BIO 340 - General Genetics: Gene Mutations

Exam Information

  • Exam on March 28.
  • Same format as previous exams.
  • 30-31 multiple choice questions.
  • Students with Disability Resource Center accommodations will receive extra time.
  • Exam 3 PDF with clicker questions and answers available on Canvas. Password: BIO340MANGONE

Translation

  • Translation: biological polymerization of amino acids into polypeptide chains.
  • Requires:
    • mRNA (messenger RNA)
    • Ribosomes
    • tRNA (transfer RNA)
    • Amino acids
  • Central dogma: DNA -> Pre-mRNA -> mRNA -> Protein

PolyA Binding Proteins (PABPs)

  • PABPs activity dictates mRNA translational levels. Interaction with:
    • eIF4E (CAP binding protein)
    • eIF4G
    • PAS (polyadenylation signal)
    • AUG (start codon)

mRNA Structure

  • 5' end: m7G cap, eIF4E, eIF4G
  • 3' end: Poly(A) tail with PABPI and PABPII
  • Only RNA polymerase II transcripts are capped and polyadenylated.
  • Ribosome binding: 40S and 60S subunits forming the 80S ribosome.

Ribosomes

  • Prokaryotes: 70S ribosome (2.5 ims 10^6 Da)
    • Large subunit: 50S (1.6 ims 10^6 Da), contains 23S rRNA (2904 nucleotides) + 5S rRNA (120 nucleotides) + 31 proteins
    • Small subunit: 30S (0.9 ims 10^6 Da), contains 16S rRNA (1541 nucleotides) + 21 proteins
  • Eukaryotes: 80S ribosome (4.2 ims 10^6 Da)
    • Large subunit: 60S (2.8 ims 10^6 Da), contains 28S rRNA (4718 nucleotides) + 5S rRNA (120 nucleotides) + 5.8S rRNA (160 nucleotides) + 46 proteins
    • Small subunit: 40S (1.4 ims 10^6 Da), contains 18S rRNA (1874 nucleotides) + 33 proteins
  • Svedberg (S): unit of sedimentation behavior in sucrose gradient during centrifugation.

tRNA Binding Sites on the Ribosome

  • Ribosome sites: E (exit), P (peptidyl), A (aminoacyl).
  • mRNA binds to the 30S subunit.
  • tRNA with growing polypeptide attached binds to the P site.

mRNA Translation Reaction

  • Three steps:
    1. Initiation:
      • mRNA is loaded into the ribosome.
      • tRNAs and other factors are recruited.
      • AUG start codon is sensed.
    2. Elongation:
      • Ribosome reads mRNA from 5' to 3'.
      • Amino acids are added to the growing peptide chain.
    3. Termination:
      • Process terminates at UAA, UAG, UGA.
      • Peptide chain is released.

Initiation of Translation

  • Step 1: Formation of IF-2/tRNA^Met and IF-3/mRNA/30S subunit complexes.
  • Step 2: Complexes combine with IF-1 and GTP to form the 30S initiation complex.
  • After IF-3 is released, the 50S subunit joins, GTP is cleaved, and IF-1 and IF-2 are released, forming the 70S ribosome.

Elongation of Translation

  • Charged tRNAs enter the A site.
  • Peptidyl transferase catalyzes peptide bond formation between amino acid on tRNA at A site and growing peptide chain bound to tRNA in the P site.
  • Uncharged tRNA moves to the E (exit) site.
  • tRNA bound to the peptide chain moves to the P site.
  • The sequence of elongation and translocation is repeated.

Polypeptide Chain Termination

  • Release factor 1 (RF-1) binds to the UAG termination codon in the A site of the ribosome and tRNA^Phe leaves the E site.
  • Release of the nascent polypeptide and RF-1, with transfer of tRNA from the P site to the E site.

Protein Structure

  • Primary structure: amino acid sequence.
  • Secondary structure: regular chain organization pattern (alpha-helix, beta-sheet, bend/loop), stabilized by hydrogen bonds.
  • Tertiary structure: 3D folding, with non-polar residues buried inside and polar residues exposed. Many proteins are organized into multiple domains (compact globular units).
  • Quaternary structure: association between polypeptides (multiple subunits or monomers). Hemoglobin is an example with two alpha-chains (141 amino acids each) and two beta-chains (146 amino acids each).

Post-Translational Modification of Proteins

  • Many amino acids can be enzymatically modified after incorporation into proteins.
  • Reversible phosphorylation of Serine, Threonine, Tyrosine serve as regulatory switches.
  • Amino-terminal acetylation prevents degradation.
  • Glycosylation makes proteins more hydrophilic or hydrophobic.

Gene Mutations

  • Mutation: alteration in DNA sequence.
  • May be single-base pair substitutions, deletions/insertions, or major chromosomal alterations.
  • Occur in somatic or germ cells.

Types of Gene Mutations

  • Somatic mutations: in any cell except germ cells, not heritable.
  • Germ-line mutations: in gametes, inherited.
  • Autosomal mutations: on autosomes.
  • X-linked and Y-linked mutations: on X and Y chromosomes.

Classification of Mutations

  • Spontaneous mutations: natural and random, linked to normal biological or chemical processes; low rates.
  • Induced mutations: from extraneous factors (radiation, UV light, chemicals).

Luria-Delbrück Fluctuation Test

  • Demonstrated that mutations are not adaptive but occur spontaneously and randomly.
  • Showed that in bacteria, genetic mutations arise in the absence of selection, rather than being a response to selection.
  • Resistance to bacteriophage T1 arises randomly in bacterial populations.
  • Most mutations are not directed at specific genes by selective forces; they are random with respect to genotype.

Classification of Mutations by Phenotypic Effects

  • Loss-of-function mutations
  • Gain-of-function mutations
  • Visible (morphological) mutations
  • Nutritional (biochemical) mutations
  • Behavioral mutations
  • Regulatory mutations

Examples of Mutations

  • Normal: THE ONE BIG FLY HAD ONE RED EYE
  • Missense: THQ ONE BIG FLY HAD ONE RED EYE
  • Nonsense: THE ONE BIG ***
  • Frameshift: THE ONE QBI GFL YHA DON ERE DEY
  • Deletion: THE ONE BIG HAD ONE RED EYE
  • Insertion: THE ONE BIG WET FLY HAD ONE RED EYE
  • Duplication: THE ONE BIG FLY FLY HAD ONE RED EYE
  • Expanding Mutation (Generations 1-3): THE ONE BIG FLY; FLY FLY; FLY FLY FLY

Neutral Mutations

  • Occur in protein-coding or non-coding regions of the genome.
  • Do not affect gene products or gene expression.
  • Have a neutral effect on the genetic fitness of the organism.

What are Mutations?

  • Mutations occur in every cell.
  • Cells have DNA repair mechanisms that can fail or be overwhelmed, accumulating mistakes over time.
  • Mutations are inherited changes in genetic material, providing new genetic variation.

β-Thalassemia

  • Genetic disease arising from many mutations in the β-globin gene.
  • Single nucleotide changes or insertions/deletions.
  • Autosomal recessive blood disorder resulting from a reduction or absence of hemoglobin.
  • Causes an imbalance in globin chains, leading to weakness, delayed development, jaundice, and enlarged organs.
  • Affects people from the Mediterranean, North Africa, Middle East, Central Asian, and SE Asian countries.
  • Severity varies from mild to extreme.

Sickle Cell Anemia

  • Autosomal recessive genetic disease.
  • β-globin gene (chromosome 11q) mutation: GAG -> GTG at the 6th codon.
  • Glutamic Acid -> Valine at the 6th amino acid.
  • α2β2 = normal hemoglobin; α2βS = sickle trait (heterozygote); α2βS2 = sickle cell disease (homozygous recessive).
  • Red blood cells become sickle-shaped, causing clumps that block blood flow.

Symptoms of Sickle Cell Anemia

  • Painful episodes (crises) affecting bones, the long bones and the chest.
  • Common symptoms include abdominal and bone pain, breathlessness, delayed growth and puberty, fatigue, fever, blindness, stroke and skin ulcers.

Sickle Cell Anemia vs. Sickle Cell Trait

  • Sickle cell anemia: inherit two copies of the sickle cell gene.
  • Sickle cell trait: don’t have the condition, but have one gene that causes it.
  • Both can pass the gene on to children.

Inheritance of Sickle Cell Anemia

  • If one parent has sickle cell trait (HbAS) and the other is normal (HbAA), no children will have sickle cell anemia; 50% chance of sickle cell trait.
  • If both parents have sickle cell trait (HbAS), there is a 25% chance of sickle cell anemia, a 50% chance of sickle cell trait, and a 25% chance of being unaffected.

Genetic History of Sickle Cell Anemia

  • Mutation occurred thousands of years ago in Africa, the Mediterranean, the Middle East, and India.
  • A deadly form of malaria was very common in these areas.
  • Children with one sickle hemoglobin gene (sickle cell trait) had a survival advantage.
  • As populations migrated, the sickle cell mutation spread.
  • In the United States, ~2.5 million have the trait, ~70,000 have the disease.

Treatments for Sickle Cell Anemia

  • Relieve pain, prevent infections, eye damage, and strokes.
  • Pain medicine: acetaminophen, NSAIDs, narcotics.
  • Blood transfusions.
  • CRISPR Cas9.

CRISPR Cas9 for Sickle Cell Anemia

  • Casgevy is the first FDA-approved therapy utilizing CRISPR/Cas9.
  • Patient's hematopoietic (blood) stem cells are modified.
  • Modified blood stem cells are transplanted back, engrafting in the bone marrow and increasing fetal hemoglobin (HbF).
  • Increased HbF levels prevent sickling of red blood cells.
  • Trials showed elimination or reduction in severity of pain crises.

CRISPR/Cas9 Mechanism

  • CRISPR (Clusters of Regularly Interspaced Short Palindromic Repeats).
  • Cas genes.
  • Stages:
    • Foreign DNA acquisition.
    • CRISPR RNA processing.
    • RNA-guided targeting of viral element.
  • Can be used for deleting or inserting genes.

Spinal Muscular Atrophy (SMA)

  • Rare disease affecting about 35,000 patients worldwide.
  • Caused by a genetic defect in the SMN1 gene (Survival of motor neuron 1).
  • Lower levels of the protein result in loss of function of neuronal cells.
  • Onset: first 6 months, Death: < 2 years.

Trinucleotide Repeat Diseases

  • Mutant genes contain tri-nucleotide repeat sequences.
  • Normal individuals have fewer than 30 repeats.
  • Over 20 disorders exhibit over 200 repeats.
  • Examples: Fragile-X syndrome, Myotonic dystrophy (MD), Huntington’s Disease (HD).

Huntington’s Disease

  • Progressive degenerative condition; autosomal dominant.
  • Genetic defect that causes brain nerve cells damage.
  • Victims show symptoms later in life.

Early Symptoms of Huntington’s Disease

  • Slight, uncontrollable muscular movements.
  • Stumbling and clumsiness.
  • Lack of concentration.
  • Short-term memory lapses.
  • Depression.
  • Changes of mood, sometimes including aggressive or antisocial behavior.

How Huntington’s Disease Progresses

  • Change in one’s physical state: involuntary movements, slurred speech, weight loss.
  • Change in one’s emotional state: mood swings, depression, easily frustrated.

Incidence of Huntington’s Disease

  • Only one in 10,000.
  • 30,000 known cases in the U.S.
  • 150,000 people (US) who are at risk.
  • Death 10-20 years after initial symptoms.
  • The suicide rate is 12.7%.

Huntington’s Disease Genetics

  • Trinucleotide repeat disorder caused by the length of a repeated section of a neuronal gene Huntingtin (HTT) exceeding a normal range.
  • Wild-type contains 6-35 glutamine residues, affected individuals contain greater than 36 glutamine residues (up to 250).
  • HTT gene located on chromosome 4 at 4p16.3.
  • HTT contains a sequence of three DNA bases—cytosine-adenine-guanine (CAG)—repeated multiple times.
  • CAG is the genetic code for glutamine, resulting in a polyglutamine tract.

Huntington’s Disease Outcomes

  • <28 CAG repeats: Normal range; individual will not develop HD
  • 29-34 CAG repeats: Individual will not develop HD but the next generation is at risk
  • 35-39 CAG repeats: Some, but not all, individuals in this range will develop HD; next generation at risk
  • >40 CAG repeats: Individual will develop HD

Huntington’s Disease Mechanism

  • A sequence of 36 or more glutamines results in a protein with different characteristics.
  • The altered form, called mHtt (mutant Htt), increases the decay rate of certain types of neurons.

Huntington’s Disease Cures

  • Currently there is no known cure.
  • Pharmaceutical drugs have been used to control the symptoms.
  • High calorie diet.
  • Potential cures:
    • Inhibiting protein expression of mutant allele (siRNA).
    • Transplantation of embryonic striatal tissue into the degenerated tissue.