Psychology 5137 Topic

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Psychology 5-137Topic 3

• Gene Structure Function
• Epigenesis

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Outline

• DNA RNA
• Gene structure and function
• Human Genome
• Genetic Variation
• Genetic Regulation and Epigenesis

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Properties of Genetic Material

• Specify a code for protein synthesis (i.e., code
  for an the sequence of amino acids in a
  polypeptide chain.)
• Duplicate or replicate during both mitosis and
  meiosis

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Deoxyribonucleic Acid (DNA)

• Double stranded
• Strands are held together by (hydrogen) bonds
  that form between the nucleotide bases of the DNA
  molecule
• Adenine (A) ltgt Thymine (T)
• Guanine (G) ltgt Cytosine (C)

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DNA
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Transcription

• One of the two DNA strands is transcribed to a
  single-stranded nucleic acid called ribonucleic
  acid (RNA) RNA has the same bases as DNA except
  uracil (U) substitutes for thymine (T).

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Example
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TTT
TCC
Non-transcribed DNA strand
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AAA
AGG
Transcribed DNA strand
Transcription
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UUU
UCC
mRNA
Translation
Phenylalanine
Serine
Amino Acid
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Translation

• The basic informational unit is 3 nucleotide
  bases (called a codon). Each codon specifies a
  single amino acid.
• There are 44464 possible sequences but only 20
  possible amino acids.

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Gene

• A sequence of DNA (a locus on a chromosome) that
  is involved in (codes for) the synthesis of a
  functional polypeptide (proteins consist of one
  or more polypeptides).

Modern Definition (circa 2006) A locatable
region of genomic sequence, corresponding to a
unit of inheritance, which is associated with
regulatory regions, transcribed regions and/or
other functional sequence regions
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Length of Human Genome

• 3,000,000,000 bases of DNA
• 1 kilo base (kb) 1000 bases
• 1 mega base (Mb) 1,000,000 bases
• 1 giga base (Gb) 1,000,000,000 bases
• Average protein has 400 amino acids, requiring
  1200 DNA bases or 1200bp

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Non-coding DNA

• 98 of human DNA does not code directly for
  protein
• Pseudogenes (evolutionary relics)
• Repetitive DNA
• Interspersed
• Minisatellite repeats (10-30 bp)
• Microsatellite repeats (lt 10 bp)
• Regulatory regions
• Intragenic

VNTR
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Gene Structure

• Typical gene is composed of multiple
• exons Expressed sequences of DNA that are
  translated into protein
• introns - Intervening DNA sequences that are not
  translated

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Gene Structure
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b-hemoglobin
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Human Genome Project

• 1990 - international collaboration conceived as a
  15 year effort
• 1994 - genetic (linkage) map published (marker
  density of less than 1/Mb) (1 year early)
• 2001 publication of draft sequence published in
  Science (Celera) and Nature (HGP)
• 2003 HGP sequencing declared complete on 14
  April 50 year anniversary of Watson Crick
• 2006 Last human chromosome (1) sequenced 224
  MB, 3141 genes, 991 psuedogenes

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Relative Genome Size
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Human Genome Project

• Byproducts
• Mapping of other organisms (nematode, fruit fly,
  mustard plant, mouse, rat and chimp genomes have
  been sequenced)
• Automated, high throughput DNA processing
• Near Future
• Identify human genes (genomics) and their
  products (proteomics)
• Identify genetic variation
• HGDP ? HAPMAP Project
• SNP Consortium
• Gene regulation development

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Genetic Variation

• Genetic variation between individuals refers to
  differences in the DNA sequence
• Originally arose through (gametic) mutation.
• An estimated 99.8 - 99.9 of our DNA is common
• But then .1 of 3,000,000,000 3 million
  differences!

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The Genetic Basis for Human Variation
Derived from dbSNP release 128 http//www.ncbi.nlm
.nih.gov/SNP/
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Sources of Genetic Variation
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Types of Genetic Variation

• Base Substitution Single Nucleotide
  Polymorphisms (SNPs)
• Silent (e.g., AAA to AAG ? Phe)
• Missense (e.g., sickle-cell disease T?A)

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Sickle-cell Mutation
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Types of Genetic Variation

• Base Substitution
• Silent (e.g., AAA to AAG ? Phe)
• Missense (e.g., sickle-cell disease)
• Nonsense or chain terminations (e.g., some
  thalassemia) (e.g., TGC?TGA Cysteine?Stop)
• Noncoding region (significance ?)

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HapMap Discovery Increased SNP Density and
Validated SNPs
10 million rs SNPs
5 million validated rs SNPs
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PKU Mutations
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Sources of Genetic Variation

• Chromosomal Variation
• Nondisjunction Aneuploidy
• Structural Variation
• Rearrangements
• Deletions/Insertions
• Copy-number variants (CNV)

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Types of Genetic Variation Chromosomal
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Sources of Genetic Variation

• Chromosomal Variation
• Nondisjunction Aneuploidy
• Structural Variation
• Rearrangements
• Deletions/Insertions
• Copy-number variants (CNV)

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Redon, R. et al. (2006). Nature, 444 444-454.
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Sources of Genetic Variation

• Chromosomal Variation
• Nondisjunction Aneuploidy
• Structural Variation
• Rearrangements
• Deletions/Insertions
• Copy-Number Variants (CNV)
• Genetic Polymorphisms between-individual
  variation in DNA sequence at a certain
  chromosomal location or gene (most common allele
  lt99).

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Types of Genetic Variation

• Base Substitution
• Silent (e.g., AAA to AAG ? Phe)
• Missense (e.g., sickle-cell disease)
• Nonsense or chain terminations (e.g., some
  thalassemia) (e.g., TGC?TGA Cysteine?Stop)
• Noncoding region (significance ?)
• Frameshifts change in reading frame due to
  insertion or deletion

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Types of Genetic Variation

• Chromosomal/Structural Variations (or
  rearrangements) in the amount of genetic material
  inherited
• Polymorphisms Variations in the DNA sequence
• SNPs (10,000,000)
• VNTR (STR, SSR)

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Types of Genetic Variation Variable Number of
Tandem Repeats (VNTR)

• Microsatellite Small number of bases (lt10)
  repeated a variable number of times (usually lt
  100)(gt100,000)

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Types of Genetic Variation Variable Number of
Tandem Repeats (VNTR)

• Microsatellite Small number of bases (lt10)
  repeated a variable number of times (usually lt
  100)
• Trinucleotide Repeats
• Huntington Disease CAG in coding region

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Huntingtons disease is an example of a
microsatellite triplet repeat in a coding region
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Types of Genetic Variation Variable Number of
Tandem Repeats (VNTR)

• Microsatellite Small number of bases (lt10)
  repeated a variable number of times (usually lt
  100)
• Trinucleotide Repeats
• Huntington Disease CAG in coding region
• Fragile X CGG in a non-coding region
• Neurodegenerative
• Expansion, most likely either through father (HD)
  or mother (FraX), accounts for anticipation

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Types of Genetic Variation Variable Number of
Tandem Repeats (VNTR)

• Microsatellite Small number of bases (lt10)
  repeated a variable number of times (usually lt
  100)
• Trinucleotide Repeats
• Huntington Disease CAG in coding region
• Fragile X CGG in a non-coding region
• Neurodegenerative
• Expansion, most likely either through father (HD)
  or mother (FraX), accounts for anticipation
• Minisatellite 15-70 bases usually repeated 100s
  of times

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Detecting DNA Polymorphisms

• Restriction Fragment Length Polymorphisms (RFLPs)
  -- digest with restriction enzyme, sort by gel
  electrophoresis, label with probe.

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Restriction Enzyme

• Recognize a short sequence of DNA (4-8 bases) and
  cleaves the DNA at or near that site.
• ECO-RI recognizes the 6 base sequence GAATTC

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Sickle Cell Mutation
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Detecting DNA Polymorphisms

• Restriction Fragment Length Polymorphisms (RFLPs)
  -- digest with restriction enzyme, sort by gel
  electrophoresis, label with probe.
• Microsatellite Repeat Polymorphsims -- amplify
  using Polymerase Chain Reaction (PCR), sort via
  electrophoresis, need only fluorescent stain not
  radioactive probe

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Detecting DNA Polymorphisms

• Restriction Fragment Length Polymorphisms (RFLPs)
  -- digest with restriction enzyme, sort by gel
  electrophoresis, label with radioactive probe.
• Microsatellite Repeat Polymorphsims -- amplify
  using Polymerase Chain Reaction (PCR), sort via
  electrophoresis, need only fluorescent stain not
  radioactive probe
• Single Nucleotide Polymorphisms (SNPs) --
  amenable to computerized automated processing
  (DNA chips).

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Reading Assignment 1

• All scores between 22 and 25 (out of 25)
• Possible 12 points for each question, the 25th
  point given only if you got 12 on both (i.e.,
  nobody can get a 24)
• Questions graded on things like could they
  generate discussion, are they already answered in
  the article, etc.

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Outline

• DNA RNA
• Gene structure and function
• Human Genome
• Genetic Variation
• Genetic Regulation and Epigenesis

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Genetic Effects A Simple Model
Inherited DNA Sequence
Cellular Environment
Gene Expression
Phenotypic Effects
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Types of Regulation

• Long-term turning on/off genes
• X-chromosome inactivation
• Epigenetic processes
• Short-term regulation
• Promoter, enhancer sequences alternative
  splicing
• Jacob-Monod model

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• (Mary) Lyons Hypothesis
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• Only one X chromosome is active in somatic cells
• Inactivated X can be either the maternal or
  paternal chromosome, random
• Inactivation occurs early in embryonic
  development
• Inactivation is permanent in all daughter cells
  of somatic cells

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Agouti Mouse
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Epigenetic Mechanisms
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Robert M Sapolsky (2004). Mothering style and
methylation. Nature Neuroscience  7, 791-792
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Types of Regulation

• Long-term turning on/off genes
• X-chromosome inactivation
• Epigenetic processes
• Short-term regulation
• Promoter, enhancer sequences alternative
  splicing
• Jacob-Monod model

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Gene Structure
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Jacob-Monod Model (1961)

• E. coli live in mammal digestive tracts
• Lactose-rich environment early
• Lactose interacts with relevant gene to initiate
  transcription

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Summary

• DNA
• function
• structure
• Gene
• function
• structure
• regulation
• Polymorphism
• definition
• arise
• detection

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Summary

• Genetic Variation
• Chromosomal (aneuploidy, structural)
• Polymorphisms (SNPs, microsatellites)
• Detecting Genetic Variation
• RFLP ? cut, sort, label
• Microsatellites ? PCR expand, sort, label
• SNPs ? automated
• Regulation
• Long-term expression - methylation
• Short-term promoters, splicing
• Responsive to environment

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Gene Regulation

• Transcriptional Control
• Promoters -- initiate transcription
• Enhancers -- increase transcription rate
• Silencers -- reduce transcription rate

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Genetic Regulation (Epigenesis)

• Methylation
• X chromosome inactivation
• Imprinting (next topic)
• Transcription Factors
• Promoters, Enhancers, Silencers
• Operator Model
• Posttranscriptional Control
• Alternative splicing

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Mutations (Germline)

• Types
• Base Substitution
• Silent (e.g., AAA to AAG ? Phe)
• Missense (e.g., sickle-cell disease).
• Nonsense or chain terminations (e.g., some
  thalassemia)
• Frameshift (e.g., from insertion or deletion)
• Trinucleotide Repeats
• Regions
• Coding, exonic, region
• Regulatory region, e.g., in promoter region
• Non-coding, e.g. intronic, region

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Unstable Trinucleotide Repeats

• Examples
• Huntington Disease CAG in coding region
• Fragile X CGG in a non-coding region
• Key Features
• Neurodegenerative
• Expansion, most likely either through father (HD)
  or mother (FraX), accounts for anticipation

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PKU Mutations
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Genome sizes
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Sickle-cell Mutation
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Gene Structure
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How Does the Human Genome Stack Up?
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Sickle-cell Mutation
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Classification of SNPs

• SNPs may occur at any position in the above gene
  structure and
• based on its location it can be classified as
  intronic, exonic or
• promoter region etc.
• Coding SNPs can be further subdivided into two
  groups
• Synonymous when single base substitutions do not
  cause a change
• in the resultant amino acid
• Non-synonymous when single base substitutions
  cause a change
• in the resultant amino acid.

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• Flow Chart
• dbSNP - Search dbSNP to find SNP records for a
  gene
• Step By Step Guide
• dbSNP
• Enter "BRCA1 Gene Name" in the search box
• Click on "Limits"
• Go to "Function class" and select "coding
  nonsynonymous"
• Go to "Organism(s)" and select "Homo Sapiens"
• Go to "Validation" and select all options except
  "no info"
• Click on "Details" and review "Query Translation"
  
• Click on "GO"

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A big breakthrough RFLPs
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HapMap Discovery Increased SNP Density and
Validated SNPs
10 million rs SNPs
5 million validated rs SNPs
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Development of a genome-wide SNP map How many
SNPs?
Nickerson and Kruglyak, Nature Genetics, 2001
10 million common SNPs (gt 1- 5 MAF) - 1/300 bp
Feb 2001 - 1.42 million (1/1900 bp) Nov 2003 -
2.0 million (1/1500 bp) Feb 2004 - 3.3 million
(1/900 bp) Mar 2005 - 5.0 million (validated -
1/600 bp)
When will we have them all?
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SNPs

• A Single Nucleotide Polymorphism is a source
  variance in a genome. A SNP ("snip") is a single
  base mutation in DNA.
• SNPs are the most simple form and most common
  source of genetic polymorphism in the human
  genome (90 of all human DNA polymorphisms).
• There are two types of nucleotide base
  substitutions resulting in SNPs
• Transition substitution between purines (A, G)
  or between pyrimidines (C, T). Constitute two
  thirds of all SNPs.
• Transversion substitution between a purine and a
  pyrimidine.

slide 2, summary 1.2
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Sequence Variation and SNP Distribution

• Sequence variation (quantity of SNPs) can be
  measured in nucleotide diversity
• the number of base differences between two
  genomes over the total number of bases compared.
• SNP Distribution is not uniform for any of the
  three categories
• Over a complete genome (1/3 in coding, 2/3 in
  non-coding).
• Over all the chromosomes (fewer SNPs in sex
  chromosomes).
• Over a single chromosome (SNPs ofteb concentrated
  around a specific location).

slide 3, summary 1.2
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Variation Types

• Macro
• Chromosome numbers
• Segmental duplications, rearrangements, and
  deletions
• Medium
• Sequence Repeats
• Transposable Elements
• Short Deletions, Sequence and Tandem Repeats
• Micro
• Single Nucleotide Polymorphisms (SNPs)
• Single Nucleotide Insertions and Deletions
  (Indels)

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(a) LAC Operon Turned Off
(b) LAC Operon Turned On
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Microsatellite Markers Tandem Repeats
ATTGTTATCCGCTCACAATTCCACACAATCACACACACACACACACACAC
ACAGAGTGAGCTAACTCACATTAATTGCGTTGC
12 repeat units
ATTGTTATCCGCTCACAATTCCACACAATCACACACACACACACACACAC
ACACACACACACACAGAGTGAGCTAACTCACATTAATTGCGTTGC
18 repeat units

• frequently found within the genome
• highly polymorphic

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Sources of Genetic Variation

• Chromosomal Nondisjunction structural
• Polymorphisms
• SNPs (10,000,000)
• Tandem repeats
• Microsatellite (gt100,000)
• Trinucleotide repeats Huntington Disease

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Sources of Genetic Variation

• Chromosomal Nondisjunction structural
• Polymorphisms
• SNPs (10,000,000)
• Tandem repeats
• Microsatellite (gt100,000)
• Trinucleotide repeats Huntington Disease
• Minisatellite

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Types of Genetic Variation Chromosomal
Iafrate et al. (2004). Nature Genetics
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Genetic Effects A Simple Model
Inherited DNA Sequence
Cellular Environment
EPIGENESIS
Gene Expression
Phenotypic Effects
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Genetic Effects A Simple Model
Inherited DNA Sequence
Cellular Environment
Gene Expression
Phenotypic Effects
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II
Lecture Introduction to DNA methylation and its
impact on gene regulation