Week 1, Hilary Term
Biology Hon. Mods: Cells and Genes 1.4 Genes 2
Human Sciences Prelims: Genetics and Evolution
Lecturer: Rosalind Harding email: rosalind.harding@zoo.ox.ac.uk
http://www.stats.ox.ac.uk/~harding
• Texts:
– Hartwell et al. Genetics: From Genes to Genomes. 2 nd
McGraw Hill, 2000, 2004.
ed.,
• Chapter 3. Extensions to Mendel: Complexities in relating genotype to phenotype.
• Chapter 9: Deconstructing the genome: DNA at high resolution.
– Griffiths et al. An Introduction to Genetic Analysis, 2000
• Lecture Notes:
– Weblearn
– http://www.stats.ox.ac.uk/~harding
1. A garden
2. Phenotypic traits in alternative forms
3. Some breeding experiments!
Long Short
Stems Stems
Dominant Recessive
We can explain how these dominant and recessive traits are inherited
P AA X aa
Gametes A Only a Only
Sperm produced
F1 A a (Zygote)
1/2 A
1/2 A
1/4 AA
1/2 a
1/4 A a
1/2 a 1/4 A a 1/4 aa
Overall F2 ratio 1AA : 2A a : 1 aa
Eggs
Produced
What is the link between genes as units of inheritance and “alternative forms”?
ƒ
First we need to examine the relationship between genotypes and discrete ‘Mendelian’ phenotypes.
ƒ We need to know more about these traits at molecular and biochemical levels.
Question: How are wrinkles and other
Mendelian traits produced?
Dominant allele R
Functional starch branching enzyme RR or Rr unbranched starch
Recessive allele r branched starch
Inactive starch branching enzyme
Round rr unbranched starch unbranched starch Wrinkled

Answer: Traits are derived from the function of gene products
Dominant allele L
Functional
Gibberellin-20-Oxidase
LL or Ll
Long
Stems
Inactive Gibberellin Active Gibberellin
Recessive allele l
Inactive
Gibberellin-20-Oxidase ll
Inactive Gibberellin Inactive Gibberellin
Short
Stems
Discrete character differences are often determined by differences in the products of single genes
• Enzymes
• Receptors
• Structural proteins
Long Short
Stems Stems
Gene for cystic fibrosis (a genetic disease) encodes a cell membrane protein
Round Wrinkled
But Mendel’s expected ratios of offspring phenotypes only work in special cases! Why?
We need to consider extensions to Mendel’s explanations:
ƒ For single-gene inheritance (this lecture)
ƒ Linkage (lectures later this term)
ƒ Multifactorial inheritance (lectures later this term)
Mendelian phenotypes are special because they reveal the link between single genes and discrete phenotypes. Until the last decade, almost all genetic mapping of genes to phenotypes was of single genes to Mendelian phenotypes.
Quantitative and other complex traits were almost always too difficult!
• Consider Human blood groups
• The gene for the ABO blood type has three alleles: I A ,
I B , i
• I A specifies an enzyme that adds sugar A I specifies an enzyme that adds sugar B i
B does not produce a functional sugar-adding enzyme.
•
Genotypes → Phenotypes (antigens)
I A I A , I A i A
I B I B , I
I A I B
B i B
AB ii O
• In 1944 the actress Joan Barry
(blood group A) sued Charlie
Chaplin (blood group O) for support of her child (phenotype B).
• The blood group evidence was presented in court.
• The jury supported Joan Barry’s claim. Were they right?
The mother must be I A i
The child must be I B i (inheriting i from mother)
The child must have inherited I B from the father.
Charlie Chaplin was ii and therefore could not have been the father.
Example of co-dominance: In Type AB, both A and B antigens are present

ƒ Heterozygotes can be distinguished from both homozygous
ƒ Co dominance: the F1 hybrid resembles both parents in that the
ƒ alternative traits are both visible.
Incomplete dominance: the F1 hybrid resembles neither pure -
ƒ Cross red snapdragons with white snapdragons and the hybrid progeny are pink. Red requires a double dose of a redproducing enzyme, one copy results in only enough pigment to make flowers look pink, white results when there is no functional enzyme.
When yellow mice are mated they produce 2 yellow to 1 agouti offspring.
Heterozygotes survive. Homozygotes A y A y die before birth.
The A y allele is dominant for yellow coat colour, but is a recessive allele in terms of lethality.
Q. What is the origin of alternative alleles?
A. Mutation
Only germ-line mutations are inherited, somatic mutations are not.
The wildtype allele dictates the most common phenotype in a natural population.
Mutation can produce many forms that are likely to be rare unless humans intervene by artificial selection.
Typically, low frequency alleles (<1%) are called mutants.
Mutant alleles may be recessive or dominant.
Mutations in the DNA are the source of new alleles
• Mutation is the process whereby genes change from one allelic form to another. The creation of entirely new alleles can occur.
• Genes mutate randomly, at any time and in any cell of an organism.
• Mutations occur during normal replication; can also occur due to a mutagen, and due to erroneous repair following a exposure to a mutagen
• Substitutions
• Insertions
• Deletions
Many mutations at the DNA level have no consequences for the individual’s phenotype. (Such mutations can become common.)
Mutations are revealed in an individual’s phenotype if the function of genes and their products are affected.
loss-of-function (many places in a gene where a mutation can knock out function) gain-of-function (relatively rare)

Loss-of-function mutations often cause recessive traits
Wildtype phenotype Wildtype phenotype Mutant phenotype
Phenotype is associated with presence of protein, but not sensitive to quantity.
But, loss-of-function mutations can cause dominant traits
• Example:
– Normal gene, function and phenotype: the gene encoding type I collagen, a structural protein required in large amounts to make bone
– Loss-of-function mutant allele causes the brittle bone disease, type I osteogeneis imperfecta, in heterozygotes. So the disease allele is dominant because in this case the phenotype is sensitive to quantity.
Gain-of-function mutations are rare, likely to be dominant
Wildtype phenotype
Mutant phenotype
Mutant phenotype
Presence of any of the mutant affects expression of the phenotype
Allele 1 – HbA: normal haemoglobin
Example: Sickle-cell anaemia
CCTG A GGAG
Pro-Glu-Glu
Allele 2 – HbS: sickle-cell haemoglobin
CCTG T GGAG
Pro-Val-Glu
The example of sickle-cell anemia, a recessive disease phenotype
• A substitution in the DNA changes the amino-acid coding from a glutamic acid (glu) to a valine (val)
• Presence of Haemoglobin S (HbS) causes red blood cells to sickle (gain-of-’function’)
• Sickle-cell disease only occurs in HbS homozygotes – this disease phenotype is recessive. (The phenotype of HbS heterozygotes is called sickle-cell trait .)
• However, HbS heterozygotes living in regions of endemic malaria are greatly protected from malarial disease – the gain-of-function attributed to the HbS allele as malaria resistance is dominant in the phenotype.
• The presence of two or more allelic forms in a species, the variation is correctly referred to as polymorphism
(“many morphs”) when the alternative forms are common , i.e. wildtype alleles

Polymorphism at the phenotypic level
Mendel’s studies of phenotypic polymorphisms provided evidence for allelic variation in single genes.
He used the phenotypes as markers to trace the hereditary processes of segregation and assortment.
Purple White
Round Wrinkled Yellow Green
Long Short
Stems Stems
• Studies of protein polymorphism of Drosophila and
Humans in the 1960s found high levels of heterozygosity in populations.
• But much genetic variation in the genome, in each case, remained undetected. Why?
– degeneracy in genetic code (64 codons, 61 code for
20 amino acids)
– substitution of similar amino acids might give an
“identical” protein as far as the method of detection is concerned
– also, can only observe those proteins (allozymes) which can be separated on a gel after extraction and electrophoresis, and then can be detected.
ƒ Aim to (i) separate the different alleles of a single locus and (ii) visualize them.
ƒ Ultimately the differences reside at the level of DNA sequence
ƒ Electrophoretic separation can be applied to both protein and DNA variation
ƒ Allelic variation can be either:
ƒ co-dominant (both alleles visible), or
ƒ dominant (only one allele visible)
-ve
Principles of allele detection by electrophoresis
Co-dominant Dominant
A
1
/A
1
A
2
/A
2
A
1
/A
2
A
1
/A
1
A
2
/A
2
A
1
/A
2
-ve
+ve
+ve
‘Polymorphisms’ at the DNA level
ƒ Restriction Fragment Length Polymorphisms (RFLPs)
ƒ Minisatellites or VNTRs (Variable Numbers of Tandem
Repeats)
ƒ Microsatellites or SSRs (Simple Sequence Repeats)
ƒ Single Nucleotide Polymorphisms (SNPs)
Many are classic polymorphisms in the sense of being common ‘alternative forms’, but much of the DNA variation discovered within the genomes of single individuals is better regarded as potential polymorphism: provided there is no reason apparent to consider it functionally deleterious it may become common.
Genomicists look at two basic features of genomes:
1. Sequence
2. Variation, also referred to as polymorphism

• Tasha was chosen as an example of a highly inbred dog, i.e. high homozygosity = low heterozygosity, to make assembly of the genome from
DNA sequences easier.
• Polymorphisms were also discovered from:
– Variable alleles (of DNA nucleotides) within Tasha’s genome
– By comparing Tasha’s genome with sequences from a poodle
– By comparing bits of sequences across several breeds, including wolves and a coyote
• In recent decades geneticists turned to molecular variation, first proteins, then DNA variation
• Many DNA polymorphisms are found by Genome projects
– up to 10-fold sequence coverage in order to identify sequence errors and help with assembly provides a resource for SNP discovery.
• Polymorphic markers of known location in the genome are used for:
– Gene mapping
– Association of genes to phenotypes
– Genetic Identity
– Population Genetics
• Allelic variants differ in terms of restriction sites; restriction sites are DNA motifs recognized (cut) by restriction enzymes
• Restriction fragments have different lengths
• In the past, electrophoretic separation based on different molecular weights of DNA fragments produced by restriction digest and subsequent detection by Southern blot
• Now, identified by automated DNA sequencing and computer analysis
5’
3’
Detecting RFLPs:
(1) Cut the DNA into different length
Taq I
TCGA
AGCT
3’ 5’
5’ 3’
Taq I
TCGA
AGCT
3’ fragments
Taq I
5’ 3’
5’
3’
TCGA
AGCT
5’
Restriction site is missing
Detecting RFLPs:
(2) Separate the alleles by electrophoresis
• VNTRs: Variable Number Tandem Repeats or
Minisatellites
• Allelic variants differ in terms of number of DNA repeats – up to thousands of copies of 7-300 bp motifs
• Electrophoretic separation of VNTRs based on different molecular weights of DNA fragments
• Very high levels of polymorphism exist and are detectable
• Alec Jeffreys developed DNA finger-printing based on minisatellites

Alec Jeffreys
• SSRs: Simple Sequence Repeats or
Microsatellites
• Allelic variants differ in terms of number of DNA repeats - SSRs: 1-6 bp motifs
• Detection by PCR and sequencing of SSRs
• DNA profiling based on SSRs now replaces DNA fingerprinting.
GA GA GA GA GA
GA GA GA
GA GA GA GA
GA GA GA GA GA GA
ƒ In humans, expect one nucleotide difference every
~1 kb
ƒ Two phases: SNP discovery by sequencing, and
SNP genotyping (scoring known SNPs in large samples)
ƒ SNP genotyping requires complete knowledge of the genomic sequence
ƒ
Neither labour intensive nor prohibitively expensive to analyse samples - but development and set up costs can be high
ƒ The present and future of polymorphism detection
Single nucleotide polymorphism
Allele 1
ACTTGAT T ACAGGAC
Allele 2
ACTTGAT G ACAGGAC
Detection of SNPs by Hybridisation
Oligonucleotide complementary to one allele
TGAACTA A TGTCCTG
Allele 1 ACTTGAT T ACAGGAC
Completely base-paired
TGAACTA hybrid is stable
A TGTCCTG
Allele 2 ACTTGAT G ACAGGAC
X
TGAACTA A TGTCCTG
Single mismatch hybrid is unstable
Detection of SNPs by Hybridisation
Oligonucleotide complementary to SNP site
TGAACTA A TGTCCTG
Allele 1 ACTTGAT T ACAGGAC
Completely base-paired
TGAACTA hybrid is stable
A TGTCCTG
Allele 2 ACTTGAT G ACAGGAC
Single mismatch hybrid is unstable

Detection of SNPs by Hybridisation
T
A
C
C
A A G
C
C
T
T -CCGGC-
G
-GGCCGoligonucleotide hairpin
ACTTGAT T ACAGGAC
-CGGC TGAACTA A TGTCCTG GGCCG-
Allele 1 hybrid fluoresces
Allele 2 ACTTGAT G ACAGGAC mismatch hybrid
- no signal
• Aims
– To discover SNPs
– To genotype SNPs in different human populations
– To define haplotypes and their population frequencies
– To discover associations between haplotypes and common diseases
Q. What was this lecture about?
A. The genetic basis of polymorphism and how to detect it.
Next lecture (Monday Wk 2)
Hardy-Weinberg
A model for the distribution of polymorphism in populations
Application of electrophoresis to DNA and RNA variation for identifying cloned genes
First, separation of fragments according to size by migration in a gel (electrophoresis).
Southern blotting: Transfer
DNA fragments to a filter and use a probe for detection of variation
Northern blotting: Transfer
RNA fragments
Western blotting: Transfer protein and visualize specific proteins with specific antibodies.
Detection of SNPs by Hybridisation