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Molecular Basis for
Relationship between Genotype and Phenotype
genotype
DNA
DNA sequence
transcription
RNA
translation
protein
function
phenotype
organism
amino acid
sequence
Tissue-specific Regulation of Transcription
Regulated transcription depends on:
- specific enhancer for gene(s)
- enhancer-specific activator proteins
- correct interaction between enhancer and activator
Tissue-specific regulation requires that the enhancer-specific
activator is present only in cells of that tissue type.
ectopic expression: expression in an abnormal location
“Master Switch” Gene
Eye formation requires over 2000 genes.
eyeless (ey) mutation causes small rudimentary
eyes to form in Drosophila melanogaster.
Small eyes (Sey, Pax-6) in mouse causes similar
phenotype.
Aniridia gene in human (lack of normal iris) shows
considerable homology to ey gene.
Comparison of ey+ and ey Phenotypes
Wild-type eyes
eyeless (ey) eyes
size of ey eyes
“Master Switch” Gene
Wild-type eyeless (ey) gene can be induced
to be expressed ectopically.
eyeless (ey) gene codes for a helix-turn-helix
transcription protein.
Enhanceosomes and Synergistic Effect on Transcription
Enhanceosome: protein
complex of trans-acting
factors bound to
appropriate DNA
sequences.
Proteins interact
synergistically to elevate
transcription rate.
In b-interferon gene transcription, TFs recruit a coactivator (CBP)
which is needed for transcription to occur normally.
Formation of the enhanceosome and activation of RNA polymerase
by coactivator are necessary for efficient transcription.
Transcription of b-interferon gene is activated during viral infection.
Molecular Basis for
Relationship between Genotype and Phenotype
genotype
DNA
DNA sequence
transcription
RNA
translation
protein
function
phenotype
organism
amino acid
sequence
Restriction Mapping
DNA is restriction digested with restriction
enzymes, individually (single-enzyme digest)
and in combination (double digest).
The restriction fragments are subjected to
electrophoresis.
The fragments are identified, either using UV
absorbing dye or labeled probe.
Double digest determines if fragment produced
by one enzyme contains restriction sites for
the other enzyme.
Fragments are aligned by size.
Enzyme 1: 8 kb, 6 kb, 3 kb or 3 kb, 6 kb, 8 kb
6 kb, 8 kb, 3 kb or 3 kb, 8 kb, 6 kb
8 kb, 3 kb, 6 kb or 6 kb, 3 kb, 8 kb
Enzyme 2: 10 kb, 7 kb or 7 kb, 10 kb
Double Digest: 3 kb fragment is split into
2 kb and 1 kb fragments.
Restriction Fragment Length Polymorphism (RFLP)
Individuals can be identified according to RFLP
genotype. RFLP locus could be linked to a gene, and
thus be used as a diagnostic marker.
Use of Restriction Fragment Length Polymorphism
I.
Marker locus
II.
Diagnostic
A. Medicine
B. Forensics
III.
Assessment of Genetic Variation
A. Within and between populations
B. Within and between species
Restriction Mapping versus RFLP Mapping
I. Restriction Mapping
A. Based on physical analysis of DNA
B. Based on restriction sites with no variation
C. Mostly short-range (fine-scale) maps
II. RFLP Mapping
A. Based on recombination analysis of matings
B. Based on restriction-site variation between
homologous chromosomes
C. Mostly longe-range (coarse-scale) maps
Other Useful Approaches
1. Single Nucleotide Polymorphisms (SNPs)
Individuals differ in single nucleotides (every 11 to
300 bp in interval).
2. Simple-Sequence Length Polymorphisms (SSLPs)
Very short repetitive DNA sequences are more
polymorphic than RFLP sequences. These are
also called Variable Number Tandem Repeats
(VNTRs)
- Minisatellite Markers
- Microsatellite Markers
Simple-Sequence Length Polymorphisms
1. Minisatellite DNA
These are 1 to 5 kb in length consisting of repeats
15 to 100 nucleotides in length and are identified
by Southern analysis.
2. Microsatellite DNA
These are tandem repeats of dinucleotides,
commonly stretches of CA.
5’ C A C A C A C A C A C A C A 3’
3’ G T G T G T G T G T G T G T 5’
These are identified by gel electrophoresis of PCR
products.
Refer to Figure 4-15, Griffiths et al., 2015.