. Alternative resolutions
1.11
Pathway Dissections
As an example, consider the following biochemical pathway:
1
2
3
4
5
6
Phe → Tyr → p-hydroxy- → 2,5-dihydroxy- → HA → MA → CO2 + H2 O
4
Tyr →→ Melanin
Defects in this pathway can cause disease:
1 Phenylketonuria: Phe is converted to a toxic chemical
2 Tyrosinosus: Tyr levels are elevated, causing congenital abnormalities
3 Tyrosinemia: death at 6 months due to liver damage
4 Albinism
In the Mendelian model of inheritance, one gene is responsible for the
inheritance of one trait. In 1902, Garrod studied cases of alkaptonuria, in
which homegentissic acid accumulates in the blood of affected individuals. He
found that this toxic chemical doesn’t accumulate in the blood of unaffected
individuals, even if artificially introduced.
34
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
1.11.1
One Gene-One Protein
In the 1940s, Beadle and Tatum explored this hypothesis through mutants of
Neurospora crassa, which they found to be a great model organism because:
1. Life cycle well known
2. Easy to induce mutations
3. Very little required to grow: biotin, glucose, and salt
Their experiment went as follows. After growing some haploid spores,
they irradiated them with X-rays to induce mutations. These mutant haploid
individuals were mated to the opposite mating type and then each spore
produced was grown on complete media.
The screen for mutants began at this stage. Each product spore was
tested by trying to culture it on minimal media. Most will grow since most
won’t have any mutations. However, the ones with induced mutations will
not be able to grow.
Sequential tests of mutants were used to determine the protein for which
it is deficient. The mutants were tested first on plates of vitamins, plates of
amino acids, etc. Then, say the mutants were only able to grow on plates
of amino acids. The specific amino acid required was determined by plating
the mutants on 20 plates each containing one amino acid.
These experiments were used to prove the “one gene-one protein” hypothesis, which was later modified to “one gene-one polypeptide” and sometimes
“one gene-one RNA.”
1.11.2
Arg Mutants
For example, consider the mutants argE, argF, argG, argH, four genes (not
necessarily proteins) found to be required for Arg synthesis. The following
pathway is known:
→ornithine→citrulline→arginosuccinate→Arg
The result of growing mutants on minimal media plus a relevant nutrient
is summarized below:
1.12. COMPLEMENTATION TEST
35
Nutrient wt argE
nothing +
ornithine +
+
citrulline +
+
arginosuccinate +
+
Arg +
+
argF
+
+
+
argG argH
+
+
+
This implies the pathway below:
argE
argF
argG
argH
→ ornithine → citrulline → arginosuccinate → Arg
1.12
Complementation Test
A Complementation test reveals if two recessive mutations are at the same
locus or two different loci. In other words, the complementation test shows
if two mutations are two genes or two alleles of the same gene.
To carry out the test, simply create a carrier of the two recessive mutations, called a “transheterozygote,” by crossing two single carriers.
Progeny with wild-type phenotype imply that the mutations “complement” one another and therefore are different genes.
Progeny with the mutant phenotype imply that the mutations “fail to
complement” one another and therefore are two alleles of the same gene.
A Complementation group is a group of mutations that identify the
same gene and fail to complement one another.
For example of the results of a complementation test, let us consider
Drosophila mutations. Transheterozygotes were constructed and observed
for pairs of each of nine mutations.
white
garnet
ruby
vermilion
cherry
coral
apricot
buff
carnation
white
+
+
+
+
garnet
+
+
+
+
+
+
+
ruby vermilion
+
+
+
+
+
+
+
+
+
+
+
cherry
coral
apricot
+
+
+
buff carnation
+
-
36
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
Therefore, we can deduce that garnet is a single gene, ruby is a single
gene, carnation is a single gene, and white, cherry, coral, apricot, and buff
are five alleles of a single gene.
1.13
Zebrafish
1.13.1
Modeling Development
Zebrafish is an excellent model organism for vertebrate development because
it satisfies each of the following criteria:
1. Easy cultivation (doesn’t easily lose mutants)
2. Short generation time
3. Easy to house
4. Accumulated knowledge present
5. Specifics
In this case, “specifics” refers to the fact that it is a vertebrate and it is
transparent, so it is easy to observe every cell. In addition, the yolk is in the
middle of the developing organism, which means the cells don’t have to be
clouded by microyolks and are clearly visible.
1.13.2
Development
Zebrafish develop mature bodies very quickly:
Time (hours) Stage
3 Blastula
..
.
24 All organs and systems in place
72 Functional maturity
However, they reach sexual maturity only after 8-10 weeks.
1.13. ZEBRAFISH
1.13.3
37
Alternate Complementation Test
There are many zebrafish mutations that are recessive lethal, so an altered
version of the complementation test is used. When heterozygotes are crossed,
1/4 of the progeny are expected to show the lethal phenotype.
1.13.4
Haploid Embryos
Zebrafish embryos are normally diploid when fertilized, but special haploid
embryos can be contructed to more closely examine the organism’s genetics.
The genetic material of sperm is destroyed by irradiation, but the sperm
retains the ability to stimulate an ovum to begin development into a zygote.
One such haploid embryo only has genetic material from the mother.
Analysis of F2 progeny is usually normal but mutations show up in the F3
generation.
1.13.5
Expression Screen
To differentiate genes that are expressed in every cell from genes expressed
only at certain times or in certain cells, the technique of in situ hybridization
is used. A riboprobe complementary to a certain mRNA of interest is contructed and introduced into target cells. The probe will bind a homologous
mRNA sequence, and detection of the probe implies the location of gene
expression.
Obesity is a characteristic where screening is effective. Obesity is a genetic
trait that causes zebrafish to continue eating whatever is present, whether or
not it is of optimal nutritive value.
1.13.6
Half Tetrads
In Zebrafish, after meiosis I, ova are stalled in meiosis II until union with
a sperm. If an irradiated sperm fertilizes this egg, a pair of Gynogenetic
diploid individuals (all DNA from mother) will result. Homologous chromosomes separate, but the second meiotic division is blocked, forcing sister
chromatids to remain in the same cell and form 2 diploid progeny with only
maternal chromosomes. Crossing-over occurs normally in these individuals.
Most progeny will be of parental ditype. The frequencies are assigned
variables as follows:
38
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
PM
MM
× mm
:
M
MM
× mm
:
p MM
q mm
r Mm
s Mm
F1NoXO (PD)
F1XO (T)
Therefore, since half tetrads are countable in Zebrafish, 2q represents
the number of PD progeny. Let N be the total number of cells that result.
Then, N −2q represents the number of T progeny, which are all recombinant.
Therefore, we can define recombination frequency as:
RF =
N − 2q
= 1 − 2q/N
N
However, it is somehow more accurate to introduce a factor of 1/2 since only
one of the chromosomes is involved in the crossing-over event. Therefore,
define the recombination frequency as:
RF = 1/2 − q/N
This kind of makes sense because it limits the range of RF to the usual range:
0 ≤ RF ≤ 0.5. But note that this number isn’t really a percent; in fact, it is
in units of chromatids per cell.
This RF value allows you to map the centromere distance. Genes very
close to the centromere will have very nearly 100% PD half tetrads.
1.14
Chromosomal Rearrangements
1.14.1
Origins of Mutations
• Replication errors (rare)
• Environment:
– Chemicals
– Transposable elements
– Radiation (point mutation or ds break)
1.15. DELETIONS
1.14.2
39
Types
• Point, or one base, mutation
• Rearrangements
• Transposable elements
1.15
Deletions
1.15.1
Overview
Deficiencies, also known as deletions, happen when chromosomal chunks are
removed.
These can be
• Interstitial (middle of chromosome)
• Terminal (end of chromosome)
and either
• Intragenic (1 gene)
• Multigenic (> 1 gene)
Deletion homozygotes (Df-/Df-) are often nonviable in multigenic deletions.
However, deletion carriers could have normal phenotype and are of some
use to geneticists. Deletion carriers may be nonviable if the gene
• Dosage is important
• Other allele is recessive (Pseudodominance). For example, retinal
blastomas occur in development if a pRB gene is deleted.
Let denote a centromere. For example, consider the two chromosomes:
1. ABCDEFG
2. ABFG
40
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
Since there is no pairing partner for CDE in the deletion mutant chromosome, there will be no base pairing in the deletion region, and consequently
1. A Deletion loop structure will form
2. No recombination occurs in the deleted region
3. Less recombination will occur between B and F
1.15.2
Allele Screens
Mutants are crossed with deletion mutants to more easily reveal the presence
of recessive point mutations in novel progeny.
One way to map deficiencies is to look for missing chromosomal bands.
The smallest overlapping region with a common genotype implies the location
of the gene on the chromosome.
Question 1 What is the resolution of this technique?
Question 2 Does genomics make allele screens obsolete?
In many ways, the high resolution of genomics does make allele screens
obsolete. However, it is nice to do a reality cross-check of genomics with
allele screen data.
1.15.3
Deletion Characteristics
1. Deletion loops
2. Recessive lethality
3. Lack of reversion
4. Reduced RF in animals not plants*
5. Pseudodominance
*The pollen in non-polyploid plants is not viable with deletions (however,
the embryo sacs, the plant female gametophytes, are). This suggests natural
selection has evolved this as a method of avoid errors and perpetuating fit
species.
1.16. DUPLICATIONS
1.16
41
Duplications
There are two types of duplications: tandem or non-tandem. Tandem duplications occur next to one another in sequence. Non-tandem duplications
appear in some other part of the genome.
For example, a tandem mutation might cause the sequence
ABCDEFG to change to
ABCBCDEFG
or
ABCCBDEFG.
As another example, consider the bar mutation in Drosophila chromosome
region 16A. As the result of a sloppy crossing-over event, the bar mutation
can be placed onto one chromosome twice. If this chromosome is replicated
and preserved, further crossing over can result in an individual who has
one chromosome with three alleles and another chromosome with one allele,
which results in the “double bar” phenotype (frequency 1/1600).
Once again, loops may form as a result of duplications that disrupt continuous base pairing.
Duplications can be detrimental if the gene’s dosage is important, or if,
for a non-tandem duplication, the gene is relocated to a position under the
control of another promoter.
1.17
Inversions
The thing to remember with inversions is that crossing over occurs only when
the homologous chromosomes have aligned in such a way as to maximize base
pairing.
1.17.1
Pericentric Inversion
Pericentric inversions occur when the centromere is involved in the loop
structure that forms and so any crossing-over events that occur will generate
chromatids with centromeres attached. Initially, the chromosomes might
have been inverted to the following configuration:
ABCDEFG
ADCBEFG
42
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
Recombination at the point between B and C (see figure) will result in the
following four strands of DNA:
ABCDEFG
ABCDA
ADCBEFGH
HGFEBCDEFGH
1.17.2
Viable
Not Viable
Viable
Not Viable
Paracentric Inversion
Paracentric inversions occur when the centromere is not involved in the
loop structure that forms, and so an Acentric chromosome fragment is
formed along with a Dicentric chromosome fragment (see figure).
There are two bad things about paracentric inversion:
1. Large portion of DNA is lost in the acentric chromosome fragment
2. Dicentric chromosome fragments are pulled apart at a random location
by the spindle during anaphase, resulting in a possible loss of genes
1.17.3
Diagnostics for Inversions
1. Inversion loops
2. Reduced RF
3. Reduced fertility
4. Inverted chromosomal landmark / cytology
1.17.4
Utility of Inversions
Since it is impossible to pure breed homozygous lethal mutants, the only
way to maintain the mutation is a lot of effort selecting offspring of carrier
heterozygous crosses.
Use a Balancer chromosome to overcome this shortfall. With multiple
inversions, these specially engineered chromosomes have special features:
1. No crossing over
2. Dominant marker mutation
1.18. TRANSLOCATIONS
43
3. Recessive lethal mutation
The benefit to using a balancer chromosome is that heterozygotes for the
novel mutant can be easily and continuously bred, since the homozygous balancer progeny will die as a result of the balancer’s recessive lethal mutation.
The other heterozygote progeny will be the only survivors.
These chromosomes can be used in genetic screens to look for new mutants.
1.18
Translocations
Translocation events occur when part of one chromosome becomes a part of
another, non-homologous chromosome. They come in two flavors: reciprocal
and non-reciprocal. Reciprocal translocations involve a two-way exchange of
genetic material, whereas non-reciprocal translocations are a one-way donation. Of primary interest are reciprocal translocations.
1.18.1
Overview
Translocation of normal chromosomes (N1, N2) result in the production of
two translocated chromosomes (T1, T2). Note that the chromosome number
comes from the origin of its centromere.
N1
N2
T1
T2
ABCDEF
GHIJKL
ABCJKL
GHIDEF
In prophase of Meiosis I, chromosomes will align to maximize base pairing,
so Cross structures will form when translocated chromosomes are present.
There are three options among offspring:
Alternate N1/N2 and T1/T2, frequency 50%
Adjacent I N1/T2 and N2/T1, frequency 50%
Adjacent II N1/T1 and N2/T2, very rare
Translocations are potentially harmful because they can split apart genes
or render a gene under the influence of a novel promoter.
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CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
1.18.2
Diagnostics of Translocations
1. Apparent linkage of genes usually on different chromosomes
2. Semi-sterility
3. Chromosome changes in size
1.18.3
Robertsonian Translocation
This type of translocation in humans leads to Down’s Syndrome, which usually results from trisomy 21. In this translocation, two of the Acrocentric
chromosomes, 14 and 21, swap material, generating a very small chromosome
(diacrocentric) and a chromosome much larger than either 14 or 21 individually. This creates effectively a larger dose of 21 and leads to the Down’s
Syndrome phenotype.
1.19
Ploidy
1.19.1
Terms
First, let’s establish some terminology.
Ploidy Number of chromosome sets
Euploid More than one set of chromosomes
Monoploid One set of chromosomes
Diploid Two sets of chromosomes
Polyploid More than two sets of chromosomes
Basic Chromosome Number Chromosomes per set (equivalent to “haploid number”)
Allopolyploid Containing sets of chromosomes from more than one organism
Autopolyploid Containing multiple sets of chromosomes from one organism
1.19. PLOIDY
1.19.2
45
Monoploidy
Bees and wasps are examples of organisms that are naturally monoploid.
Geneticists can generate monoploids in a genetically useful manner for
genetic screens. For example, consider the following plant monoploid genetic
screen:
1. Take pollen from a 2n plant and grow them up in an agar dish with
hormones. 1n sterile plants result.
2. Take somatic tissue from the plant, then mutagenize it and grow it out
on a selective agar dish. This creates a resistant 1n strain of plant.
3. Add colchine, a chemical that blocks spindle formation during mitosis
and causes formation of diploid progeny like in zebrafish half-tetrads.
1.19.3
Polyploidy
Not many animals are naturally polyploid, but some examples are leeches,
flatworms, goldfish, and brine shrimp, which have an unusual sexual cycle
and can sustain polyploidy.
Triploid oysters are sterile, so they have no seasonal sexual physiological
changes. Genetically engineering sustainable triploid oysters has been suggested as a potential method to enjoy the good taste of oysters (absent when
reproducing) all year long.
A third of all known flowering plants are naturally polyploid, a characteristic that has been unconsciously selected for by humans. For example,
the huge strawberries you can buy at Costco are a result of the big nuclei,
big cells, and big growth that is induced by octaploidy.
Though prevalent, it still causes some organisms, such as bananas, to be
sexually sterile.
Triploidy can be generated by fusing the gametes of a 4n and a 2n individual. The gametes will be 2n and 1n and so the progeny will be 3n. However,
these triploid individuals need to find triploid mates to reproduce. Since this
happens so infrequently, triploidy is considered to be effective sterility.
1.19.4
Autotetraploidy
Autotetraploidy may be induced in development when a failed mitotic event
causes the development of a tetraploid cell, tissue, or organ. If one of these
46
CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
shoots produces reproductive structures, diploid gametes will be produced.
This is somewhat advantageous for the plant, since it confers resistance
from recessive lethal mutations. By the same token, it increases susceptibility
to dominant lethal mutations, but since these are much less prevalent, an
overall advantage is retained.
1.19.5
Allotetraploidy
Also called amphidiploids, allotetraploids are the products of the union of
two gametes of non-identical organisms. After union of two haploid gametes,
chromosome doubling brings the cell’s total ploidy to 4.
Question 3 How and why are chromosomes doubled?
Question 4 What are the extremes of allotetraploids that can be constructed?
What breeding experiments with radically different organisms have been done?
What has been assumed? Why doesn’t this usually work?
1.19.6
Aneuploidy
Aneuploidy results when a subset of genetic material is over- or underrepresented in the genome. Hypoaneuploidy results from one less chromosome than usual, and hyperaneuploidy results from one more. This primarily
occurs as a result of nondisjunction at meiosis 2.
There are four viable trisomies in humans:
21 Down’s syndrome: mental retardation
13
18
X Superfemale: some mental retardation and sterility
Another aneuploidy is Kleinfelter’s syndrome, which is represented most
commonly by the genotype XXY, but can occur in many other combinations
of sex chromosomes.
The only viable human monosomy is for X, which results in sterility and
is called Turner’s syndrome.
1.20. ORGANELLE GENETICS
1.20
47
Organelle Genetics
There are two primary organelles with interesting genetic properties: mitochondria and chloroplasts, both of which have their own associated DNA,
but need protein products from the nuclear genome to function properly.
Organelle genomes can be either Heteroplasmic, meaning varied, within
a cell, or Homoplasmic, meaning nearly uniform.
1.20.1
Mitochondrial DNA
Characteristics of mitochondrial DNA (mtDNA) include:
1. Variable size between organisms
2. Mostly circular
In humans, there are 37 mitochondrial genes arranged into a very compact
DNA strand. Yeast, an organism with a smaller nuclear genome, has four
times as much mtDNA as humans. This implies that there is not necessarily
any correlation between nuclear genome and mitochondrial genome size.
1.20.2
Chloroplast DNA
Characteristics of chloroplast DNA (cpDNA) include:
1. Mostly homogenous between organisms
2. Generally more genes than mtDNA
3. Mostly circular
1.20.3
Bacterial Similarities
The endosymbiotic theory of organelle origin postulates that subcellular
structures were derived from smaller extracellular organisms at some point
in the evolutionary past.
Evidence of this includes these marked areas of similarities between organelle DNA and bacterial DNA:
1. Independent DNA replication
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CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
2. Sensitive to bacterial translation inhibitors
3. DNA not organized into nucleosomes
1.20.4
Four O’ Clocks
A small flowering plant, the four o’ clock proved to be one of the earliest
example of uniparental inheritance.
Consider the following two crosses:
P
Green F
P Variegated F
× Variegated M :
×
Green M
:
All Green
All Variegated
F1
F1
This implies maternal uniparental inheritance. Remember that a difference in offspring phenotype dependent on which parent had the mutant
phenotype was also observed in X-linked Drosophila traits, and investigated
using reciprocal crosses. However, since there is no difference in phenotype
between gender among the offspring in the four o’ clocks, this is clearly a
case of uniparental inheritance.
1.20.5
Xenopus
Consider the following crosses between the two Xenopus species laevis and
borealis, which are able to interbreed and have discernably different mitochondrial genomes:
P laevis F
P borealis F
1.20.6
× borealis M
× laevis M
:
:
All laevis mtDNA
All borealis mtDNA
F1
F1
Cultivating cpDNA
Organelle DNA is usually heterogenous within a cell, but by random chance
or selection can be made homogenous within a subsection of an organism.
For example, consider a variegated plant with speckled white and green
patches. If a shoot grows out from a green area, then the cultivation of a
new plant from those cells will result in an entirely green plant. The same
is true for an area that lacks the green pigment, although the plant will be
unable to reach maturity.
1.20. ORGANELLE GENETICS
1.20.7
49
LHON
A rare human disease that diminishes electron transport in mitochondria,
LHON has disastrous effects for the optic nerve and can cause blindness.
The condition only occurs if the populations of mitochondria in the cells of
the optic nerve are nearly homogenous for the mutation.
Children of an affected parent are variably affected since random chance
during development could have caused the optic nerve cells to develop with
the mutation and the germ cells to develop without the mutation.
One of the reasons LHON develops is the relatively high mutation rate
of DNA in the mitochondria, possibly due to the proximity of the DNA to
oxidative stress.
1.20.8
Chlamydamonas
In this useful model algae, there exists only 1 chloroplast per haploid cell.
Consider the following cross, where m represents the mating type (analogous to sex) and y and s are two genes of interest:
P m + y + sR
× m − y − sS
:
2 y + sR
2 y − sR
F1
This evidence implies that the s gene is inherited uniparentally and the
m strain’s chloroplast DNA is selectively degraded during the gamete union,
leaving only the m+ strain’s genotype of sR . The evidence also implies that
y is located on a nuclear chromosome.
In fact, this mitochondrial DNA of this organism’s m+ strain is selectively
degraded at gamete union, leaving only the genotypes of the m− strain.
−
1.20.9
Yeast
In yeast, if a chloramphenicol resistant haploid individual is mated with a
chloramphenicol sensitive haploid individual, a resistant individual develops.
Many new yeast can bud from this one offspring when grown on complete
media. If these are replica plated onto a chloramphenicol plate, half of the
colonies will lives and half will die. The colonies that survive will be homoplasmic chloramphenicol resistant.
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CHAPTER 1. CLASSICAL AND MOLECULAR GENETICS
1.20.10
Diagnostics
1. Consistent with maternal inheritance
2. Tissues should have defective mitochondrial function (assay with cytoplasmic hybrids)
3. Variability among progeny (even twins)
4. Mitotic segregation (in addition to meiotic)
Chapter 2
Genetics in Society
2.1
2.1.1
The Human Genome
Size
The complete human genome is a huge tome approximately 600 times the
length of the American Declaration of Independence. It can be fit into a
stack of textbooks, each of them the size of Genetics by Hartwell et al.,
approximately 22 feet high.
Should we be proud of the extent of our genome’s expansiveness? Well,
maybe not, as will be explained.
It is worthy to note that there are organisms with even larger genomes
than humans:
Amphibia
Tulip 10 times larger
Amoeba 200 times larger
Furthermore, some very closely related plants have genomes of drastically
different sizes.
This suggests that genome size is not uniformly informative about an
organism’s complexity.
In a technique used to gauge genome complexity, the following steps are
performed:
1. Physically shear DNA
51
52
CHAPTER 2. GENETICS IN SOCIETY
2. Denature DNA
3. Renature DNA slowly
4. Measure the proportion of ssDNA as a function of time
It turns out that the curve generated by these measurements is a sigmoidal binding curve whose mean value depends on the size and whose shape
depends on the complexity of the genome.
For example, the yeast genome is bigger than the E. coli genome, which,
in turn, is bigger than a viral genome. These organisms all exhibit standard
sigmoidal binding curves.
However, using the same assay on the human genome reveals that it
is much larger, and has three plateaus, which correspond to areas of the
genome with high, medium, and low complexity. Note that complexity here
is understood to mean lacking repeated sequences.
2.1.2
Repetition
The human genome contains approximately 45 percent repetitive sequences.
These can come in the form of Long Interspersed Elements (LINES) and
Short Interspersed Elements (SINES).
The main human LINE is L1, which is 6.4kb and appears about 20,000
times.
The main human SINE is Alu, which is 0.28kb and appears about 300,000
times.
Why do these repetitive sequences proliferate in our genome? Simple
population genetics says that since we depend on sex to reproduce, the fitness
of a transposon is twice that of its host.
2.1.3
Contructing Genome Maps
The idea about mapping a genome is essentially the same as mapping the a
city, county, or country. The goal of the map is to put the components of
the system in spatial relation to one another.
There are three kinds of genomic maps:
1. Chromosomal
2. Linkage
2.1. THE HUMAN GENOME
53
3. Physical
For humans, these three kinds of maps have been contructed and integrated.
2.1.4
Chromosomal Maps
The simplest type of map, chromosomal maps characterize the metaphase
spread banding patterns that arise with staining.
Proper training will let you identify chromosomes based on only banding
pattern.
Another method of chromosomal mapping is by using Spectral Karyotyping (SKY), which facilitates rapid chromosomal identification by differential
coloring.
The problem with chromosomal maps is that they only break the genome
up into 23 chunks, which isn’t a very deep classification.
2.1.5
Linkage Maps
Contructing a linkage map is a bit more complex and requires deduction
based on supposed genetic recombination events.
To construct a linkage map, unique (like Jesse Ventura) genetic markers
must be available in order to allow deduction of genetic location.
Think of genetic markers as analogous to the calculus. It makes no sense
when you initially study it, but you eventually will have a great grasp of it
with time.
Mapping involves finding enough markers then finding the distance between them all. Note that these markers are typically not genes, since there
are so few genes in the human genome.
The oldest type of marker is a Restriction Fragment Length Polymorphism (RFLP, say “rifflip”). One nucleotide can make the difference
in phenotype that causes the polymorphism, which is manifest in restriction
enzyme digests of a specific subset of genomic DNA. A mutation in a restriction site will cause the enzyme not to be able to cut there, and therefore a
different banding pattern will be observed.
RFLP pedigrees can be used to track the flow of chromosomes and genes
through generations in a family.
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CHAPTER 2. GENETICS IN SOCIETY
As useful as RFLPs are, they too do not appear frequently enough in the
genome to construct a complete map.
Simple Sequence Length Polymorphisms (SSLP, say “sisslip”) are
genetic markers that appear even more frequently than RFLPs and can be
used to construct more complete genetic maps. They consist of two unique
flanking sequences and a variable number of repetitions of a two or three
nucleotide sequence.
The number of repeats varies from individual to individual in a population, yet is transmitted with high fidelity from generation to generation.
The structure of SSLPs means that individuals can be rapidly genotyped
at a certain locus using PCR. In addition, simple pedigree analysis will reveal
the map distance between two such loci.
2.1.6
Radiation Hybrid Maps
These maps were constructed not by using pedigree analysis, but by physically shearing human DNA into bits, facilitating a “physical map” on the
order of nucleotides.
This technique is used somehow with Bacterial Artificial Chromosome contruction to facilitate an accurate genetic map.
2.1.7
DNA Fingerprinting
Best witnessed in the awe-inspiring 242 well gels that need to be run, this
technique is used to uniquely indentify an individual based on characterization of many genetic markers. The more genetic markers that are used, the
more specific the profile of the individual.
2.1.8
The Genome Projects
It was decided in the late 1980s that the federal government would spend
about $1 per base to sequence the human genome. The federal team of
researchers appointed to the task wanted 6-10 years to complete the project.
J. Craig Venter noticed the federal research teams basically laying down
on the job, and founded the biotech company Celera Genomics. Venter
claimed he could sequence the human genome in one year, and told the
federal team to instead work on the mouse genome.
2.2. HUMAN DISEASE GENES
55
The federal team’s strategy was to map, shred, map, shred, then sequence.
Then, using careful scrutiny of the constructed maps and genetic markers,
the sequenced DNA molecules could be put in relation to one another.
Venter’s idea was different. He did away with the inital mapping steps
and immediately shred the genome into small, sequencable pieces for his
method, deemed Shotgun Sequencing. The fastest computer in the world
and a complex algorithm were used to piece these sequenced pieces together,
and Venter was successful in completing the genome on time.
Venter was successful in constructing a human genome, and his model of
genome construction was used in every subsequent genome project.
However, with the genome project Nobel still unassigned, the question
of who deserves credit needs to be addressed. Some argue that Venter used
public mapping data developed by the federal project to achieve his end goal,
and so some credit needs to be awarded to the federal team. Read about the
controversy in Shreve’s Genome War and Sulston’s The Common Thread .
2.1.9
Other Projects
Gene Ontology is an online database that seeks to fully characterize what
is known about every gene, using primarily genome wide expression profiling
for large scale analysis.
The human genome was originally predicted to contain about 100,000
genes. However, these estimates were incorrect, and we now know the figure
to be close to 20,000 genes.
2.2
Human Disease Genes
Human genetic diseases, such as Huntington’s chorea, are often lethal, debilitating and awful.
2.2.1
Classes
There are three principal classes of genetic diseases:
1. Monogenic, in which a defect in one gene causes a disease
2. Diseases “with a genetic component” such as heart disease
3. Abnormal ploidy conditions, such as trisomy 21 and cancers
56
2.2.2
CHAPTER 2. GENETICS IN SOCIETY
Alkaptonura
Archibald Garrod applied Mendel’s laws in 1902 to discover that Alkaptonuria was a monogenic disease.
2.2.3
Sickle Cell Anemia
James B. Herrick in 1910 noticed the unusual red blood cells typical of Sickle
Cell Anemia. The primary symptom of this disease is widespread pain and
the only treatment is morphine, which induces addiction.
Since he realized that red blood cells were not much more than a physiological sack of hemoglobin, Linus Pauling investigated the hemoglobin of the
sickle cells and found that it has a different charge than normal hemoglobin.
In the third most famous paper in molecular biology, Vern Ingram wrote
in Nature in 1956 a paper that established that the difference Pauling noticed was due to one peptide being less negatively charged in the sickle cell
hemoglobin [6].
2.2.4
Mouse Models
Identifying human genes that follow simple Mendelian inheritance patterns
is a straightforward process.
Inheritance of human disease can be modeled in mouse, since they have
many similar proteins and a similarly sized genome.
The “batface” mutant is a point mutation that causes a developmental
defect in the mouse’s face.
With linkage mapping, such single gene mutants can be functionally
mapped within about a month. Based on rules studied in the first part
of this class, it is easy to determine the map distance of the batface gene to
other genes with which it is coinherited.
To facilitate this rapid recognition, homozygous mice are needed. Luckily
for geneticists, William Ernest Castle initiated a program of making mice
homozygous for everything (i.e. 99.98% of all loci) many years ago. After
about 40 generations of inbreeding, this level of homozygosity is acheived.
Note that these mice are homozygous for RFLPs, SSLPs, and other genetic
markers as well as functional genes.
Now, if one of these mice is bred to another inbred mouse with a different set of homozygous alleles, a nearly uniform heterozygote will result. If
2.2. HUMAN DISEASE GENES
57
this heterozygote is test crossed, recombination can be observed, and simple
counts of recombinant progeny will facilitate an estimate of map distance
from your mutant gene to the closest marker. Specifically, find the marker
with the highest frequency in mutant progeny, and that is the closest locus
to the mutant gene.
2.2.5
Human Crosses?
One problem with doing mutant analysis in humans is that it is impossible
to set up crosses due to ethical issues of common sense.
How, then, is the heritability of genetic disease analyzed in humans?
The solution is to examine large families of known genotype. It is possible
to use a likelihood ratio test statistic to get a idea of the relative likelihood of
linkage of a certain disease gene to a known marker based on simple pedigree
and genotype data.
In humans, the mutation in the CF gene was identified by comparing the
sequence to that of other animals, of which the expressed sequences should
be conserved but the noncoding regions should be not. This facilitated rapid
identification via hybridization. Also, the mutant CF allele was compared to
the wild-type human CF allele.
The point mutation responsible for the mutant phenotype 70% of the
time is an alteration of δ508Phe in the 250kb CFTR gene.
Gene therapy with lentivirus (HIV/Ebola) is used to insert a wild-type
copy of the CFTR gene in the ideal case. However, this is impossible since
the tissue that needs to be transformed, the lungs, are covered in a huge
layer of mucus.
Contrary to the proclamation of our textbook, the phenotype of CFTR
mice does not resemble human phenotype [5]. In fact, according to Fyodor Urnov, the mice have found a way to avoid the CF phenotype and live
completely healthy, even with a complete deletion of this gene.
2.2.6
Carrier Screening
A population of Ashkenazi jews were genotyped for an array of heritable
diseases that was observed to be present in the population. Many of these
horrible diseases, such as Tay-Sachs, have no cure and result in death at an
early age, so many families in which the parents were both carriers decided
not to risk having a child.
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CHAPTER 2. GENETICS IN SOCIETY
There are severe ethical and moral questions that need to be asked about
carrier screening a specific racial subset of a population, but they are not to
be answered here.
2.2.7
X-Linked SCIDs
By far the worst immunological disorder is the X-linked Severe Combined
Immunodeficiency, which manifests itself in 6-8 month old boys, rendering
their immune systems useless.
The only treatment is the expensive bone marrow transplant:
1. Anesthetize the boy
2. Surgically remove 15-30mL of bone marrow
3. Transform these cells with a virus that has a wild-type allele
4. Insert cells back into the boy
Using this trial method, six of seven babies were cured of SCID.
However, 2 of these babies got leukemia, and died. Consequently, the
trials were put on hold, since the FDA would not approve a treatment that
could cause leukemia, a cruel and unusual punishment.
More recently, trials were reinitiated with increased precautions, but another baby came down with leukemia. Trials are again at a standstill.
2.3
2.3.1
Cancer
Retinoblastoma
Contrary to what is pictured in texts, the early-onset cancer Retinoblastoma (RB) ends up causing malignant growths in the entire head, not just
the eye.
Knudson proposed the two-hit model in 1971 to statistically explain how
the retinoblastoma disorder is inherited [8]. He noticed that RB occurs sporadically in the general population but sometimes a predisposition to the
disorder is inherited as an autosomal dominant trait.
Therefore, he postulated two distinct conditions that would induce RB:
2.3. CANCER
59
1. Inheriting one “susceptibility” mutant allele, later called pRB, which
requires only one hit — knocking out the function of the other allele
— to induce cancer
2. Inheriting two wild-type alleles but having them both knocked out in
two hits by mutations
As an aside, Knudson cleverly modeled the number of cancers as a function of time for a given individual as a Poisson process, with startlingly
accurate agreement with data.
2.3.2
Alterations in Cancer Cells
There are six essential changes which a normal cell must undergo, in no
particular order, to change into a cancer cell:
1. Increased sensitivity to growth signals
2. Decreased sensitivity to anti-growth signals
3. Evasion of apoptosis
4. Tissue invasion
5. Sustained angiogenesis
6. Limitless replicative potential
2.3.3
Heterogeneity of Cancer
There are so many different types of cancer, and each one has a uniquely
aneuploid genome. In fact, one of the only things common to all cancers is
the genome instability that leads to cancer development.
For example, Henrietta Lacks was the first extensive case study of cervical
cancer. Cancer cells from her cervix were cultured for study, named HeLa,
and continue to grow in laboratories worldwide.
SKY was used to identify the chromosomes of normal versus HeLa cells.
Because of genome instability, HeLa cells were severely aneuploid with abnormally structured chromosomes.
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CHAPTER 2. GENETICS IN SOCIETY
2.3.4
Genetic Cancer Causation
There are two primary classes of cancer related genes:
1. Oncogenes: gain-of-function genes which are usually involved in cell
growth and will induce cancer with only one allele present
2. Tumor Suppressors: loss-of-function genes which are usually involved in homeostasis and will induce cancer only with two mutant
alleles present
These genetic changes result in a self-sufficiency for growth in cancer cells
that is usually only reserved for these cell types:
1. Bone marrow
2. Skin
3. Epithelium
4. Germ cells
This growth requires something obvious: cell division. Every cancer cell
must be able to divide in order to proliferate and overtake its host.
2.3.5
Cell Division Cycle
Hartwell studied budding yeast growth — specifically, cell division cycle mutants — to determine, ultimately, the molecular basis of the cell division
cycle, which, among other things, is instrumental to the development of cancer. He received the Nobel Prize in PM in 2001.
Gleaning such interesting knowledge from such uninteresting model organisms (yeast) points out the great significance and impact of basic research
on the advancement of practical knowledge.
Hartwell performed many forward genetic screens, techniques which involved two key tricks:
1. Temperature sensitive mutants
2. Replica plating
2.3. CANCER
61
Using these two methods, he could isolate yeast strains that were mutant
in the cell division cycle but not dead. The method of detection was first
published by Hartwell in 1970 [4].
The stage at which the cells were uniformly arrested is the stage when
the mutated protein is required to advance the cell cycle.
The gene cdc28 was identified as the gene responsible for initiation of
cell division, at the phase dubbed “start.” This is when the cell commits to
division, an event critical to carcinogenesis.
The human homolog of cdc28 is CDK1, a cyclin dependent kinase, so
named because its functionality is regulated by cyclins, a class of proteins
related to the cell division cycle. Since cdc28 and CDK1 are so perfectly
conserved, yeast modeling of the human cell division cycle is very effective.
2.3.6
Oncogenes
Mutations in the signaling pathway from the cell growth receptor to the
gene promoter binding elements are the primary cause of the prolific and
detrimental growth characteristic of cancer.
Receptor Tyrosine Kinases (RTKs) such as Epidermal Growth Factor Receptor (EGFR) fall into a class of genes called Proto-Oncogenes, or
genes which, when mutated, act dominantly to cause cancer. These mutations usually take the form of alterations or deletions to binding domains
that cause the protein to become constitutively active, losing regulation of
their extracellular or intracellular domains. When these genes are mutated,
they are referred to as Oncogenes. An example of a proto-oncogene is the
human Ras, which becomes an oncogene when it loses its GTP hydrolysis
ability.
For example, in Burkitt’s Lymphoma, a translocation results in the
generation of a chimeric oncogene whose coding region is normal but whose
transcription is constitutively on. The affected gene is called Myc, is responsible for signaling cell division, and is usually activated only briefly. Myc
affects cell division in all cell types but only creates a tumor in white blood
cells since they are proliferating.
In summary, there are three primary proto-oncogene classes:
1. Growth factor receptors
2. Signaling proteins
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CHAPTER 2. GENETICS IN SOCIETY
3. Promoter binding elements
In Chronic Myelogenous Leukemia, another condition initiated by
a genomic translocation, increased myelination results in cancerous development. This is one of the only cancer types that is treatable with a rationally
developed chemical, Gleevec. By chemical optimization and progressive elaboration of structure, the drug has been perfected and is effective in reducing
the mortality rate for this type of cancer to 5% for those people who take
400mg of this drug per day.
In some types of breast cancer, another RTK, her2, can be targeted by
herceptin, a mouse monoclonal human antibody which engages the human
immune system to attack the cancer. This is also an effective and highly
specific treatment.
In contrast, most chemotherapy drugs such as Cisplatin and Taxol indiscriminantly tear apart the body, destroying any cells that they encounter.
These treatments have large, unpleasant side effects and are highly unspecific.
2.3.7
Tumor Suppressors
The function of Tumor Suppressor proteins was first described by Ephrussi
in 1969 [3].
Examples of tumor suppressors include:
1. p53
2. pRB
Every cancer has both alleles of both genes inactivated.
Question 5 Is this really valid? There must be exceptions.
This suggests another interpretation of “two-hit” models of cancer: tumors
will only form when both tumor suppressors have been knocked out and at
least one oncogene has been turned on.
There are many ways for genes to become mutant:
1. Polycyclic Aromatic Hydrocarbons in tobacco smoke bind DNA
in lungs
2. Aflatoxin is found in moldy peas and binds to liver cell DNA
2.4. GENETICS OF HUMAN DIVERSITY
63
3. UV light directly fuses two adjacent Thymine bases
4. DNA is naturally methylated in eukaryotes, but this modification increases the rate of mutation for Cytosine bases
Thus, a clear connection is established between what happens to you and
what happens to p53, which ultimately means your rate of carcinogenesis.
When p53 is mutated, it is the exception among tumor suppressors since
the mutation acts genetically dominant, whereas most tumor suppressors
can act with only one functional allele. Haploinsufficiency in p53 occurs as a
result of p53’s usual action as a tetramer. If any one of the four subunits in
the full complex are mutated, then a nonfunctional p53 complex will result.
This results in effectively invalidating 15/16 of all p53 complexes formed.
The reason that pRB heterozygotes are actually more susceptible to cancer than wild-type individuals is because the pRB protein is involved in mutation suppression. In the heterozygote, only half as much pRB is functional,
so mutations occur at twice the normal rate. If one of these mutations is in
the other pRB allele, complete loss of pRB is sustained.
In breast cancer, estrogen acts as a potent carcinogen and morphogen of
the breast. Some drugs such as Premarin have been developed to combat
breast cancer, but evidence of their effectiveness is minimal.
In some inherited forms of breast cancer, the genes BRCA1 and BRCA2
are mutated. The only treatment for these inherited types and other spontaneously generated breast cancers are masectomy and ovarectomy. For some
reason women who have these mutant alleles and who exercise in their adolescent years are much less susceptible to this inherited form of breast cancer.
2.4
2.4.1
Genetics of Human Diversity
Introduction
The genetics of human diversity can be summarized by the fact that the
physical differences between individuals can be largely attributed to differences in DNA. One interesting example of a physical difference is race, which
is visually easy but genetically difficult to distinguish.
The study is motivated by two recent events:
1. Harvard Professor Summers’ suggestion that women are inherently less
likely to be apt mathematical thinkers than men
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CHAPTER 2. GENETICS IN SOCIETY
2. The first drug that has been developed for deployment to a specific
race, “Bidil”
Duster offers a good review of the subject in his paper “Race and reification in science” [2].
These are a few of the issues involved with the inheritance of race:
1. What is the nature of the inheritance of the phenotype of interest?
2. What is inherited along with the phenotype of interest?
3. Is race one among these?
4. If the inheritance of this phenotype is relevant to public health, what
are the social and medical effects of this knowledge?
5. What general statements can and can not be made on the basis of race?
2.4.2
Examples
For example, in the United States, sickle cell hemoglobin is an African American disease; 80,000 members of this population have the disease. In contrast,
effectively no non-African Americans have this disease. This creates a clear
distinction for this disease and a race which is not recapitulated for other
diseases.
Another relevant example is Acute Macular Degeneration (AMD),
which causes affected individuals to be unable to see anything in the middle
of their field of view and see the periphery without focus. Since this disease
is prevalent in the rapidly growing elderly population, it is of utmost importance to public policymakers. A large number of families were genotyped and
the gene responsible for the disease’s inheritance was found to be proximal
to cytological band 1q32. The two SNPs that were most probably involved
with the disorder were found on complement factor H. Individuals with these
SNPs were found to be, on average, 7.4 times more likely to contract AMD.
Humans are the only animal that drink milk past infancy. The ability to
digest milk beyond infancy is genetically inherited, with Northern Europeans
seemingly the only population that persisted with the lactase Haplotype.
This suggests that the European lactase gene had, at some point in the
past, some selective advantage based on the additional nutrients it supplied.
However, an alternate interpretation is that perhaps Europeans became the
2.4. GENETICS OF HUMAN DIVERSITY
65
only culture that drank milk precisely because they had the expression of
lactase as adults.
2.4.3
Race-Based Medicine?
Between individuals, the incidence of different bases is ≈ 1×10−4 . Therefore,
two randomly selected people will have, on average, 2-3 million bases different. Jorde and Wooding make the point that race and ancestry are correlated
but may not necessarily be used as correlates of other genetically inherited
traits, such as disease loci [7]. They conclude that genotyping for specific disease loci is a much more effective technique for establishing genotype than
racial profiling.
In his studies in 1972, Richard Lewontin found that most of the genetic
variation (≈ 85%) between individuals is explained by differences within a
race, and only a small amount (≈ 15%) is explained by variations between
races [9]. In 2004, the human genome project confirmed this on a large scale.
Question 6 How did they “confirm”? What was the definition of race?
Answer: Lewontin defined it as one of three categories of origin: Africa,
Europe, Asia.
Since humans are so attuned to deduce intrinsic differences in things from
extrinsic differences in things, we don’t intuitively realize the similarities
between perceived races.
In fact, most of the variation in the human species is within populations
in Africa. That is, at some point in the past, a common ancestor of modern
humans left Africa and formed colonies in other parts of the world relatively
recently.
In response to the issue at hand, race is genetic, but due to the quantitative nature of its inheritance, there is no simple law that governs it. The
social fear is having the genotype of someone else of your race be applied
to you even though that would be totally fallacious. Apparently USA Today writers and insurance companies would prefer this cheaper alternative
to universal genotyping.
A large number of people were genotyped for a recent study. A computer
blindly clustered the genotypes and succeeded at classifying them, on average,
into the three categories of origin mentioned above. However, there are a few
problems with this study:
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CHAPTER 2. GENETICS IN SOCIETY
1. Only a highest likelihood tree was constructed. Most individuals are of
intermediate genetic race.
2. Pharmacogenomics such as the advent of the first race-based drug,
BiDil, is promoting racial drug administration when we know that most
disease alleles inherited independently of race.
One example of this fallacious application of race to treat disease is the
inhertied mutation in Angiotensinogen, which is associated with a 10-20%
increase risk of heart disease. This gene is clearly not inherited with race.
2.5
Inheritance of Quantitative Traits
Qualitative traits such as blood type are easy to genetically characterize since
they follow simple Mendelian inheritance laws.
On the other hand, quantitative traits, such as hair, eye, or skin color,
differ in that they are controlled by many loci.
2.5.1
Pseudoscience
Some complex behaviors are commonly explained by attributing their inheritance to genetics, but this could not be further from the truth. The “folk
wisdom” that says certain abilities “run in the family” is merely a pseudoscience. For example, the New York Times ran a story about how it seemed
the ability to start businesses “ran in the family” for a certain group of people. Also, the great musician Suzanne Vega claimed her daughter must be
prolific musically due to genetics. There has even been a publication of a
“God gene,” VMAT2, which apparently is associated with increased spirituality.
These examples are all total BS, and can all be attributed to nature versus
nurture.
As an analog, consider Fermat’s Last Theorem, which has gone unproven
for centuries but was finally proven in the last decade. It states that there
are no solutions to the equation
xn + y n = z n
for n > 2. It is a simple concept to understand, but certainly not to prove.
That said, it is apparent that you don’t have to fully understand something
to argue about it. Most ignorance is willful.
2.5. INHERITANCE OF QUANTITATIVE TRAITS
2.5.2
67
Quantitative Traits
Quantitative traits will be normally distributed in a population. This result
is very easy to show, using the central limit theorem, even with a simple trait
that depends on only three loci.
Certainly, height is a quantitative trait. Also, it is clear that language
is not a quantitative trait, since any child may be taught any language. Is
there any validity to the claim that neuroticism is a quantitative trait?
As an analogous example, consider the disease pellegra, which in 1910
was deemed Mendelian in the U.S. South. Later, it was determined that the
disease was actually caused by a niacin deficiency.
This suggests a simple test to see if a characteristic is a quantitative trait.
Simply put the progeny of people at the extremes of the quantitative spectrum in the same environment, and see if they develop similarly or extremely
differently. However, for humans, this test is not feasible.
There are three things essential to quantitative traits:
1. Metric
2. Meristic, the ability to break it down into components or segments (ie
loci)
3. Threshhold
This means a meaningful method of measurement is necessary for quantifying quantitative traits.
For example, an inebriometer was built to quantify how tolerant flies were
to alcohol.
Going back to our question about neuroticism, it is apparent upon close
inspection that it is in fact pseudoscience. Two alleles are present in the general population for the serotonin receptor, which is the supposed implicator
of neuroticism. But how does one measure neuroticism? Some attempted to
by using a questionnaire with such subjective questions that the results were
totally unscientific. There was one study of fMRI of anxiety in response to
stress which had a very inconclusive result but was cited as though it was
truth. This is getting out of hand.
In contrast, in mouse models, it is easy to measure anxiety objectively.
There are two metrics:
1. Open field activity
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CHAPTER 2. GENETICS IN SOCIETY
2. Open field defecation
In 1998, Dean Hamer tried to argue that there probably existed genes
for any human behavior X. However, this is pseudoscience since, although
there are genetic components to almost everything in human life, there is
no objective metric for most of these quantitative traits and the effects of
environment on development are far too complex to assign a simple genetic
model of inheritance.
The principal reasons Hamer misunderstands the reality of genetics is
that most of the behavioral traits have
1. No good metric
2. No distinction between “genetic” and “inevitable”
3. Hundreds of other genes may affect the phenotype of one gene which
is “associated” with a certain phenotype
The government and insurance companies will misunderstand this and
we will all be screwed!
One quantitative trait that is easy to examine in mice is body weight.
Cross a fat mouse with a small mouse and then mate the brothers and sisters
of that cross. Genotype the grandchildren for a bunch of loci and see which
ones are inherited with fatness. About 100 loci were identified across the
mouse genome as being inherited with fatness. The effect of each of these
genes is negligable for the entire phenotype of fatness. This is precisely
why statements such as “gene X predisposes people to condition Y ” are
meaningless, or, at the very least, need to be taken with a grain of salt.
An interesting phenomenon is the Norm of Reaction, in which some
environmental stimuli has an effect on development, gene expression, and ultimate adult phenotype. For example, in flies, the size of the eyes depends on
the temperature to which the fly is exposed during development. In humans,
we have already discussed the example that shows women who exercise in
their teens are less susceptible to inherited forms of breast cancer.
For all of these reasons, and due to the lack of a definition, it is meaningless to claim a trait is X% determined by nature and Y % determined by
nurture.
Similarly, the following problems exist in the claim that there is a gene
X “for” a quantitative trait Y :
2.6. BACTERIAL GENETICS
69
1. “For” is a misleading word used in the wrong context
2. The number of loci involved is usually enormous
3. The norm of reaction must be considered
4. The morality of these statements can never be judged by science a la
Gould
2.6
Bacterial Genetics
The study of “bacterial” genetics is, in many respects, nominal only. So
much about bacteria has taught us about ourselves through the unity of life.
Bacterial genetics is an excellent example of how basic research can have a
profound effect on our understanding of more complex biological and medical
concepts.
2.6.1
Classical Genetics
In his Nobel lecture, T.H. Morgan asked, in a physical sense, “What are the
genes?” His own response in the same lecture was that “a geneticist has not
much concern” for the physical nature of the gene.
The experimental methods and theory of classical genetics were unaffected
by and uninterested in physical genetics.
2.6.2
Rise of Molecular Genetics
In the mid-1930s, geneticists’ views began to change, and they realized that
finding the physical nature of the gene was of interest.
Remember that Crick originally proposed the theory of
DNA −→ RNA −→ Protein
and attached the name “central dogma” to it because there was no evidence
for it at the time.
All the evidence for the central dogma was worked out in the two decades
between 1945 and 1965, using the experimental models of bacteria and phage.
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CHAPTER 2. GENETICS IN SOCIETY
2.6.3
Bacterial Virus Resistance
Fifty years ago, biology did not recognize humans to be fundamentally the
same as bacteria. That is, they did not recognize the fact that both were
organisms that used DNA as the genetic material with the same genetic code
and produced other organisms like themselves.
The Bacteriophage viral life cycle starts when a virus finds a suitable
bacterial host. It injects its viral DNA through the bacterial membrane into
the bacterial cytosol, where it is made into a transcript and then translated
into self-assembling viral proteins. These proteins cooperatively assemble
more viruses using the bacterium’s proteins and energy, and finally lyse the
bacterium when enough viruses have been made. The cycle repeats when
one of these new viruses finds a suitable bacterial host.
In the early 1930s, biologists observed that, when treated with phage,
some bacteria resist infection. During the next two decades, many biologists
devoted their lives to finding out how and why.
The essential experiment to prove that bacterial virus resistance is inherited was first performed by Salvador Luria and Max Delbrück. They studied
bacterial virus resistance at Cold Spring Harbor, NY, after fleeing from World
War II in Europe.
The prevailing belief of the conventional bacteriologist during their initial
work was that exposure to antibacterial agents caused some bacteria to acquire the characteristic of resistance, which somehow could be passed down
to subsequent generations. However, this hypothesis reeked of Lamarckism,
so was rejected by Luria.
At a faculty dance, Luria found the inspiration for the design of the
critical experiment in the faculty gambling on the slot machines.
There were two hypotheses, of which one needed to be proven true and
the other false:
1. Somehow the phage induces resistance in the bacteria
2. Some bacteria are already resistant to phage because they have inherited a resistance gene
The experiment was performed with two experimental setups:
1. One bacterium was assigned to each test tube
2. Many bacteria were assigned to each test tube
2.6. BACTERIAL GENETICS
71
Many test tubes of each setup were grown up. The result was that, after
treatment with phage, there was great variation in the growth of the clonal
tubes which sprang from the single cell. In contrast, there was somewhat of
a uniform growth in tubes which sprang from the large sample of cells.
These results implied that some bacteria were “born” with the innate
ability to resist viral infection. That is, they have a gene for viral resistance
that they inherited from the chromosomes of their parents and will pass on
to the chromosomes of their progeny.
2.6.4
One Gene-One Enzyme Hypothesis
The one gene-one enzyme hypothesis, first proposed by Beadle and Tatum,
was verified by the critical experiments on Neurospora crassa. This was the
experiment that proved the interrelation of biochemistry and genetics.
A student of Tatum’s, Joshua Lederberg, tried to apply the results of
Beadle and Tatum’s fungi investigations to bacteria. His experiment involved
two sets of bacteria:
1. Leucine and thiamine auxotrophs
2. Biotin and cysteine auxotrophs
He mixed these two strains of bacteria, and looked for new phenotypes.
Luckily, he saw that new phenotypes did arise, and that this spontaneous
phenotype generation required physical contact of the bacteria. This physical
contact, initially discovered by Lederberg around 1945, was later dubbed
Conjugation.
Before 1962, the biological community thought bacteria carried out sexual
reproduction. That’s right. They had no idea there was a fundamental
difference between prokaryotes and eukaryotes.
Streptomycin was discovered in 1962 and it served as the first successful
treatment for tuberculosis. It is a chemical agent that kills a bacterium very
efficiently when it tries to replicate.
Hayes used streptomycin in the Lederberg experiment to show the difference between sexuality and conjugation. Effectively, his experiment set up
two sets of bacterial crosses where he found that a specific strain needed to
have the streptomycin resistance gene in order to eventually confer resistance
onto the entire colony. Another result that the experiment showed was that
the F factor codes for the pilus building and genetic transfer phenotype in
bacteria.
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CHAPTER 2. GENETICS IN SOCIETY
2.6.5
The Operon
Before Jacob and Monod formulated the operon model of gene regulation,
the existing hypothesis was that lactose acts positively via an inducer which
activates ribosomes.
A first experiment performed was genetic bacterial crosses involving the
creation of artificial diploids for the LacZ and LacI genes. The cross was as
follows:
z + i+ M
× z − i− F
:
z +/− i+/−
The simplest possible explanation in this experiment is that if gene I
codes for an inducer then it will always be active, causing lactase synthesis,
regardless of the presence of sugar.
However, the data contradict this simple interpretation. The amount of
β-galactosidase made was measured as a function of time after mating. For
the control sample in which lactose was present, the bacteria made a lot of
β-gal over a period of time. For the experimental sample of bacteria with no
lactose, the unexpected result that they produced a little β-gal for two hours,
then the amount produced leveled off. So, at the end of the experiment the
double heterozygote was not making any β-gal, so the inducibility seems to
be dominant.
There are a couple problems in this interpretation:
1. Normally you would deduce that i− is a loss-of-function gene. In this
i− is a gain-of-function mutation.
2. Why does the leveling off take 2 hours to effect?
The investigators used a control experiment to determine a key fact that
help them formulate the operon model. They reversed the direction of the
cross:
z + i+ F
× z − i− M
:
z +/− i+/−
The result of this cross was that there was no initial β-gal synthesis without lactose. But, the same result was expected, since the resultant bacteria
have the same genotypes!
2.6. BACTERIAL GENETICS
73
The key fact to realize is that only DNA is transferred in bacterial conjugation, not cytoplasm. Therefore, something in the i− cytoplasm makes the
transient β-gal synthesis happen.
Jacob and Monod used this chain of experimentation and logic — later
referred to as the PaJaMo experiment — to deduce the operon model: the I
gene is a repressor, not an inducer!
2.6.6
Cis-Trans Test
Ed Lewis developed the complementation test, also known as the cis-trans
test. Genes cis to one another are on the the same chromosome, and genes
trans to one another are on different chromosomes.
Jacob and Monod constructed partial diploids in bacteria which could be
tested in this way.
The operon hypothesis predicts that the repressor binds DNA, so if the
DNA is mutated, the repressor should be unable to bind.
To prove the validity of the hypothesis, a double dominant i+ i+ bacterial
strain was constructed. Then, statistically, if the phenotype is constitutive
galactosidase activity, it is more likely that the region of DNA where the
repressor binds is mutated than both the repressor genes being knocked out.
2.6.7
Determining the Suppressor
The suppressor was originally thought to be a RNA. An experiment was
perforemed by biochemists in which they inhibited protein synthesis yet still
observed repression. They deduced that the repressor must therefore be
RNA.
A more conclusive genetic experiment revealed that the suppressor really
was a protein. Geneticists induced a “nonsense suppressor” mutation in the
stop tRNA in one strain of E. coli. Then, they created many constitutively
active (i− ) strains of E. coli. These were then mated to the nonsense suppressor strain, and one of the strains was seen to restore somewhat of the
wild-type phenotype. Therefore, they deduced that the suppressor must be
a protein.
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CHAPTER 2. GENETICS IN SOCIETY
Chapter 3
Analysis Techniques
3.1
3.1.1
Themes in Genetics
Introduction
One of the major themes of genetics is the distinction of the discipline from
biochemistry. Though biochemistry and genetics are completely useful on
their own, the union of the two provides greater results than the sum of the
parts.
It has been suggested that proteins have been, or can be, generated,
which can act to dictate changes in DNA. Thus, such a system would be a
form of accelerated evolution and could be, possibly, controlled by artificial
selection. However, the real question for us humans before we undertake such
an endeavor, is whether such a system is evolutionarily stable.
3.1.2
Distinctions
Morgan thought of genetics as an abstraction without any sort of chemical
reality.
We shall think of genetics as a science that defines functional questions
that should be answered by other disciplines, for example biochemistry.
There is also another section of genetics called reverse genetics, which has
only been around since the integration of genetics with biochemistry. Reverse
genetics is concerned with the identification of the function of a biomolecule
that has been isolated by biochemists.
75
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CHAPTER 3. ANALYSIS TECHNIQUES
3.1.3
Relevance of the Mendelian Test
The operational definition of a gene is “a difference that makes a difference.”
What this really means is “a gene where a change in the allele can affect a
change in a trait.”
Thus, genetics is actually the study of breeding behavior and embryonic
development, not necessarily how genes affect physical appearance.
An alternate segregational definition of alleles is based on the three classes
you get out of a simple Mendelian monohybrid cross.
Consider the following questions:
1. You have isolated a purebreeding wild-type and a purebreeding mutant
strain of some organism. Is a single gene responsible for this difference?
2. You have isolated two purebreeding mutant strains m1 and m2. Are
the genes responsible for the difference in phenotype actually two alleles
of the same gene?
3. Linkage mapping. Is intragenic recombination different than intergenic
recombination?
You can test these questions with the complementation test. Cross the
purebreeding mutants with one another. There are two possible results:
One Gene m1/m2 No wild-type progeny will ever be bred from these individuals.
Two Genes m1 + / + m2 Of the sixteen genotypic classes that may result
from breeding, some will be wild-type.
The important distinction in this example is that breeding behavior can
be used to determine genotype, rather than simple appearance.
Now consider the third question. Pseudoalleles are parts of a gene that
can recombine. An organism with two mutant alleles in a gene with two
pseudoalleles can therefore theoretically restore wild-type functionality by
simple recombination. This observation stimulates the operational definition
of a gene as a unit of segregation rather than a unit of function.
3.2. VIRAL GENETICS
3.2
3.2.1
77
Viral Genetics
Introduction
Viral genetics is worthy to study because high resolution genetic maps can
be constructed, as will be explained.
3.2.2
T4 Plaque Morphology
T4 is a bacteriophage of which its plaque morphology can be easily studied in
culture. Additionally, its plaque morphology was found to be a genetically
determined trait. A Plaque is essentially a hole in bacterial growth on a
plate where a virus has been successful in infecting and replicating in the
bacteria, forming about 1 × 106 virus particles. Plaque formation can be
influenced by such factors as the host bacterial strain and temperature.
In 1948, Hershey carried out the following experiment with T4 and two
strains of Escherichia coli. He worked with rapid lysis (r) mutants and saw
the following viral plaque size:
T4
rII+
rII−
E. coli B
Small
Big
E. coli K12
Small
None
It was about this time that Tom Kline offered us the insight that “If
you’re not confused, you’re not learning genetics.”
3.2.3
Viral Complementation
The equivalent of a viral complementation test is acheived with a High
Multiplicity of Infection (MOI) experiment in which many copies of two
viral strains are introduced to the nonpermissive host.
If there are two complementation groups for these genes, rIIA and rIIB,
then we can establish complementation with the cis-trans test.
Let us first assume that we have isolated a new rD mutation that is
dominant. Is it in the same gene as rIIA, which is a loss of function gene?
We can use the cis-trans test to find out.
We know that a high MOI with rD and wild-type or even rIIA virus
results in the dominant rD mutant phenotype. Do a high MOI experiment
with rD and rIIA to create a virus with these two loci in cisto find out if
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CHAPTER 3. ANALYSIS TECHNIQUES
they are in the same gene. If they are, then the rIIA should knock out the
function of the gene, negating the fact that the rD mutation is present on
the same allele. Since a wild-type allele should also be present, wild-type
phenotype will be observed. In contrast if they are on different genes, then
the dominant rD mutant phenotype should show up.
The point of course is twofold:
Different Genes Not doing something is recessive to doing something
Same Gene Doing nothing is dominant to doing something
This implies the generalized complementation test:
Different Genes cis = trans
Same Gene cis 6= trans
3.2.4
Reverting the Dominance
As discussed in the previous section, dominant mutations are recessive to
recessive alleles at different loci of the same gene (think about it).
You can establish that restoration of wild-type phenotype from dominant
mutant phenotype is actually due to intragenic function deactivation by performing the technique known as Reverting the Dominance. For example,
consider the following series:
Phenotype Allele 1 Allele 2
D
D
+
+
Dr
+
r
Dr
Dr
Note that D is dominant, r is recessive, and + denotes wild-type. The
dominance can be reverted if one r is inactivated.
3.3
3.3.1
Importance of Mutations
Benzer’s Deductions
Seymour Benzer made the following deductions about chromosomes from
genetic experiments with T4:
3.3. IMPORTANCE OF MUTATIONS
79
1. Contiguous, linear
2. Since recombination in genes is observed just like recombination between genes, Cistrons (the old word for “gene”) can be adjacent
3. The smallest non-zero genetic distance was found to be 0.02cM, about
2-3 bases
3.3.2
Deletion Mapping
Deletions were initially inferred from shortening of map distances between
known genes.
Sydney Brenner and Francis Crick used Benzer’s map to learn about the
genetic code’s features:
1. Triplet
2. Reading frame
3. Degenerate
Take, for example, the following system, consisting of gene A which is
upstream of gene B:
• Frameshift mutation in A causes the genotype A− B + . Transcription is
started anew at the promoter of gene B so its product is unaffected.
• Deletion mutant that fuses the A and B genes results in the genotype
A− B + .
• The previous deletion mutant, plus a frameshift mutation in gene A
results in the genotype A− B − . Frameshifted transcription proceeds to
gene B since it is fused with A and so none of the bases of B are read
in the proper reading frame.
• Deletion mutant that fuses genes A and B, and also a point mutation in
gene A results in the genotype A− B + . Point mutations do not alter the
reading frame of B even though the genes are fused. In fact, it is likely
that the point mutation does not result in a functional or structural
change in A.
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CHAPTER 3. ANALYSIS TECHNIQUES
• Deletion mutant that fuses genes A and B, and also a nonsense mutation in gene A results in genotype A− B − . Suppressing this mutation
(i.e. with stop tRNA mutation) results in restoration of A− B + genotype.
• Only the nonsense mutation in gene A results in the genotype A− B + .
Nonsense suppression results in genotype A+ B + . Since in this case
genes A and B are not fused, the nonsense mutation only lasts until
the end of the gene. Transcription is begun anew at the promoter of
gene B.
The real point of these deletion mapping experiments is not on the mutations themselves but rather how to infer how the normal system works from
the information you gain by examining the mutants. You need an observable,
heritable difference to define a gene.
3.3.3
Mutagenesis
Mutations are defined by the following characteristics:
1. Molecular nature of change
2. Extent of gene it changes (point versus chromosomal)
3. Effect on organism (phenotype, lethality, sterility, etc.)
4. Function change
In 1934, Muller defined two categories of mutations that fundamentally
could describe all mutations:
Loss-of-Function
1. Usually recessive (if not, haploinsufficient)
2. Amorphic or null
3. Hypomorphic or “leaky,” in an allelic series that gets progressively
more defective
Gain-of-Function
1. Usually dominant
2. Hypermorphic, or too much
3. Neomorphic, or gaining a new function or site of action
4. Antimorphic, or antagonizing the wild-type function with a dominant negative phenotype
3.4. MUTATION CLASSES
3.4
Mutation Classes
3.4.1
Kline’s Sex Lethal
81
First, remember how to revert the dominance. That is, induce a dominant
mutation in a gene, then revert the dominant phenotype back to wild-type
by causing a recessive mutation in the same gene that is “dominant” to the
original dominant mutation.
Kline’s first major discovery, the Sex Lethal gene, is a good example
of an odd mutation. When this mutation is expressed as a gain-of-function
mutation, it is of male lethal and female rescued phenotype. As a loss-offunction mutation, it is of male normal and female lethal mutation. How
shall we make sense of these results?
Perhaps this is regulated by dosage of the X chromosome in embryogenesis?
An intersting fact is that stop codons in flies aren’t as strictly enforced.
Really, the stop “codon” is determined by the bases in the general area of
the stop triplet. Additionally, in 75
Another thing to note is the distinction between dosage and phenotype.
For example, consider the white eye mutation. Is this a hypomorphic loss of
function mutation, or possibly a null mutation, or...?
The answer to all of these questions can be determined with genetic analysis.
3.4.2
Amorphic
Amorphic mutant alleles has no effect on phenotype. Increasing the wildtype dosage generates more wild-type phenotype. Increasing mutant dosage
generates no change in phenotype.
For example, consider the white eye mutation w in fruit fly (Table 3.1).
3.4.3
Hypomorphic
Hypomorphic mutant alleles result in partial loss-of-function. Increasing
the wild-type dosage generates more wild-type phenotype. Increasing the
mutant dosage also generates more wild-type phenotype.
For example, consider the hypomorphic mutant allele wa in fruit fly (Table 3.2).
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CHAPTER 3. ANALYSIS TECHNIQUES
Genotype
+/w
w/df
w/w
w/w/w
Phenotype
Wild-type
White eyes
White eyes (same)
White eyes (same)
Table 3.1: Phenotypes of various genotypes related to the white eye mutation in fruit flies. The amorphic mutation w is a recessive loss-of-function
mutation.
Genotype
wa /df
wa /wa
wa /wa /wa
Phenotype
Loss-of-function
More function
Even more function
Table 3.2: Phenotypes of various genotypes of the hypomorphic loss-offunction mutant allele wa in fruit fly.
3.4.4
Hypermorphic
Hypermorphic mutant alleles result in a gain-of-function phenotype. Increasing the wild-type dosage generates more mutant phenotype. Increasing
the mutant dosage also generates more mutant phenotype.
For example, consider the Vulval Precursor mutation lingf in C. elegans
(Table 3.3).
3.4.5
Antimorphic
Antimorphic mutant alleles result in a gain-of-function phenotype that suppresses normal function. Increasing the wild-type dosage generates more
wild-type phenotype. Increasing the mutant dosage generates more mutant
phenotype.
For example, consider the ovoD mutation in the oogenesis pathway in
fruit flies (Table 3.4).
3.4. MUTATION CLASSES
Genotype
+/+
lingf /+
lingf /df
lingf /lingf
lingf /+/+
83
Phenotype
B
Most B, Some A
B
All A
All A
Table 3.3: Phenotypes of various genotypes of the Vulval Precursor mutation
in C. elegans. The lingf allele is a dominant gain-of-function hypermorphic
allele.
Genotype
ovoD /+
ovoD /df
ovoD /+/+
Phenotype
Abnormal oogenesis
More abnormal
More normal
Table 3.4: Phenotypes of various genotypes related to the oogenesis pathway
in fruit flies. The antimorphic mutant allele ovoD is a dominant gain-offunction allele.
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CHAPTER 3. ANALYSIS TECHNIQUES
Genotype
AntpNS /+
AntpNS /df
AntpNS /AntpNS
AntpNS /AntpNS /+
AntpNS,− /+
AntpNS,− /AntpNS,−
Phenotype
Antenna changes to a leg
Antenna changes to a leg
More mutant
Same as AntpNS /AntpNS
Wild-type (dominance reverted)
Leg changes to antenna (embryonic lethal)
Table 3.5: Phenotypes of various genotypes related to the ectopic expression
of antenna genes in the leg. The neomorphic mutant allele AntpNS is a
dominant gain-of-function allele. Reverting the dominance at this locus yields
the loss-of-function AntpNS,− allele.
Class Type
Amorph Loss
Hypomorph Loss
Hypermorph Gain
Antimorph Gain
Neomorph Gain
↑wt
↑wt
↑wt
↑mut
↑wt
0
↑mut
0
↑wt
↑mut
↑mut
↑mut
Example
w
wa
lingf
ovoD
AntpNS
Table 3.6: Summary of the five mutant classes and their characteristics.
3.4.6
Neomorphic
Neomorphic mutant alleles result in a novel gain-of-function phenotype.
Increasing the wild-type dosage does not change the phenotype. Increasing
the mutant dosage generates more mutant phenotype.
For example, consider the antenna to leg mutation in fruit flies (Table 3.5).
This is an example of Ectopic expression, gene expression at the wrong time,
place, or sex.
3.4.7
Summary
The five mutant allele classes we have discussed are summarized in Table 3.6.
3.5. CONDITIONAL MUTATIONS
3.5
3.5.1
85
Conditional Mutations
Introduction
Conditional mutations come in a variety of flavors:
1. Auxotroph
2. Host range (i.e. virus)
3. Sex limited (i.e. ovoD , Sxl)
4. Suppression, either intragenic or intergenic
Analysis of gene and allele specific interactions between genes leads to the
conclusion that there are four distinct categories of intergenic interaction. We
will now discuss these categories of intergenic complementation and examples
of genes from each category.
3.5.2
Gene and Allele Specific
One example of a gene and allele specific interaction involves heterodimers.
Consider the two genes a and b. If we mutate gene a to allele a1 , we see a
mutant loss-of-function phenotype. If we mutate gene b to allele b1 , we see
a mutant loss-of-function phenotype. But, if we combine these mutations
in a single organism with genotype a1 b1 , then we see the wild-type phenotype. Such a result implies that these two genes are gene and allele specific
interaction partners that probably form a heterodimer in vivo.
Another example of a gene and allele specific interaction may occur in a
single gene that forms a homodimer. Consider the following example with
gene c. Mutating gene c to allele c1 results in a mutant loss-of-function
phenotype. Mutating gene c to allele c2 results in a (probably the same)
mutant loss-of-function phenotype. However, if we combine these mutation
in a single organism with genotype c1 c2 , then we see the wild-type phenotype.
Such a result implies that this gene acts as a homodimer in a gene specific,
allele specific manner in vivo.
An interesting consequence of the previous example is the complication of
the complimentation test. If we have two mutant strains X and Y , we would
cross them in the trans complementation test to see if the mutations are at
the same locus. Usually, if the mutations complement one another, we infer
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CHAPTER 3. ANALYSIS TECHNIQUES
that they are in different genes, and if the mutations fail to complement, we
infer that they are in the same gene.
However, this example is different, and nicely illustrates why the full cistrans test is needed sometimes. If we have the previous case, that is, X = c1
and Y = c2 , then their trans configuration will be X/Y = c1 /c2 and this will
show up as a wild-type phenotype because of the gene’s gene specific, allele
specfic homodimeric interaction. Without performing the cis version of the
complementation test, we would infer that these mutations are in different
genes, and this is certainly not the case.
Of course, generating the cis version is slightly more difficult. That is,
an organism with the c1,2 allele must be created, then crossed to a wild-type
organism with the normal c allele. This will result in mutant progeny with
genotype c1,2 /c, so we in the end infer that these two mutations in cis are
dominant to the wild-type and in the same gene.
3.5.3
Gene Specific, Allele Nonspecific
One example of a gene specific, allele nonspecific interaction involves the sex
lethal mutation SxlML and the daughterless mutation da. A da/damother
will produce only male progeny, with the female progeny of genotypes da/da,
da/+, and +/+ all dying.
So, what’s going on here is that counting the number of X chromosomes
is the method of sex determination in Drosophila. The molecular mechanism
behind this counting requires both da + and Sxl+ . But, since SxlML is a
dominant gain-of-function gene that is constitutively active, it doesn’t matter
what allele a female gets at the da locus.
The SxlML allele is able to rescue females in this gene specific, allele nonspecific interaction. It is so called because the products of the two genes da
and SxlML interact, but it doesn’t matter which allele of da the organism has,
since SxlML is constitutively active. This implies da usually has a regulatory
function for SxlML .
3.5.4
Gene Nonspecific, Allele Specific
Consider the rII locus as an example of a gene nonspecific, allele specific
interaction. With the rIIAam allele, suppression of the UAG stop codon
results in functional rII product. However, with the rIIAdiff allele, suppression
of the UAG stop codon results in nonfunctional rII product. This is a gene
3.5. CONDITIONAL MUTATIONS
87
nonspecific, allele specific interaction because nonsense suppression affects all
genes, not just this one specifically, and only one specific allele of this gene.
Consider another example, involving the hairy wing hw suppression locus
in fruit flies. The secret to this mechanism is a Gypsy Enhancer Boundary that separates an enhancer from its target promoter. Specifically, when
the hairy wing suppressor protein binds the gypsy element, it prevents DNA
folding and the enhancer cannot access the promoter, causing no transcription to occur. First, generate a dominant cut mutant, which is adjacent to
a gypsy enhancer boundary element. Then, suppress the mutation using the
hairy wing suppressor, which prevents the enhancer from accessing the cut
gene’s promoter, thus restoring wild-type functionality at the cut locus since
no transcription of the dominant mutant allele occurs. Finally, mutation of
the hairy wing suppressor gene will result in restoration of mutant cut phenotype. This interaction between the cut gene and the hairy wing suppressor
gene is said to be gene nonspecific, allele specific because the suppressor can
act on many different genes (any one with an adjacent gypsy enhancer boundary) but only when the allele that can bind the gypsy enhancer boundary is
present.
3.5.5
Temperature Sensitive
Temperature Sensitive mutations are useful because their protein products are active at one temperature and inactive at another.
Temperature sensitive mutations are particularly useful by developmental geneticists, because they allow pinpointing the interval of necessity of a
certain gene. By using temperature shifts, we can examine what the mutant
allele does during specific period of development.
For example, consider the two sets of experiments in which organisms are
initially grown at the permissive termperature TP of a hypothetical gene’s
protein product X, then shifted to the nonpermissive temperature TNP , or
vice versa TNP →TP (Figure 3.1).
First, let us consider the TNP →TP set of experiments. At time A, the
temperature shift to TP results in 100% wild-type progeny, which reveals
that X is not necessary before time A. At times C, D, E, the temperature
shift to TP results in 0% wild-type progeny, which implies that gene X must
be necessary before time C. Notice that at time B the curve is rapidly
decreasing. This is the initial time point where X is necessary.
Now, let us consider the TP →TNP set of experiments. At time A, B, C,
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CHAPTER 3. ANALYSIS TECHNIQUES
100
Shift to
permissive
temperature
Shift to
non-permissive
temperature
0
Percent Wild-Type Progeny
Temperature Sensitive Mutation Survival Curves
0
A
B
C
D
E
Time After Fertilization of Temperature Shift
Figure 3.1: Each curve represents a different set of experiments. In the shift
to nonpermissive experiments, organisms are grown from fertilization at the
permissive temperature, then shifted to the nonpermissive temperature at
various time points, measuring the number of progeny that survive for each
shift. Likewise, in the shift to permissive experiments, organisms are initially
grown at the nonpermissive temperature, then shifted to the permissive temperature.
3.6. MUTAGENESIS
89
the temperature shift to TNP results in 0% wild-type progeny, which reveals
that X must be necessary after time C. At time E, the temperature shift
to TNP results in 100% wild-type progeny, indicating that X is not necessary
after time E. Notice that at time D the curve is rapidly increasing. This is
the time point after which X is no longer necessary.
The end result of this analysis is that X activity is necessary for wild-type
development in the period that begins at time B and ends at time D. This
method of analysis with temperature sensitive mutations reveals the specific
developmental stage(s) in which a gene product’s activity is necessary. Pulsing temperature peaks is a method that can be used alongside this analysis
to get a higher resolution of temporal sensitivity.
3.6
Mutagenesis
3.6.1
Introduction
Spontaneous mutation has a rate which has been judged optimal by natural
selection. Some genes are more prone to mutations than others.
Geneticists have even isolated some Mutator mutations that drastically
increase the rate of mutation throughout the genome. These genes are usually
involved in genome maintenance activities, such as nucleotide repair mechanisms. Conditional mutator mutations are key genetic tools for mutagenesis.
Chemical agents can also be used to be used to generate mutations:
• Base analogs
• Intercalating agents
• Base modifying agents
These agents are most effective against sperm cells.
3.6.2
Radiation
Nonionizing Radiation comes from ultraviolet light and damages DNA
by making thymidine dimers. Most cells have multiple independent repair
pathways:
1. Light-dependent repair
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CHAPTER 3. ANALYSIS TECHNIQUES
2. Light-independent repair
However, these repair pathways are error prone and may lead to mutation.
Ionizing Radiation comes from X, γ, or cosmic rays and usually ends
up causing a double strand break. These breaks can be repaired by one of
two mechanisms:
• Homologous recombination
• Double strand joining (Non-Homologous End Joining)
There has been speculation that double strand joining is an artifact of
evolution that is left over from a previous era and is no longer useful since it
is rather carcinogenic. One idea to combat cancer is suppressing the NHEJ
machinery.
Ionizing radiation is the principal source of variation that is responsible
for mutations that generate the evolution of drug resistant bacteria.
3.6.3
Mobile Genetic Elements
In the 1920s, some mutations that were discovered were hard to maintain
and deemed too unstable to keep in the lab. Geneticists couldn’t figure
out why these certain traits displayed non-Mendelian forms of inheritance.
Furthermore, the geneticist that was doing most of the research, Barbara
McClintock, was hard to follow intellectually. Not many people realized the
significance of her work until geneticists began to see these Transposons in
other organisms than maize, her model organism.
Bacterial insertion sequences of about 1-5kb are used to:
1. Insert into the organism
2. Catalyze non-homologous recombination
3. Hop into the DNA of a chromosome and grab some DNA
4. Hop out
These insertion sequences are parasitic genetic elements that encode transposase and antitransposase. Many copies hopping around the genome puts
the host at risk of spending too much energy on these parasites, so they have
3.7. GENETIC MOSAICS
S|
M
P
91
M~
Normal
Dysgenic
P~
Normal
Normal
Table 3.7: Fruit fly crosses involving genes for P-elements. P flies are permissive for transposition, M are not. Dysgenic flies have a high mutation
rate, multiple chromosomal rearrangements, and sterility.
evolved a mechanism of limiting their reproduction to an optimal compromise
between energy use of the host and reproductive potential of the parasite.
It is by using these P-Elements that bacteria accomplish the rapid invention and spread of antibacterial resistance genes.
Consider the fruit fly crosses in Table 3.7. One of the progeny classes
suffers from PM hybrid dysgenesis, in which there is a high mutation rate,
multiple chromosomal rearrangements, and sterility. I guess dysgenesis can
be explained by thinking that P strains are usually stable, but when transposable elements are introduced to a genome which is not used to them, it
collapses.
3.6.4
3.7
3.7.1
Balancer Chromosomes
Genetic Mosaics
Introduction
Consider the eye in fruit fly as a model for development. It is a great model
organ because mutations in the eye still generate progeny that are viable and
fertile.
However, there are some mutations that even this ideal model system
isn’t good enough to handle. Consider the eyeless gene, which results in
no eyes, but also is pleiotropic so it affects the phenotype of many other
characteristics of the fly.
It is pleiotropic mutations like the eyeless gene that affect the viability
and fertility of the organism that motivate the use of Genetic Mosaics as
a technique of genetic analysis.
92
3.7.2
CHAPTER 3. ANALYSIS TECHNIQUES
Genetic Screens
A genetic mosaic is defined as an organism that developed from a zygote
with one genotype, but developed into an organism that contains cells of
more than one genotype. Contrast this defintion with that of a Chimera,
an organism created from multiple zygotes.
Consider some new mutation of interest m. Can we control its loss of
heterozygosity in the organism?
The first Scientific American article about genetic mosaics was posted in
1914 about a fruit fly Gynandromorph, a half male, half female organism.
This example was caused by a replication error early in development.
3.7.3
Mitotic Recombination
X-rays can induce Mitotic Recombination, a process in which crossingover occurs during mitosis. This is best exemplified in the following schematic,
which illustrates how a Twin Spot is generated:
G1 Heterozygote for both recessive mutations w and rst but since the mutant alleles are in trans they are phenotypically wild-type
S Chromosomes get replicated; now the individual has two copies of each
mutant allele
Crossing-Over Generates two homologous replicated chromosomes, each
with a chromatid with w and a chromatid with rst
Cytokinesis There is then a probability of 1/2 that the cells that result
from mitosis will have lost heterzygous at one of the two loci
If the LOH event occurs as diagrammed during development, a twin spot
of rst cells next to w cels should be generated at the site of mitotic recombination. Note that these processes can be induced by the FLP/FRT
yeast system that can be transgenically introduced to fly. This system causes
site-specific mitotic recombination from a 2µ plasmid.
Let us return to our example of the fly eye. It is generated from 6-23
precursor cells in the larva and eventually grows to ≈ 16, 000 cells per eye. A
mutation in the eye will only be seen if the mutation is Cell Autonomous,
that is, if the mutation’s expression is only affected by the local cellular
environment, and not neighboring cells (which may be wild-type in a mosaic).
3.7. GENETIC MOSAICS
93
Of course, a bigger effect on the final eye phenotype will be seen if the mitotic
recombination event occurs earlier in development.
Another trick is to screen for recessive mutations by puting a mutation
that slows development on the wild-type chromosome. One such allele, a cell
lethal mutation, in fly, is called the Minute. When mitotic recombination
makes a homozygous minute cell, it fails to grow around the other wild-type
cells, so it is effectively a cell autonomous lethal mutation. Thus, only the
other chromosome’s homozygote prevails in the region, and so it can be easily
identified.
The minute mutation’s growth retardation also implies that minute heterozygotes will not survive in a field of wild-type homozygotes. So, instead
of trying to make a minute heterozygote in a field of wild-type homozygotes,
make a wild-type homozygote in a field of minute heterozygotes.
3.7.4
Maternal Effect Lethality
Consider the m mutant allele, which generates phenotypically normal heterozygotes but abnormal homozygotes. Mothers with genotype m/m give
birth to dead progeny in a phenomenon dubbed Maternal Effect Lethality. This is considered a strict maternal effect since homozygous males are
phenotypically completely normal. The heterozygous progeny are rescuable
if the death is conditional.
The really bad thing about these types of mutations is that they are
so hard to detect. Arbitrarily say you induced the mutation in a male fly
(P). Mate with a wild-type female to generate males and females that are
heterozygous (F1 ). Mate these with each other to get some progeny that
are homozygous (F2 ). Finally, mate all these females (which don’t show any
visible phenotype) to see if any produce dead progeny (F3 ). This gets tedious
and time consuming very quickly, but there is a better solution.
Genetic mosaics are the solution of course. Consider a fly whose genotype m?/+ is unknown after a mutagenesis experiment. Clone its germ line
stem cells to a m/m. Then let’s also consider the ovoD cell autonomous
antimorphic mutation, which sterilizes females by blocking oogenesis. The
result is m/m individuals are OK, m/+ovoD individuals can’t make eggs,
and +ovoD /+ovoD individuals are bad. This technique allows identification
in the F2 generation. In case you haven’t noticed yet, the ovoD mutation is
used, linked to the wild-type allele, to suppress the fertility of the non-target
flies. Thus, only the crosses that need to be done can be done.
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CHAPTER 3. ANALYSIS TECHNIQUES
3.8
3.8.1
Transient Phenocopies
Motivation
In some organisms, there are no genetic tools we can use. For example,
in honeybees, a species with interesting behavioral patterns, there are no
analytical genetic tools. In planaria, a species that is interesting due to its
ability to regenerate an entire individual from half of one, there are likewise
no tools available to make mutations.
Sometimes, a quicker method of mutagenesis is needed than performing
a full-on forward or reverse genetic screen.
In these dire genetic situations, the newfound techniques of RNA Interference (RNAi ) experiments come into their own.
3.8.2
The Discovery
Essentially, what RNAi does is mimic the effect of a hypomorphic mutation
by treatment with a specific chemical that targets a given gene. It just so
happens that the specific chemical is a Small Interfering RNA (siRNA),
a double stranded RNA molecule of about 21-23b that corresponds to the
gene you want to silence. Note that this can only simulate a hypomorphic
mutation, not a null morph (although efforts are underway to prove the
existence of null morph RNAi ).
The concept of RNAi was originally discovered in frogs, where researchers
were trying to determine if it was possible to knock out an mRNA without
knocking out its corresponding gene. Their idea was to introduce antisense
single stranded RNA so that it would, presumably, base pair with the sense
message and prevent it from being translated. The concept didn’t work in
frogs, but it did work in C. elegans. Researchers were amazed. However, the
truth was far more complex and beautiful than they had anticipated.
The nematode gene they had knocked out in this manner was called par-1.
These are the experimental steps they followed:
1. Like any good scientists, they ran a control experiment with the sense
single strand RNA rather than the antisense strand, which was supposed to be the agent of suppression. They observed that either strand
was sufficient for suppression, and were dumbfounded.
3.8. TRANSIENT PHENOCOPIES
95
2. To make things even more confusing, in 1998-1999, researchers uncovered that the suppression effect was semi-heritable.
3. Furthermore, the suppression effect was observed to spread from cell
to cell.
4. Finally, a definitive experiment proved that it was double stranded
RNA that was accomplishing the par-1 suppression. They observed
about a hundredfold increase in par-1 silencing among the dsRNA samples. Since the RNA samples were all prepared in vitro, they concluded
that the “single stranded” RNA samples must have been contaminated
with a tiny bit of dsRNA, which they deduced to be the real agent of
suppression.
3.8.3
Mechanism
An enzyme called Dicer cuts dsRNA into small 21-23b pieces, then presents
them to machinery which somehow chops up nascent mRNA.
Key concepts of this system were uncovered from simple genetics experiments.
For example, one experiment had a promoter control GFP, which made
the worm constitutively green. Performing a RNAi experiment against GFP
resulted in dark worms. Then, a mutagenesis step restored the green phenotype. Researchers deduced that the silencing machinery had been mutated.
It was found that the RNAi transcriptional silencing machinery is extremely conserved among metazoans. It had already been identified in plants
as a weird phenomenon called quilling and cosuppression with trans genes.
The silencing machinery of RNAi revolves around a RNA Induced Silencing Complex (RISC), to which the 21-23b dsRNA fragments generated by dicer are presented. One such fragment combines with a RISC to
form a ssRNA/protein RISC, which is able to target mRNA via direct hybridization. It is interesting to note the specificity (21-23b) that has evolved
naturally in this mechanism. In a perfect match of ssRNA to mRNA, RISC
will cut the mRNA into pieces, terminating its viability as a message. In
a slight mismatch of ssRNA to mRNA, the RISC will merely inhibit the
message from being translated.
The observed generational stability of this suppression is explained by
RNA polymerase amplification of the initial dsRNA signal.
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CHAPTER 3. ANALYSIS TECHNIQUES
Another component of the RNAi mechanism is a RNA Induced Transcriptional Silencing Complex (RITS), an epigenetic modifier of chromatin structure, which effectively silences transcription of genes whose dsRNA
is present in the cell.
Consider as an example the Lin4 miRNA, a Hairpin loop structure that
gets cut up by dicer, presented to RISC, and then bound to the 30 UTR of
some related genes Lin14 and Lin28 , which it downregulates. Production of
this hairpin is accomplished by introducing a DNA that will be transcribed.
The DNA sequence has sense and antisense complementary regions that can
form the hairpin structure once it is translated into mRNA, which spontaneously forms dsRNA.
3.8.4
Limitations
RNAi experiments are extremely useful, but also have some limitations:
1. Highly variable (will the siRNA get into the nucleus?)
2. Off-target effects (will the siRNA be specific to the gene I want to
examine?)
3. Swamping the system (will too much siRNA be added?)
4. Hypomorphs but not null morphs (will partial loss-of-function be sufficient for our analysis?)
In addition, delivery of siRNA is not easy in many organisms, but it is in
nematode. It must be done
1. in vitro
2. Given genes for hairpin structure
3. Feed the nematode and it will go to any cell but the neuron
3.8.5
Speculation
Kline’s idea was that RNAi is probably a viral defense mechanism. Systems
are required for transposon stability. RNA is doing things normally for gene
regulation in development.
3.9. SEX DETERMINATION
97
This entire system probably evolved as a runaway consequence of RNA
pol being produced at some time. If RNA pol is making more messages
in the cytoplasm, then it is plausible that to save energy, natural selection
adopted the evolution of the RNAi machinery as a method of achieving an
equilibrium. Rather than have all messages be degraded very quickly, why
not have important messages persist in the cytoplasm for a while? Well,
the reason why not is because then RNA pol starts making antisense copies
(?) of these, which hybridize to the sense strand. With the presence of
both copies of the message in the cytoplasm, dsRNA forms spontaneously.
Perhaps this is just the natural cycle of RNA degradation, and bits of dsRNA
that are chopped up by dicer have evolved the side effect of silencing the gene
from which they were made. It makes sense that this response is seen, since
dsRNA levels would mean high message levels, and no more need for more
transcription. I need to look this stuff up and see if any of it has been
proposed yet.
3.9
3.9.1
Sex Determination
Introduction
Sex Determination is the process by which an organism chooses to be
male or female. Every organism has a slightly different mechanism of sex
determination. There is no universality of sex determination, although there
seems to be a universality of sex.
Sex determination is unknown in Zebrafish, a fact that its investigators
are trying to remedy. In Appendix C of our text, worm sex determination
is not discussed [5]. In fact, nematodes are self-ferilizing hermaphrodites,
but without males in the population, they would have no mechanism of
transporting genes from one individual to another.
3.9.2
Evolution of Sex
Of critical evolutionary significance is the 1:1 sex ratio that has evolved in
nearly all populations of sexually dimorphic species. It is a fact that had
been taken for granted for many years prior to Darwin, who was seemingly
the first to acknowledge its significance in his chapter “Sexual Selection”
in The Origin of Species [1].
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CHAPTER 3. ANALYSIS TECHNIQUES
Selection
Natural
Sexual
Favors Selective Force
Fittest The natural environment
Sexiest The social environment
Table 3.8: Contrasting natural selection and sexual selection
Another critical observation is that sexual selection is usually contrary to
natural selection (Table 3.8). Examples of Runaway Structures that have
evolved as a result of sexual selection include deer antlers, peacock tails, and
(it is hypothesized) human brains.
Why was intelligence selected for in humans? It is not immediately obvious that natural selection would prefer intelligent beings that can create
music and poetry, especially since there are so many nonintelligent ones that
survive just fine. In addition, there are some massively destructive tendencies of humans, such as nuclear weapons, that are seemingly in stark contrast
to natural selection. The theory is that evolution of the human brain has
been driven by arbitrary female choice and so has escalated beyond what we
might expect for simple natural selection.
The presence of sex among species alive today is astounding. Indeed, sex
is ubiquitous and has evolved at least 1 billion years ago. Evidence of this
claim include a 425 million year old fossilized penis, and the observation that
the genes for meiosis are among the most conserved.
3.9.3
The Effects of Sex
It is best to define the sexes in terms of gametes. In Anisogamous species,
it is easy to define sex since the usual eggs and sperm are produced. However,
in Isogamous species where there is little to no dimorphism in sex cells, it
is more difficult.
Sex has some enormous associated costs:
1. Males dilute female contribution to the next generation. Since each
parent contributes only 50% of its genes to the next generation, each
individual needs to produce 2 offspring to break even.
2. Sex is dangerous (i.e. STDs, vulnerability) and requires a lot of energy
(i.e. pollen)
3.9. SEX DETERMINATION
99
3. Sexual conflicts within a species. The Haig Hypothesis asserts that
a male wants his offspring to get its resources from the female, and a
female is unsure which male is best.
4. Sex breaks up winning gene combinations
But sex also has many benefits:
1. Reduces the mutational load. When a gene is lost in an asexual population, those organisms can’t recover it (Muller’s ratchet). Therefore,
there is a loss of genes without sex and recombination. An example of
this is the human Y chromosome, which long ago became recombinationally isolated from the X.
2. Males are handy for getting rid of bad genes in the population.
3. Frees good mutations from bad backgrounds.
4. Makes new alleles combinations quickly. This benefits of this offset
the drawbacks of breaking up winning gene combinations, because the
ideal gene combination is constantly changing in the amorphous natural environment. An example of this is parasites, particularly human
pathogens. Because the population is variable enough, no one parasite is able to kill everyone, such that even for HIV, about 10% of the
population does not develop AIDS.
Therefore, genes must determine the frequent 1:1 sex ratio that is found
in most populations, and the sexual dimorphism that is present in most
populations.
3.9.4
Environmental Sex Determination
An interesting example is the marine worm Bonnellia viridis, which is one
of the most sexually dimorphic species. Females of the species are about
100mm in length, whereas males are only 1mm in length, little more than
packages of DNA. If an embryo of the species lands on a rock, it generates a
female. However, if an embryo of the species lands on a female, it generates
a male. Strange, but even stranger is that the sex ratio is still probably 1:1.
The preceding example is an organism that uses Environmental Sex
Determination. Other examples are turtles and alligators that determine
sex based on the temperature of the egg, and tropical fish that remain females
as long as there is a dominant male in the school.
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3.9.5
CHAPTER 3. ANALYSIS TECHNIQUES
Genotypic Sex Determination
This contrasts Genotypic Sex Determination, in which sexual differentiation is caused by the segregation of alleles, genes, or chromosomes.
Both of these types of sex determination contrast Aristotle’s version of
the theory, which asserted that the sex of the offspring are determined by
the heat of the act. The more heated, the more likely it was to be male.
However, this assertion is inconsistent with the observation of the 1:1 sex
ratio, which we know was observed even in Aristotle’s time since records of
the census persist. Again, humans knew about the 1:1 sex ratio but did not
realize its significance until Darwin.
But still, why is there a 1:1 sex ratio if males have so much extra reproductive capacity? The answer is, only a minority of males reproduce. For
example, Ghengis Khan is the direct ancestor of ≈ 0.5% of all people alive
now.
Darwin claimed that the 2 sexes contribute equally to the next generation.
This has evolved because the minority sex always contributes slightly more to
the next generation. For example, in a population that had more males than
females, a preference to give birth to a female in the next generation would
give those genes an advantage in that next generation. Fisher examined the
skew of the 1:1 distribution in strange organisms.
An interesting consequence is that there are slightly more males at birth
than females. The reason for this is so there will be a precise 1:1 ratio at
reproductive age. Males are more vulnerable, since they have only one X
chromosome, so they die early to produce the 1:1 ratio at reproductive age.
3.9.6
Mechanisms of GSD
Motivated by the deduction of a triploid mother as a result of seeing an
intersex progeny fly, sex determination in flies was found to be a function of
the Sex to Autosome Ratio (X:A). Triploid XXX:AAA flies are female,
but diploid XX:AAA flies are intersex. These individuals are Phenotypic
Mosaics.
Thus, in many species, this X:A ratio determines sex and affects the
choice to develop into one sex or the other. Yet, the genetic material is the
same, so how is this done? An interesting experiment that sheds some light
on the situation is the production of genotypic sexual mosaics. A half XX,
half XO fly develops into a half female, half male gynandromorph. Yet, a half
3.9. SEX DETERMINATION
101
haploid X, half diploid XX fly fully develops into a female. This is because
sex is determined by the X:A ratio.
Another example of this method of sex determination is in C. elegans,
where the number of X chromosomes determines hermaproditic or male development. There is a 1:1 sex ratio of hermaphrodites to males, but in a
population of only hermaphrodites, males will arise at a low rate due to
nondisjunction events in hermaprodite self-crosses. Again, the ratio seems
to determine sex. Males are produced with an XX:AAA ratio of 2/3 = 0.67,
whereas females are produced with an XXX:AAAA ratio of 3/4 = 0.75.
However, it must be noted that these ratio methods of determining sex are
certainly the minority. Humans are an example of a more universal method
used to determine sex: the Dominant Masculinizer, which in humans is
the SRY gene on the Y chromosome. The Y chromosome was discovered
in 1929, and in 1959, aneuploid humans were discovered. Kleinfelter’s syndrome patients, sterile males, were found to be XXY, and Turner’s syndrome
patients, sterile females that mostly abort, were found to be XO.
An interesting example of a dominant masculinizer is the autosomal M
gene in the house fly. Males develop from M/m genotypes and females develop from m/m genotypes. This can be explained by noticing that Muller’s
ratchet is probably at work here, but has not gone so far as to produce
dimorphic sex chromosomes that are recombinationally isolated yet.
Another interesting example are honeybees, which have strange sex determination methods. Male drones develop from unfertilized haploid eggs,
and fertilized eggs turn into diploids that can either become fertile females
(queens) or sterile females (workers). When forced to, a queen will mate with
her sons, which we know leads to increased homozygosity, and we observe
that it sometimes produces a diploid male. Therefore, we deduce that sex
detemination is controlled in honeybees based on if the individual is heterozygous (female) or homo(hemi)zygous (male). Therefore, this is just an
obscure method of genetic sex determination.
Strangely, a phenomenon called Maternal Effect GSD also produces
the 1:1 sex ratio. In this mechanism, F/f mothers produce other females,
and f /f females produce males. Since all males are f /f , we have that:
P F/f ~ × f /f |
P
f /f ~
× f /f |
:
:
1 F/f ~
1 f /f ~
2 f /f |
F1
F1
Why is there so much variation among sexual differentiation methods?
102
CHAPTER 3. ANALYSIS TECHNIQUES
One clue comes from some arthropods, which have sexual life cycles that
can be disturbed by a parasite. That bacteria infects the arthropod, and
perverts the sex ratio to favor females in the population since the bacteria
can only survive in the cytoplasm. The answer probably is that sex has been
reinvented over the millenia to confuse parasites such as this. It has been
demonstrated that reinvention is quite easy in nematode and can be done
with only 2 mutations.
3.10
Dosage Compensation
3.10.1
The Lion Hypothesis
Dosage Compensation, the phenomena accomplished in most species by
X Inactivation, is the phenomena where all the genes on one of the female’s
X chromosomes are transcriptionally inactivated and all genes on the other
X chromosome are upregulated.
Why aren’t all X-linked genes dominant? It might seem that this would
be a logical solution to complement X-inactivation, since then no harmful
recessive genes would be exposed after X-inactivation. However, this can be
explained by invoking non-autonomity. Natural selection in the body selects
wild-type cells and the body has considerable ability to compensate for a
loss of cells, so if there are any recessive lethal mutations on one of the X
chromosomes, only cells which inactivate that chromosome will survive.
The Lion Hypothesis is the articulation of the theory that one X is
always inactivated. This can be demonstrated by putting an autosomal gene
on an X chromosome, then observing its inactivity in 50% of cells produced.
The reciprocal experiment can be performed by putting an X-linked gene
onto an autosome, then observing the result that it is always active.
3.10.2
Defining the X Chromosome
The X Inactivation Center (Xic) is the genetic element on the X chromosome responsible for defining it as a target for the X-inactivation machinery.
Searl’s Translocation between X and 16 in mouse has been used to
demonstrate that as long as the normal X is inactivated, the dosage remains
fine. If the translocated X chromosome, which includes the Xic but some elements from 16 substituted for X elements, becomes inactivated, then dosage
3.10. DOSAGE COMPENSATION
103
becomes unbalanced since there are X chromosomal elements that were not
inactivated on chromosome 16. The point is that early in embryonic development, there is a 1:1 ratio of cells with translocated X inactivated to cells
with normal X inactivated. But, since translocated X inactivation is unstable, the organism selects cells with normal X inactivation, which is what is
principally seen in the developed organism.
X-inactivation can also be used as a genetic tool. By putting a gene on
the X chromosome, you know it will be inactivated 50% of the time, so you
can study its null morph phenotype if it is a cell autonomous mutation.
3.10.3
Noncoding RNA
Other genetic elements involved in X-inactivation are the X controlling element (Xce), of unknown function to me at this time, and the X Inactive
Specific Transcripts (Xist), which encodes a noncoding RNA that coats
the inactive X chromosome only in cis. How is this done? First, it was
demonstrated that this functionality is only in cis by knocking out one X
chromosome’s Xist promoter, which induced the other X to be the one that
always inactivates. Additionally, sufficiency was shown by putting Xist on
an autosome, which will inactivate that autosome if early enough in development. Therefore, Xist is involved only in the establishment of the inactivation
phenotype.
In fly, dosage compensation is achieved by the msl gene and the rox1,2
genes. Knocking out both rox genes induces loss of dosage compensation.
This illustrates the biological theme of regulatory complexes with noncoding
RNAs.
3.10.4
Exceptions
Embryonic stem cells are totipotent cells that are good for studying dosage
compensation.
It should be noted that not all genes on the X chromosome are inactivated.
For example, in humans, 10% of genes are not inactivated. Consider two
genes a, which exhibits dosage compensation, and b, which does not, because
there is a copy on the Y chromosome (and is not inactivated). Then, normal
dosage will be as in Table 3.9.
What about more than one active X chromosome? Experiments have
shown that about half of X chromosomes inactivate (Table 3.10).
104
CHAPTER 3. ANALYSIS TECHNIQUES
Female
Dosage compensation Xa/Xa
No dosage compensation Xb/Xb
Male
XaYXbYb
Table 3.9: Normal dosage for X-linked genes a and b. Gene a must have
haploid dosage, so one X is inactivated to compensate. Gene b must have
diplod dosage, so it is on a region of the X which is not inactivated, and it
is on the Y chromosome.
Genotype
X-inactivations
XY AA
0
XX AA
1
XXY AAA
1
XXX AAA
1 or 2
XXXX AAAA 2
Table 3.10: Number of X-inactivations for various normal and aneuploid
genotypes, determined experimentally.
Also, take care to differentiate SRY from dosage compensation.
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[3] B Ephrussi et al. Malignancy of somatic cell hybrids.
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[7] Lynn B. Jorde and Stephen P. Wooding. Genetic variation, classification,
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105
106
BIBLIOGRAPHY
Appendix A
Glossary
Acentric Chromosome without a centromere, 42
Acrocentric Chromosome with a very short p arm, 44
Acute Macular Degeneration Genetic disease manifest in old age which
causes partial blindness and inability to focus on objects, 64
Aflatoxin Carcinogen found in moldy peas and binds to liver cell DNA, 62
Alkaptonuria Monogenic human disease named after the black urine which
is its primary symptom, 56
Allele An instance of a gene; each gene has many associated alleles, 12
Amorphic Mutant alleles where increased dosage has no effect on phenotype, such as the white eye mutant in fly, 81
Anaphase The third phase of cell division in which chromosomes are pulled
to opposite poles of the cell, 10
Anisogamous Sexual class in which sex cells are dimorphic, 98
Antimorphic Gain-of-function alleles that suppress normal function, 82
Autosome A chromosome that do not influence sex determination, 9
Bacterial Artificial Chromosome Bacterial chromosome with a large DNA
insert, which is usually of interest to sequence or map, 54
107
108
APPENDIX A. GLOSSARY
Bacteriophage Virus that infects bacterial hosts to replicate, 70
Balancer Genetically useful, artificially constructed chromosome that has
a dominant lethal mutation, a recessive lethal mutation, and multiple
inversions that results in very little crossing-over, 42
Burkitt’s Lymphoma Cancer of white blood cells initiated by a translocation and consequent upregulation of Myc, a cell division signal, 61
Cell Autonomous Mutation class in which only one cell of an organism
needs to be mutant for the mutant phenotype to appear, 92
Chimera Organism created from multiple zygotes, 92
Chromatid One of the two segments of DNA joined by a centromere near
the center of the replicated chromosome, 10
Chromatin The complex of DNA and protein that comprises the nuclear
genetic material, 9
Chromosome Linear array of genes and noncoding regions, 9
Chronic Myelogenous Leukemia , 62
Cistrons The old word for “gene”, 79
Coefficient of coincidence Quantity used to gauge interference; defined
as nObserved Doubles /nExpected Doubles , 26
Complementation group Group of mutations that identify the same gene
and fail to complement one another, 35
Complementation test For two genetic mutants, a test that reveals if they
share the same genetic locus, 35
Conditional Type of mutation (very useful for geneticists) that doesn’t
affect all progeny all the time, 85
Conjugation Process of bacterial DNA exchange, first discovered by Lederberg, 71
Cross Cytological structure microscopically visible which indicates formation of translocated chromosomes, 43
109
Deletion loop Structure formed
pairing, 40
Ω
in deletion mutants that optimizes base
Dicentric Chromosome with two centromeres, 42
Dicer Nucleic acid cutter which slices big dsRNA molecules into small 2123b pieces that can be presented to RISC in the mechanism of RNAi ,
95
Dominant Masculinizer Genetic element of the Y chromosome (often SRY)
that induces male development, 101
Dominant Allele which effectively silences a relatively recessive allele; dominance ensures expression of the allele in phenotype, 12
Dosage Compensation Inactivation of X chromosome in females and consequent overproduction of transcripts in active chromosome, 102
Ectopic Gene expression at the wrong time, in the wrong place, or in the
wrong sex, 84
Environmental Sex Determination Method of sexual differentiation that
is induced by host, temperature, neighbor density, presence of males,
or some other non-genetic factor, 99
FLP/FRT Mitotic recombination system that can be used transgenically in
fly; FLP is a recombinase and FRT is the DNA sequence it recognizes,
92
Gene Ontology Database which attempts to characterize function, location of expression, etc. of every gene, 55
Gene conversion Unidirectional transfer of genetic information which can
sometimes be provoked by heteroduplex DNA, 31
Gene The unit of inheritance; physically manifested by DNA in chromosomes, 12
Genetic Mosaic An organism useful for genetics experiments in which each
cell does not contain the same genetic material, 91
110
APPENDIX A. GLOSSARY
Genotype A description of the inherited genetic content; usually written as
the alleles present in the individual, 12
Genotypic Sex Determination Method of sexual differentiation that produces the 1:1 ratio and is induced by segregation of alleles, genes, or
chromosomes, 100
Gynandromorph Half male, half female organism that was first publicized
in 1914, 92
Gynogenetic diploid Genetically useful individual in which all of the DNA
comes from the mother; created by fusing a normal egg with an irradiated sperm and applying pressure (Zebrafish) or colchisine (plants),
37
Gypsy Enhancer Boundary Protein that binds to certain DNA elements
in fly that can prevent enhancer activation when the hairy wing suppressor protein binds, 87
Haig Hypothesis Essentially the principle of sexual selfishness as an evolutionary driving force, 99
Hairpin Substrate (miRNA) of RNAi machinery that binds to UTRs and
silences transcription, 96
Haploinsufficiency Not being able to sustain normal phenotype with only
one functional allele, 19
Haplotype Large portion of chromosome which is inherited together, 64
Hemizygote In males, the condition of a phenotype being determined by a
single X-linked allele, 16
Heteroduplex DNA in which the two strands of the helix consist of different, but homologous, sequences, 31
Heteroplasmic Cytoplasmically varied; refers to the many genetic varieties
of the same type of organelle present in a cell, 47
Heterozygous Individual with two different alleles, 13
111
High Multiplicity of Infection Experiment to establish complementation
in which many copies of two viral strains are introduced to a nonpermissive host, 77
Holliday model Theory of crossing-over that postulates formation of a specific structure that results from a single strand nick, 32
Homologous Chromosomes Chromosomes that match in size, shape, and
order of genes, 9
Homoplasmic Cytoplasmically uniform; all the organelles of a given type
in one cell are genetically uniform, 47
Homozygous Individual with two of the same alleles, 13
Hypermorphic Gain-of-function alleles, 82
Hypomorphic Partial loss-of-function alleles, 81
Interference The phenomenon of single crossover events somehow inhibiting double crossover events; quantified via 1−Kc = 1−nObserved Doubles /nExpected Doubles ,
26
Interphase The phase of the cell cycle between mitosis events. It consists
of the phases G1, S, and G2., 9
Ionizing Radiation Comes from X, γ, or cosmic rays and usually ends up
causing a ds-break, 90
Isogamous Sexual class in which sex cells are the same, 98
Law of Independent Assortment Pairs of alleles separate at meiosis and
join at fertilization independent of other pairs of alleles, 13
Law of Segregation The two alleles present for each gene separate during meiosis and unite randomly with an allele from another gamete at
fertilization, 12
Linked Genes which do not assort independently of one another; marked
by observed recombination frequency of less than 1/2, 21
Lion Hypothesis Theory that one X is always inactivated, 102
112
APPENDIX A. GLOSSARY
Maternal Effect Lethality Recessive mutation that is manifest in breeding behavior if mother is homozygous: progeny are mostly dead and
sometimes rescuable, 93
Maternal Effect Method of GSD in which the mother’s genotype influences
sexual development, 101
Meiosis The process of segregating alleles into gametes, 9
Metaphase The second phase of cell division which is marked by chromosomal alignment on the “metaphase plate.”, 10
Minute Cell autonomous lethal mutation which can be used to slow development; when used on a wild-type chromosome in mitotic recombination,
the homozygous minute cells fail to grow, 93
Mitosis The process of chromosome separation in somatic cells that produces two identical daughter cells, 9
Mitotic Recombination Crossing-over during mitosis, which can be induced by x-rays and is manifest in a twin spot, 92
Mutator Mutation class which increases the rate of mutation, 89
NPD Non-parental ditype; marked by recombinant progeny, and consequently indicates separation of parental chromosomes during meiosis
I, 28
Neomorphic Gain-of-function alleles that cause novelty, 84
Non-Homologous End Joining Repair mechanism invoked after a double
strand break, an event that usually occurs from ionizing radiation, 90
Nondisjunction Nonstandard chromosomal segregation during meiosis I
which results in aneuploid progeny, 17
Nonionizing Radiation Comes from UV light and damages DNA by making thymidine dimers, 89
Norm of Reaction , 68
Octad The four pairs of identical spores that result from meiosis in Neurospora; useful for quantifying centromere map distance, 30
113
Oncogene Gain-of-function gene usually involved in cell growth which will
induce cancer with only one allele, 60
Oncogene Mutant genes which act dominantly to cause cancer, 61
P-Elements Genetic elements that are permissive to transposition, 91
PD Parental ditype; indicates no recombination, 28
Paracentric inversion Inversion event where the centromere is not involved
in the loop structure that forms; the two chromosomes generated from
crossing-over will be dicentric and acentric, 42
Pericentric inversion Inversion event where the centromere is involved in
the loop structure that forms; chromosomes generated from crossingover will result in chromatids with centromeres attached, 41
Phenotype The morphological, physiological, or biochemical manifestation
of an inherited characteristic that is observable to the geneticist, 12
Phenotypic Mosaic Individuals that do no appear uniform (i.e. gynandromorphs, intersex), 100
Plaque Morphological feature of T4 bacteriophage that is genetically determined and can be mapped with very high resolution, 77
Pleiotropic Gene whose product contributes to more than one phenotype,
15
Polycyclic Aromatic Hydrocarbons Class of compounds in cigarette smoke
that binds to DNA in lung epithelial tissue, 62
Prophase The intial phase of cell division which is marked by chromosome
condensation and nuclear envelope breakdown, 10
Proto-Oncogene Class of genes which when mutated may lead to cancer,
61
Pseudoallele , 76
Pseudodominance Expression of a recessive allele caused by deletion in
the homologous chromosome, 39
114
APPENDIX A. GLOSSARY
Punnett square A genetics problem solving device constructed by first
drawing a table with haploid gamete genotypes on the periphery and
then writing the genotypes of progeny that would result from the meeting of these games in the center, 13
RNA Induced Silencing Complex (RISC) Machinery of RNAi into which
siRNA is incorporated then used to silence a corresponding mRNA, 95
RNA Induced Transcriptional Silencing Complex (RITS) Epigenetic
modifier of chromatin structure in RNAi , which effectively silences transcription of genes whose dsRNA is present in the cell, 96
RNA Interference (RNAi ) Complex method of epigenetic silencing regulated by double stranded RNA, 94
Receptor Tyrosine Kinase Growth factor receptors that are often protooncogenes, 61
Recessive lethality A fatal condition that causes developmental death in
utero only when two recessive alleles are inherited; usually deduced
from a 2:1 F2 ratio, 15
Recessive Allele which is effectively silenced by a relatively dominant allele; recessive alleles are expressed only when they are the only alleles
present, 12
Recombinant Progeny with genotypes unlike those of parents, 22
Restriction Fragment Length Polymorphism Genetic marker resulting
from a mutated restriction site, 53
Retinoblastoma First cancer whose inheritance was accurately modeled,
by the two-hit model, 58
Reverting the Dominance Technique of showing the validity of the assertion that wild-type phenotype is restored in the cis-trans test because
two mutations are in the same gene, 78
Runaway Structure An organ with seemingly no natural benefit that have
become very large or pronounced, presumably at equilibrium between
natural selection, which disfavors, and sexual selection, which arbitrarily favors, 98
115
Searl’s Translocation Demonstration of necessity of X-inactivation by swapping non-Xic genetic elements in X and 16 in mouse, 102
Severe Combined Immunodeficiency Genetic disease which renders the
immune system useless, 58
Sex Chromosome A chromosome involved in sex determination, 10
Sex Determination The molecular mechanism by which an organism becomes sexually differentiated, 97
Sex Lethal Fly gene locus where loss-of-function phenotype is male normal
female lethal, and gain-of-function phenotype is male lethal and female
rescued, 81
Sex to Autosome Ratio (X:A) Determines sex in fly, 100
Sexual Selection The mechanism of evolutionary change by which arbitrarily chosen sexy characters in males are selected by females; often
used to explain the evolution of runaway structures such as the peacock’s tail or the human brain, 97
Shotgun Sequencing Method of genome construction in which the genome
is haphazardly shred, sequenced, then pieced back together automatically with computer alignment programs, 55
Sickle Cell Anemia Monogenic human disease named after the misshapen
red blood cells characteristic of affected individuals, whose primary
symptom is widespread pain, 56
Simple Sequence Length Polymorphisms Genetic marker characterized
by unique flanking sequences and a variable number of repetitions of a
two or three nucleotide sequence, 54
Sister Chromatids Identical DNA segments connected at a centromere
that exist only after DNA replication in S phase and before cell division in mitosis, 10
Small Interfering RNA (siRNA) Double stranded RNA cut up (21-23b)
copy of a gene to be silenced in RNAi experiments, 94
116
APPENDIX A. GLOSSARY
T Tetratype; indicates crossing-over between replicated chromosomes during
metaphase I, 28
Telophase The last stage of cell division before cytokinesis; it is marked by
reformation of the nuclear envelope, 10
Temperature Sensitive Mutation class in which the protein product is
active at one range of temperatures and inactive at another, facilitating
developmental genetics experiments, 87
Test Cross Genetic cross, used to deduce the genotype of an individual with
dominant phenotype, in which the individual of unknown genotype is
crossed with a recessive homozygote, 13
Tetrad (cytology) The structure of synapsed chromosomes that forms during metaphase, 10
Tetrad Unit of progeny is many yeast strains, including S. cerevisiae, and,
functionally, Neurospora, 27
Translocation Event in which part of a chromosome is traded with, or given
to, a non-homologous chromosome, 43
Transposon Mobile genetic elements, first identified in Drosophila, that
naturally encode a transposase but can be used as transgenes, 90
Tumor Suppressor Loss-of-function gene usually involved in homeostasis
which will induce cancer only with two mutant alleles, 60
Tumor Suppressor Proteins which act dominantly to suppress tumors, 62
Twin Spot Manifestation of mitotic recombination in fly that is made available because homologous chromosomes are close to each other in interphase, 92
X Inactivation Center (Xic) Genetic element on X chromosome which
defines it as a target for inactivation, 102
X Inactivation Transcriptional silencing of all genes on one X chromosome,
102
X Inactive Specific Transcripts (Xist) Gene which encodes a noncoding RNA that coats the inactive X chromosome only in cis, 103
Index
Acentric, 42
Acrocentric, 44
Acute Macular Degeneration, 64
Aflatoxin, 62
Alkaptonuria, 56
Allele, 12
Amorphic, 81
Anaphase, 10
Anisogamous, 98
Antimorphic, 82
Aristotle, 100
Autosome, 9
Cross, 43
daughterless, 86
Deletion loop, 40
Dicentric, 42
Dicer, 95
Dominant, 12
Dominant Masculinizer, 101
Dosage Compensation, 102
double strand break, 90
Ectopic, 84
Embryonic stem cells, 103
Environmental Sex Determination,
99
Bacterial Artificial Chromosome, 54
Bacteriophage, 70
Balancer, 42
Burkitt’s Lymphoma, 61
Fisher, 100
FLP/FRT, 92
Cell Autonomous, 92
Chimera, 92
Chromatid, 10
Chromatin, 9
Chromosome, 9
Chronic Myelogenous Leukemia, 62
Cistrons, 79
Coefficient of coincidence, 26
Complementation group, 35
Complementation test, 35
complementation test, 76, 78
Conditional, 85
Conjugation, 71
Gene, 12
Gene conversion, 31
Gene Ontology, 55
Genetic Mosaic, 91
Genotype, 12
Genotypic Sex Determination, 100
Ghengis Khan, 100
Gynandromorph, 92
gynandromorph, 100
Gynogenetic diploid, 37
Gypsy Enhancer Boundary, 87
Haig Hypothesis, 99
117
118
Hairpin, 96
hairy wing, 87
Haploinsufficiency, 19
Haplotype, 64
Hemizygote, 16
Heteroduplex, 31
Heteroplasmic, 47
Heterozygous, 13
High Multiplicity of Infection, 77
Holliday model, 32
Homologous Chromosomes, 9
Homoplasmic, 47
Homozygous, 13
Hypermorphic, 82
Hypomorphic, 81
Interference, 26
Interphase, 9
Ionizing Radiation, 90
Isogamous, 98
Kleinfelter’s syndrome, 101
Law of Independent Assortment, 13
Law of Segregation, 12
Linked, 21
Lion Hypothesis, 102
Maternal Effect, 101
Maternal Effect Lethality, 93
Meiosis, 9
Metaphase, 10
Minute, 93
Mitosis, 9
Mitotic Recombination, 92
Muller’s ratchet, 99, 101
Mutator, 89
natural selection, 89
INDEX
Neomorphic, 84
Non-Homologous End Joining, 90
Nondisjunction, 17
Nonionizing Radiation, 89
Norm of Reaction, 68
NPD, 28
Octad, 30
Oncogene, 60, 61
oogenesis, 82
P-Elements, 91
Paracentric inversion, 42
PD, 28
Pericentric inversion, 41
Phenotype, 12
Phenotypic Mosaic, 100
Plaque, 77
Pleiotropic, 15
pleiotropic, 91
Polycyclic Aromatic Hydrocarbons,
62
Prophase, 10
Proto-Oncogene, 61
Pseudoallele, 76
Pseudodominance, 39
Punnett square, 13
quilling, 95
Receptor Tyrosine Kinase, 61
Recessive, 12
Recessive lethality, 15
Recombinant, 22
Restriction Fragment Length Polymorphism, 53
Retinoblastoma, 58
Reverting the Dominance, 78
INDEX
RNA Induced Silencing Complex (RISC),
95
RNA Induced Transcriptional Silencing Complex (RITS), 96
RNA Interference (RNAi ), 94
RNA polymerase, 95
Runaway Structure, 98
Searl’s Translocation, 102
Severe Combined Immunodeficiency,
58
Sex Chromosome, 10
Sex Determination, 97
Sex Lethal, 81
Sex to Autosome Ratio (X:A), 100
Sexual Selection, 97
Shotgun Sequencing, 55
Sickle Cell Anemia, 56
Simple Sequence Length Polymorphisms, 54
Sister Chromatids, 10
Small Interfering RNA (siRNA), 94
SRY, 101
STD, 98
T, 28
Telophase, 10
Temperature Sensitive, 87
Test Cross, 13
Tetrad, 10, 27
The Origin of Species, 97
Translocation, 43
Transposon, 90
Tumor Suppressor, 60, 62
Turner’s syndrome, 101
Twin Spot, 92
Vulval Precursor, 82
119
X controlling element, 103
X Inactivation, 102
X Inactivation Center (Xic), 102
X Inactive Specific Transcripts (Xist),
103