Week 1: Gene to Protein (Sickle Cell Disease)

3.1. Basic principles of heredity

3.2. Monohybrid crosses and the Principle of Segregation
note: probability as a tool in genetics 3rd edition: pp.51-53; 4th edition: pp.52-55)
Chapter 6: Pedigree analysis, applications and genetics testing
6.1, 6.2. Pedigrees

Gregor Mendel - experimented with

peas, having the monastery garden and
greenhouse for use. Peas grow relatively
rapidly (compared to animals) and also
produce many offspring which made his
experiments less time consuming
In Mendel's crosses, seed shape
determined by gene that exists as 2
alleles (specific version for a gene)
Genotype is the set of alleles that an
individual organism possess
Phenotype is the manifestation or
appearance of a characteristic
o This characteristic can be either
physical, physiological,
biochemical, or even behavioural

Note that in Mendel's experiments,

the genotype largely determined the
shape of the seeds whereas in other
situations, environmental factors could
have a huge impact on phenotypic
characteristics (such as the height of a
tree)
Remember that only alleles are pass
down (not the parent's phenotype)
Mendel realized that each variety of
peas he was studying was pure
breeding (homozygous) as all offspring
were the same as their parents

He then began to cross the different varieties

Mendel used monohybrid crosses - crossing two different pure breeding pea plants (round vs
wrinkled) - this is called the P (parental) generation

The offspring (F1 Generation) expressed only one of the phenotypes present in the parental
generation
The F1 generation was then self fertilized by each other - producing the F2 generation in which he
discovered a 3:1 ratio of round to wrinkled
This lead to the idea that each plant must possess two genetic factors encoding a character
2nd conclusion - alleles separate when gametes are
formed by the parents (one from each)
3rd - the concept of dominance and recessiveness
4th conclusion - alleles separate with equal
probability
Principle of segregation - each individual diploid
organism has two alleles for any particular
characteristic which then segregate into gametes in
equal proportions

Punnett Squares:

Backcross - crossing a heterozygous F1 generation

with a homozygous parent

In the square, each cell contains an allele from each

of the corresponding gametes
Probability as a tool in genetics:
Multiplication rule: the probability of two or more
independent events occurring together is calculated
by multiplying their independent probabilities (eg.
Probability of rolling two 4's) - the key word is and
Addition rule: the probability of any one of two or
more mutually exclusive events is calculated by
adding the probabilities of these events (eg.
Probability of rolling either a 3 or a 4)
Binomial expansion and probability: sometimes we want to know the probability of several
different combinations of outcomes occurring

Another way to determine the probability of any particular combination of events is to use the
following formula:

Where P equals the overall probability of event X with probability p occurring s times and
event Y with probability q occurring t times

The Testcross:

When one individual of unknown genotype is crossed with another individual with a known
homozygous recessive genotype for that trait
o In the instance that you have a tall pea plant but don't know it's genotype, you could
perform a testcross with a homozygous short plant
If the tall plant were homozygous: all progeny would be tall
If it were heterozygous, half would be tall and half would be short

Pedigree Analysis, Applications, and Genetic Testing

The study of human genetic characteristics presents some major obstacles:

o Controlled matings are not possible unlike with plants
o Humans have long generation times (geneticists would have to wait on average 40 years to
observe the F2 progeny
o Human family size is also generally small (not
enough sample size)
Instead, to study human inheritance, we use
pedigree charts which are pictorial representations
of a family history of the inheritance of traits
A son will always inherit his X chromosome from his
mother - if we observe that a trait is passed from

father to son, it is certain that the trait is not X-linked

Mating between closely related people is called consanguinity
Autosomal recessive traits appear with equal frequency in males and females. Affected children
are commonly born to unaffected parents who are carriers of the gene for the trait which tends to
skip generation. Traits are also more likely to appear among progeny of related parents

Autosomal dominant traits appear with both

sexes with equal frequency but usually does not
skip generations. Affected people have affected
parents. Unaffected persons cannot possibly
transmit the trait

X linked recessive Traits:

o Appear more frequently in males than in
females (males only have one X
chromosome)
o Affected males usually born to unaffected
mothers (tends to skip generations:
unaffected female to affected male to
unaffected female)
o Cannot be passed from father to son
(receives Y chromosome from dad)
o All daughters of affected man will be carriers
o Woman displaying trait must be homozygous

Y linked Traits
o Only males are affected - passed from
father to son
o If the father is affected, all of his sons will
be affected
o Do not skip generations

Class Example

Anemia is a clinical condition with signs of

abnormally low red blood cell count
RBCs contain haemoglobin which transports oxygen
Anemia can be chronic or temporary due to poor
nutrition
Hereditary anemia is due to abnormalities in
haemoglobin structure
Symptoms include:
o Fatigue, pain crises (cells dying), swelling
and inflammation, splenic sequestration,
lung and heart injury, ulcers
Tissue is being deprived of oxygen

 Haemoglobin has 2 alpha and 2 beta subunits which

make up the protein tetramer
o Thus the product of two protein coding genes
(alpha and beta globin)
The genetic cause is due to a single nucleotide base
pair substitution in the beta globin gene sequence (SNP) A is
replaced with T resulting in Valine amino acid rather than
Glutamine
o The consequence is the formation of long,
linear polymers of HG at low concentrations
o Lifespan of RBCs is reduced from 12 days to
10-20, cells are not being replaced enough
Sickle cell disease is inherited as a homozygous
recessive disease two Beta-S alleles
Mendels first principle of segregation: TWO ALLELES
OF AN ALLELE PAIR SEGRAGATE APART DURING GAMETE FORMATION

Basics of probability and inheritance:

o Sum Rule: probability of the occurrence of any of several possible mutually exclusive
events is the sum of the probabilities of the individual events
For instance, the probability of rolling a 3 OR a 4 is 1/6 + 1/6 = 2/6
o Product Rule: probability of two independent events happening simultaneously is the
product of their individual probabilities
for instance, probability of roll of a 6 AND THEN a 4 in two separate rolls is 1/6 x
1/6 = 1/36
o Another example:
the probability of having a boy and a girl can occur in two ways:
1st child is a girl, 2nd is a boy: x =
1st child is a boy, 2nd is a girl: x =
The probability is the total of these two probabilities =
Genotypic/phenotypic ratios
can also be drawn out as branch
diagrams:
1: draw out the possible
alleles for one parent with their
probabilities
2: connect each allele
individually to the other parents
possible segregated alleles
3: multiple the connect
probabilities to get the probability of
getting that particular genotype in
the offspring

With a western blot and densitometry, SCD can be distinguished by its expression of proteins

Vernon Ingram also studied SCD and found that a peptide (Valine) was substituted in place of
Glutamic acid father of molecular genetics
Dominant and recessive are relative terms
dependent upon the level of analysis, we can
distinguish levels of phenotype in the context of
SCD
o Physiological
o Cellular
o Molecular
The phenotype can also be effected by
changes in the environment:
At normal altitudes, the
heterozygous carriers do not show symptoms of SCD

However, at high altitudes, heterozygotes show a phenotype that is

intermediate between the two homozygous this is phenotypic incomplete
dominance

Week 2: Population Genetics (Malaria and the B-globin allele)

Chapter 25: Population genetics
25.1. Genotypic and Allelic frequencies
25.2. Hardy-Weinberg principle
25.4 Forces that can change allelic frequencies
we are focusing upon the role of natural selection 3rd edition: pp.695 700 ; 4th edition: pp.709- 714.

Population genetics - studies the genetic makeup of groups of individuals and how its genetic
composition changes with time
o Focus attention on mendelian population which is a group of interbreeding sexually
reproducing individuals with a common set of genes (gene pool)
o Evolution is changes in a gene pool (frequency of different alleles)
o Mutations commonly appear in higher frequency due to a selective advantage
o Mechanisms of malaria:
Symptoms include headache, nausea, high fever, vomiting and flu like
symptoms
Susceptible to other infections which may lead to death
Cause by protozoan plasmodium falciparum carried by the mosquito larvae
mature in the liver of the host animal and then RBCs

How do Sickle cells help against Malaria?

Sickled RBCs are fragile and how low lifespan in heterozygotes
This lifespan interrupts the development cycle of the plasmodium larvae, preventing
proliferation of malaria in the host
BsBs homozygous carriers would be resistant but still die early due to the detrimental effects of
SCD
Therefore, BsBa carriers live on and pass their genes (gain of Bs and Ba allele in population)
natural selection maintains both alleles in population homozygous phenotypes are still
maintained balanced polymorphism
Allele frequency: frequency of a single allele of a gene within the whole population, to calculate
this, you first need genotypic frequencies
Remember that diploids carry two alleles per gene = 2N

Genotypic frequencies:

p+q=1
frequency of two alleles in a population are unlikely to be the same
the recessive allele will never disappear from the gene pool of future generations they are
maintained
Mendel patterns of inheritance for a cross are based upon frequencies of alleles in individuals
Fitness: the relative reproductive success of one genotype compared to other genotypes
within a population
o Ex. Bb is only 80% as successful as Bb or BB at producing offspring
Fitness (W) of the recessive phenotype is 80% Wbb = 0.8
Selection coefficient (relative disadvantage ) intensity of selection against a
genotype = 1 W = 1 0.8 = 0.2

o
o

o
o

We set the fitness of the genotype with highest reproductive success to 1, every other
fitness level is relative (divide number of offspring by most successful genotype)

Selection against a dominant allele will reduce frequency of the allele in the population
if selection coefficient is higher, frequency of alleles will decrease at a higher rate
If s=1.0, allele will be eliminated in one generation

Selection against a recessive allele will also reduce frequency of the allele- very fast at
beginning, slows down, a proportion is still maintained in heterozygotes cannot be
entirely eliminated even if lethal
To determine changes in allele frequency due to selection:

Sickle cell anemia is an example of overdominance which results in a stable equilibrium

The frequencies change if the selective pressures change
o Balanced polymorphism loss of an allele in one genotype is balanced by a selective
advantage in another genotype
Evidence for this advantage:
o Overlay of maps of malaria higher incidence of Bs allele
o Molecular evidence that Bs allele independently mutated
o Frequency of carriers increases with increasing age (live longer)
B^E and B^C allele variants were also found in Asian/pacific and west African populations as well
resistant to malaria
Linus Pauling passionate about screening techniques to prevent sickle cell anemia:
o Heterozygotes should not marry each other and have children
o Heterozygotes marrying homozygotes should have few children
o Current screening techniques:
Blood smears
Protein electrophoresis
When more than 2 different types of alleles can exist at the loci (eg. 3 alleles):

X-linked loci - supposed there are two alleles at an X-linked locus XA and Xa

Can also be calculated through

Hardy-Weinberg Principle (of equilirbrium): makes several simplifying assumptions about population
and provides two key predictions if these assumptions are met

Assumption: population is large, randomly mating, not affected by mutation, migration, or natural
selection
Prediction 1: allelic frequencies of population do not change
Prediction 2: the genotypic frequencies will not change after one generation in the proportions p1,
2pq, and q2 (hardy weinberg equilibrium)
By adhering to the hardy weinberg principle, populations cannot evolve
When a population is not in hardy weinberg equilibrium, we have no basis for predicting the
genotypic frequencies
When in HW equilibrium, genotypic frequencies are determined by allelic frequencies
Single generation of random mating produces the equilibrium frequencies p2, 2pq and q2

HW law applied to multiple alleles and X-linked alleles

Forces that can change allelic frequencies:

Mutation: before evolution can take place, genetic variation must exist which arises through
mutation
o Allelic frequencies change with time as some alleles mutate into others, eventually reach
equilibrium (forward and reverse mutation rates)
Migration: the influx of genes from other populations (also known as gene flow)
o Causes gene pools of populations to become more similar
o Adds genetic variation to populations
Genetic Drift: usually due to small population size, a small deviation by chance can lead to changes
in allelic frequencies (random and unpredictable)
o Reduces genetic variation within populations, causes genetic divergence among populations
Natural selection: when individuals with adaptive traits produce a greater number of offspring
than that produced by others in the population
o If genetic, they are inherited by the offspring, reproductive advantage allows populations to
become better suited (adapted) to their environments

CHI SQUARE TESTS: a goodness of fit chi square test is used to determine whether the differences
between the observed and the expected numbers of each genotype arise through chance

25.4 Forces that change allelic frequencies:

Processes that bring about change in allelic frequency include mutation, migration, genetic drift, and
natural selection
Mutation:

Allelic frequencies change with passage of time, eventually they reach equilibrium and are determined
by forward and reverse mutation rates
At equilibrium, Hardy-Weinberg law tells us that genotypic frequencies will remain the same
Long periods of time are required for population to reach mutational equilibrium

Migration: prevents populations from being genetically different from one another and introduces
genetic variation within populations

Genetic Drift:
Because no population is infinitely large, allele frequency will deviate by chance
Limited sample size can be referred to as sampling error
Natural Selection: when individuals with adaptive traits produce a greater number of offspring than that
produced by others in the population
Fitness is the relative reproductive success of a genotype

Week 3: Dihybrid patterns of inheritance

Multiple genes determine phenotypic traits (cats and humans are not peas)
Dihybrid ratio: 9:3:3:1
there can be multiple types of mutations at a single gene
example: multiple alleles contributing to fur coloration in cats, different dominance
frameshift mutation deletion of a nucleotide causes the reading frame to be read differently, often
results in a loss of function allele
number of alleles determines number of possible genotypes

Wildtype allele functional protein most common phenotype

Loss of function allele: no longer produced, non functional, or produced at lower levels
Wiltype usually dominant
o Half of protein is sufficient to achieve normal phenotype: happlosufficiency (ie tyrosinase)
o Or not sufficient haploinsufficiency (tailless cats)
Dominant alleles can also be gain of function mutations (ie huntingtons disease)
Law of segregation: allele pairs separate independently during gamete formation
o Mendel proved this using monohybrid crosses
Law of independent assortment: inheritance pattern of one trait will not affect pattern of another
trait
For two alleles, the Mendelian ratio is 9:3:3:1

Gene interactions may produce novel phenotypes that will modify the 9:3:3:1 ratio
epistasis: when one gene masks or modified the phenotypic expression of another gene

Complementation: 9:7 phenotypic ratio

o Mutations in two different genes product the same mutant phenotype
o Also a duplicate recessive epistasis
When a loss of function mutation leads to a lethal phenotype, we get a 2:1 ratio

Week 4: Quantitative Genetics

Week 5: Chromosomal Theory of Inheritance

Studies reveal that multiple genes are on each chromosome

Haploid (N) one copy of genetic material
Diploid (2N) two copies of genetic material; homologous pair consists of a homolog from each
chromosome
Ploidy number of complete chromosome sets
Eukaryotes gametes are haploid, adults are diploid
Chromosomes can be classified by their length and position of centromere (attaches to spindle
fibers during mitosis and meiosis)

Painting probes or g banding can be used to distinguish the 23 pairs of chromosomes we have
Can also be separated into two categories:
Autosomes: present in the same copy number in both males and females number of
morphology is species specific
Sex-chromosomes: different copies in males vs females
Chromosome number and structure varies between species:
Humans have 46 total chromosomes, as do sable antelopes
12 chromosomes in the Datura stramonium
12 variant phenotypes (arisen from different trisomies)
Sex chromosomes are represented differently in the two sexes
In the meiosis of grasshoppers, independent assortment was observed
Parallel to Mendels patterns of inheritance
Review of mitosis:
Def: replication of identical cells one cell produces two genetically identical cells
All cells in body reproduce and multiply by mitosis
A failure to separate is called nondisjunction
Homologues: two chromosomes carrying the same gene sequences
Meiosis: germ line cells undergo meiosis in order to produce haploid gametes
1st law: principle of equal segregation
2nd law: principle of independent assortment
Anaphase 1: reductional division homologues segregate (explains 1st principle)

Pairing permits exchange of DNA between homologous pair in prophase

Nonsister chromatids in bivalent undergo breakage and reunion at the chiasma (crossing
over)
Pairs attach to spindles independently of one another (independent assortment)
Anaphase 2: equational division, paired sister chromatids separate
Evidence that genes are transmitted on chromosomes:
Wild type flies have red eyes carrying w+ allele
o White allele is a mutation w
o Red eyed female crossed with white eyed male
Observed 237 red eyes flies compared to 3 white eyed flies
o After F1 cross:
3470 red (1011 male, 2459 female) : 782 white (all males)
Morgan proposed that eye colour gene is on X chromosome males are
only hemizygous one allele
If different ratios are seen for the two sexes, the gene is likely on a sex
chromosome
If reciprocal crosses give different results, the gene is likely on a sex
chromosome
Non-disjunction of X chromosome: homologues fail to segregate in meiosis 1 and/or sister
chromatids fail to segregate in meiosis 2

Week 6 Chromosome Structure and Gene Regulation

If genes are on chromosomes, then what exactly are chromosomes?

Chromosomes are compacted and organized within the nucleus this compaction/coiling changes in
different stages of the cell cycle and changes when genes are transcribed into RNA
Chromosomes consist of DNA and histone proteins
o In interphase, the chromosomes are unwound to allow transcription where in the
metaphase they are tightly coiled

we see no transcription occurring at the telomere or centromeres which are tightly compacted
these are classified as heterochromatin highly compact throughout cell cycle, low gene
expression, low gene density
o rather than constitutive, facultative heterochromatin actually converts between the hetero
and euchromatin

euchromatin composes most of the chromosome is gene rich and relatively

transcriptionally active (turned on/off dependent on cell type)
histone tails are modified to alter chromatin structure changes the ability of transcription
factors to access DNA
o may provide binding sites for other proteins
non-histone proteins are also important (scaffold proteins) - contain 20 000 to 100 000 bp
Under interphase:
o 1: chromatin structures changes to allow transcription chromosomal puff
o 2: when chromatin is in open conformation DNAse can cut DNA into small pieces
Sensitivity of DNA to digestion by DNase 1 is correlated with gene expression
Chromatin changes structure by relaxing decreased association with histone proteins
Modifications in the histone protein include:
o Acetylation
o Methylation

Phosphorylation
The centromere:
o Functional definition attachment of microtubule spindle fibres for chromosome
movement in mitosis and meiosis
o Commonly see repeated sequences not protein coding, not exclusive to centromere
o Centromeres are structurally important:
Lost centromeres results in degraded chromosome fragments
Too many may also result in a break in mitosis or meiosis
The telomere:
o Natural ends of linear chromosomes prevent chromatin degradation and
inappropriate attachment
o Also repeated sequences
In between: genes
o Unique sequence DNA: single copy genes, protein coding genes, only about 4-5% in
humans is actually protein coding
o Lots of moderately and highly repetitive DNA ie ribosomal DNA and tRNA genes
Remember females have two x chromosomes whereas males only have one and a shorter y
chromosome
o dosage compensation - way to equalize gene expression
either increase expression from y chromosome, decrease the two x
chromosomes, or turn off expression from one X chromosome
involves modifications to chromosome structure and gene expression
o this demonstrates epigenetics - heritable changes in gene expression that does not
involve a change in DNA sequence
o Barr and Bertram found a condensed mass in the nuclei of female cells in cats, not males
Was suspected to be to the x chromosome
o Sometimes genes are on the X-chromosome
o Early in development, one X chromosome is randomly inactivated
Females that are heterozygous for X-linked traits are genetic mosaics
Xist RNA coats inactivated X chromosome which attracts protein modification
factors
Compaction occurs by modification of histones that is initiated by the
Xist RNA
In each cell of the embryo, number of Xics are counted
One of the X chromosomes is targeted for inactivation
Examples of mosaics in humans for X-linked genes include:
Red green colour blindness
Anhidrotic ectrodermal dysplasia
Remember RNA polymerase and general transcription factors initiate transcription
o Cis sequences DNA sequences that bind to DNA such as enhancers and promoters
To inhibit - silencers

Trans factors proteins that bind to DNA such as transcription factors, activator
proteins, mediators, etc
to inhibit repressor proteins
o In a barr body, these proteins can not bind
The ability of protein binding is determined by chromatin structure
Modifications to histones:
o Methylation: may activate or repress gene transcription
o CpG islands are found in promoter sequences
Methylation inhibits the binding of an activator protein to the enhancer element
Methyl-CpG binding recruits other proteins that cause region to become more
compact

Acetylation: usually stimulates gene transcription

Deacetylation is associated with silencing (no transcription) as seen in heterochromatin

Genomic Imprinting the expression of genes is determined by whether the gene is inherited
from the mother or the father
o Igf2 mice inheriting the deletion from mother were normal, mice inheriting from
father were only 60% of normal birthweight

The methylated state is heritable during mitosis

It is reset prior to meiosis

In comparison to sex-linked differences, genomic imprinting is determined by the sex of the parent
carrying the mutant allele, not the individual inheriting the mutation
Prader- Willi syndrome infants are small and weak with poor feeding in first 6 months, after 12 months
they eat uncontrollably and leads to diabetes and obesity paternally inherited
Angelman syndrome- motor and mental retardation maternally inherited