-Progressive degenerative neurological disease

-Characterized by involuntary jerky movements called "chorea"

-Memory loss, depression, dementia

-Onset: ~40

-Death by early 50s

-No known cure

-Single gene

-Dominant

-Fully penetrant

-Dominant

-BRCA 1 - susceptibility gene

-Incompletely penetrant: 80% chance of developing breast cancer, 50% chance of developing ovarian cancer

1) To promote cell cycle/cell division

2) To prevent cell cycle/cell division

(BRCA 1 falls into neither of these categories)

directly controlling the cell cycle and cell division.

(Cancer is largely a disease where cells lose growth control)

-A single unit of heritable information

-A gene is comprised of the transcribed region of DNA plus all of the DNA control sequences necessary to get the transcript expressed in the proper expression pattern (i.e. at the correct level, in the correct cell type, at the correct time in development, under the proper environmental conditions)

-Genetic makeup of an organism

-Description of all alleles than an organism possesses

-An observable trait

-Dependent on both genotype and environment

-Most often protein

-Increasingly RNA

-Carry out functions in cells

-Generally not transcribed

-Generally 5' to the start of transcription

-Provide information about when, where, and at what level a gene is to be transcribed

Spatial: genes turn on in a subset of cells of the organism, e.g. brain specific, liver specific

Temporal: genes turn on at a particular time in development, e.g. embryonic vs. adult

20,000

(Human haploid genome contains 3.2 X 10⁹ bp)

20

(4³ = 64 possibilites, 61 codons are amino acids, 3 are stop codons)

1) Base (adenine, guanine, cytosine, and thymine)

2) Sugar (deoxyribose)

3) Phosphate

Nucleotides are composed of these 3 things

1) Hydrogen bonds between pyrimidines and purines

2) Two chains - double helix

3) Antiparallel - 5' to 3' directionality of two chains run in opposite directions

4) Sugar-phosphate backbone is on the outside, bases in middle

5) DNA has a net negative charge

1) Sugar: ribose for RNA, 2' deoxyribose for DNA

2) Uracil (a pyrimidine) is utilized in place of thymine

3) Although RNA can form Watson-Crick hydrogen bonds, RNA is generally single stranded

1) DNA replication errors - one copying error per billion nucleotides synthesized

2) Chemical damage to DNA - spontaneous breakdown, environmental damage

2 possibilities

1) Between genes --> no phenotypic effect

2) Within a gene --> can cause phenotypic effects

-------2 possibilities

------1) in control region

------2) in transcribed region

------------2 possibilities

------------1) in coding region - open reading frame (ORF)

------------2) in transcribed, but untranslated regions (UTR)

1) Silent (synonymous) - no change in amino acid

GGG (glycine) --> GGA (still glycine)

2) Missense (non-synonymous) - change in amino acid; altered protein

CTT (leucine) --> CGT (arginine)

3) Nonsense - amino acid to stop codon; truncated protein

TGG (tryptophan) --> TGA (stop)

1) Overexpression, misexpression

2) New activity

3) "Dominant-negative"

independent assortment

(2ⁿ possible combinations for n homologues)

-Genetic variation

-Environmental variation

Variance components: (V_p=V_g+V_e)

-Heritability (H=V_g/V_p) = slope of parent offspring regression

Measures genetic variation

1) Homogenize individuals

2) Apply homogenate to electrophoresis gel

3) Run gel; proteins migrate according to charge

4) Apply a histochemical stain to gel

5) Alternative genotypes and alleles directly visible

1) Extract DNA

2) Cleave with restriction endonuclease

3) Run out on gel

4) Stain / add probe

5) Compare fingerprints

Current forensic technologies focus on extremely variable repeat sequences in human genome

common diseases should be caused by common genetic variants

(this is not reasonable, however, because natural selection selects against common genetic variants that cause disease)

1) Selection

2) Migration

3) Mutation

Non-random, linear pressure - change in allele frequencies in a directional manner

1) Genetic drift (random change in allele frequency from one generation to the next)

2) Recombination

1) Diploid sexual population

2) Infinite population size

3) Random mating

4) No selection

5) No migration

6) No mutation

Directional selection

(Mutations work conversely - add variation)

1) Terrestriality

2) Bipedalism

3) Encephalization

-African origin of H. sapiens

-2nd, 3rd wave of migration

-H. sapiens displaces resident H. erectus and Neanderthals

-Like African replacement model

-Involves hybridization between H. sapiens and others

-Parallel origin of H. sapiens in different regions

-Requires gene flow

-Greater diversity in Africa

-Most recent common ancestor 100,000-200,000 years ago

-Very distinct from human mtDNA

-No gene flow

-Only one "gene"

-85% within population

-8-9% between populations within races

-6% between races

Homozygous mutant mice (htt/htt) are embryonic lethal - die during gestation

But heterozygous (htt/HTT⁺) mice are completely normal. This demonstrates that htt is recessive to HTT⁺. --Evidence that HD disease alleles are new activity gain-of-function

1) Drugs to promote clearance of aggregated nuclear HTT protein

2) Drugs to block "new" activities of HTT aggregates

3) Drugs to inhibit the proteases that cleave mutant HTT (protease cleavage is necessary for disease development)

4) Allele-specific gene silencing strategies -- difficult

Markers --> Linkage --> Phenotype

Want to find genetic variant(s) which explain additive variance underlying a trait

-Amplify small regions (100-10,000 bp) of DNA in vitro (in a test tube)

-Can get 10⁶-10⁹ fold amplification

1) Enzyme to polymerize DNA (DNA polymerase)

2) Template (DNA to copy)

3) Oligonucleotide primer - need a free 3'-OH to begin synthesis

4) Deoxynucleotide triphosphates - dATP, dCTP, dGTP, dTTP (dNTPs)

(DNA synthesis always proceeds 5' to 3')

1) Two single-stranded oligonucleotide primers

-complementary to sequences flanking the microsatellite

-arranged in an orientation so that their 3' ends point toward each other

2) Double-stranded DNA template

3)Deoxynucleotide triphosphates

4) Thermostable DNA polymerase

1) Denature DNA (95^oC) - separates two strands

2) Anneal primers (50^oC) - usually 20-25 bases long

3) Add nucleotides (dNTPs) and DNA polymerase

4) Repeat: denature, anneal, polymerize - amplification of the DNA between the primers

The tendency for trait to be co-inherited - results from physical organization of chromosomes

(Markers that are far away on chromosomes will have less linkage disequilibrium / more recombination than markers that are close to each other)

1) Recognition of damage

2) Removal of damaged region

3) Resynthesis of DNA to replace the removed region (using DNA polymerase)

DNA replication errors

(able to distinguish newly replicated strand from template strand)

microsatellite

-Microsatellites are not accurately replicated by DNA polymerases

-Need to the MMR machinery to correct these errors

1) Randomly glue the pieces back together using non-homologous end joining (NHEJ) - active in G1 of the cell cycle

2) Use the sister chromatid to serve as a template for putting the pieces back together - recombination repair - active in G2 of the cell cycle

1) enzymes

2) regulatory molecules

3) structural proteins

Patients exhibit extreme sun sensitivity and have a high rate of skin damage and skin cancers (1000 fold increased rate)

Autosomal recessive inheritance

-Patients exhibit brittle hair, fish-like scales on the skin, developmental and mental retardation

-Defects in sulfur metabolism

-Autosomal recessive inheritance

-No increased rate of skin cancers

-Patients are dwarfs often exhibit facial and limb abnormalities

-Mental retardation

-Premature aging

-Early death due to neurodegeneration

-No increased rate of skin cancers

XPB and XPD - 3 activities

1) Basal transcription - defective in TDD

2) NER - defective in XP

3) Transcription-coupled DNA repair - defective in CS

Partial loss of function

HTRA1 (heat shock serine protease / HTRA serine peptidase)

Encodes a protein of 480 amino acids

-Control region mutations can affect transcription

-Can affect level - too much RNA produced

-Can affect spatial/temporal pattern - where/when RNA is transcribed

-In control regions, there are binding sites for sequence-specific DNA binding proteins

-Some of these sequence specific DNA binding proteins function as activators (increase rate of  transcription)

-Other sequence specific DNA binding proteins function as repressors (decrease transcription)

Parkinson's

Diabetes

Alzheimer's

Hepatitis

Cystic fibrosis

Huntington's disease

1) Totipotent - give rise to all cells - embryonic and extraembryonic (e.g. placenta)

2) Pluripotent - give rise to all cell types of the embryo/adult

3) Multipotent - give rise to a subset of cell types, e.g. hematopoetic stem cell give rise to all blood cells

1) Produce different cell types during development

2) Replenish cells in the adult

1) Renewable - replace themselves

2) Stable - not change over time

3) Expandable - produce large numbers

4) Differentiation - reproducible, controllable, functional in vivo

1) Addresses ethical concerns since ES cells are generated from adult cells - no embryos are destroyed

2) Addresses issues of rejection - since could generate ES cells for each individual

Of the following reprogramming factors, which is essential to form iPS cells?

Oct4, Sox2, c-myc, Klf4

1) Germ-line gene therapy - transgene present in germ cells (eggs and sperm), thus alleles that are passed on to progeny are changed - not being pursued in humans due to ethical concerns

2) Somatic cell gene therapy - cure genetic defect in an affected somatic cell type - is being pursued in humans, but results are mixed

-Lungs accumulate thick mucus secretions

-Leads to blockage of airways and secondary infections - mostly bacterial

-Chronic, persistent, and serious lung infections

-Underhydration of lung airway

-Leads to changes in viscosity and/or ionic properties of the airway surface mucus leading to reduced ability to clear inhaled bacteria

-Recessive

-Single gene

-Loss-of-function alleles

-One of the most common genetic disorders among individuals of W. European origin

1) Use an adenovirus to deliver the CFTR gene - not effective, get immunological response

2) Use liposome mediated gene transfer to deliver the CFTR gene - more effective than Adv - low level correction that is extremely transient

3) Use a recombinant adeno-associated virus to deliver the CFTR gene - partial correction of chloride transport abnormalities

4) Use compacted DNA to deliver the CFTR gene

What are two molecular causes of SCID (severe combined immunodeficiency)?

(Lack of function of B and T lymphocytes - compromised immune system)

1) Mutation in ADA gene - encodes adenosine deaminase - in the absence of this enzyme, a metabolic intermediate builds up in cells that is toxic to B and T cells

2) Mutation in γ_c-cytokine IL2 receptor - protein necessary for growth and differentiation of T cells

Polynucleotide kinase 3' phosphatase

Kinase - enzyme that adds a phosphate

Phosphatase - enzyme that removes a phosphate

4 mutations: 2 missense, 1 duplication (frameshift), 1 splicing defect

Loss-of-function mutations

1) Run protein extract on an SDS-polyacrylamide gel

2) Transfer the proteins to a nylon membrane

3) Use an antibody against PNKP - protein that very specially recognizes PNKP, but not other proteins

4) Use x-ray film to visualize results

Collect tissue. Fix with formaldehyde.

Section tissue into thin sections (15 microns).

Attach sections to glass microscope slides.

mRNA on the slide is detected via hybridization.

Labeled single-stranded probe - often called antisense strand.

Label allows visualization of where probe hybridizes to mRNA.

True or false?

Mutation is a deterministic force at the level of individuals within a population

Signal peptide - directs the protein to the secretory pathway (for proteins that will be transported outside the cell)

IGFB domain - insulin growth factor binding domain - characterized by 12 Cys residues

Serine protease domain

Mutation does not change the amino acid sequence of the protein - not located in the coding region

Located in the control region - thus affects transcription - high level of transcription - SRF repressor unable to bind