MCB 150 - unit 3

Point mutation

A mutation affecting a single base pair in DNA (includes base substitutions and small insertions/deletions).

Base substitution

A point mutation in which one base pair is replaced with another.

Silent (same-sense) mutation

A base substitution that changes the nucleotide sequence but not the amino acid sequence; usually at the third base of a codon because the genetic code is degenerate.

Missense mutation

A base substitution that changes one amino acid in the polypeptide; may not alter function if the new amino acid has similar chemistry (charge, polarity, side chain), e.g. His -> Arg.

Nonsense mutation

A base substitution that changes a codon for an amino acid into a stop (termination) codon, producing a truncated polypeptide; can cause a null phenotype.

Transition mutation

A base substitution that keeps the same nucleotide family (purine -> purine or pyrimidine -> pyrimidine).

Transversion mutation

A base substitution that changes nucleotide family (purine to pyrimidine or the opposite way around).

Frameshift mutation

Insertion or deletion of a number of nucleotides that is NOT a multiple of 3, shifting the reading frame of all downstream codons; usually abolishes protein function and often causes truncation.

In-frame insertion/deletion

An insertion or deletion of a multiple of 3 nucleotides; it does NOT shift the reading frame (it adds/removes whole amino acids), so it is not a frameshift.

Insertion (chromosomal)

A large section of DNA is gained.

Deletion (chromosomal)

A large section of DNA is lost.

Translocation

A section of DNA moves to a different (nonhomologous) chromosome, or is exchanged between nonhomologous chromosomes.

Duplication

A section of DNA is copied so that it is present more than once.

Inversion

A section of DNA is flipped 180 degrees (reversed in orientation) within the same chromosome.

Noncoding region mutations

Can alter gene expression if they occur in regulatory regions such as the promoter, changing the rate of transcription and its regulation.

Germ-line mutation

A mutation in an egg/sperm cell or in a precursor cell that makes gametes; passed to offspring, and all of the offspring's cells carry it.

Somatic mutation

A mutation in any non-germ-line cell (e.g. skin, muscle); can occur in early or late development; not passed to offspring.

Mosaic

An individual with somatic regions that are genetically different from each other.

Spontaneous mutation

A mutation resulting from abnormalities in normal biological processes (e.g. DNA polymerase errors, free radicals, spontaneous base changes).

Induced mutation

A mutation caused by environmental agents that enter the cell and alter DNA; raises the mutation rate above the spontaneous rate.

Ionizing radiation

Radiation such as X-rays and gamma rays that penetrates deep into biological material and damages DNA by creating free radicals and breaking DNA strands.

Nonionizing radiation

Radiation such as UV light that penetrates only the surface of biological material and causes thymine (pyrimidine) dimers.

Thymine dimer

Two adjacent thymine bases that become covalently cross-linked, distorting DNA and blocking proper replication.

Ames test

A test of whether a substance is mutagenic: a Salmonella typhimurium strain that cannot make histidine (point mutation inactivates a histidine biosynthesis enzyme) is exposed to the substance, and the rate of reversion back to histidine production is compared with the spontaneous reversion rate.

Why rat liver extract is used in the Ames test

Salmonella lacks the complex metabolism of mammals, so rat liver extract is added to simulate mammalian metabolism (it converts some compounds into their mutagenic forms).

Direct repair

Repairs damage by reversing it in place (e.g. removing a mutated attachment such as a methyl group, or photolyase reversing thymine dimers) without cutting out the base or the DNA backbone.

Nucleotide excision repair (NER)

A region of several nucleotides in the damaged strand is removed, and the intact strand is used as a template to resynthesize a normal complementary strand; found in all organisms.

Bacterial NER proteins

UvrA, UvrB, UvrC, UvrD; they repair thymine dimers, chemically modified bases, and missing bases.

UvrA-UvrB complex

Two UvrA + one UvrB form a complex that tracks along DNA and senses distortions; when damage is found the UvrA proteins are released and UvrC binds UvrB.

UvrC

Makes incisions in the damaged strand on both sides of the lesion, then is released.

UvrD

A helicase that binds UvrB, separates the strands, and removes the cut damaged segment; then UvrB and UvrD are released.

Final steps of bacterial NER

DNA polymerase (Pol I in E. coli) fills the gap using the undamaged strand as a template, and DNA ligase seals the nick.

Proofreading

The 3' -> 5' exonuclease activity of DNA polymerase removes a mismatched nucleotide right after it is added, reducing replication errors (~100-fold).

Mismatch repair (MMR) system

Scans newly synthesized DNA for mismatched or hemimethylated DNA and fixes the error in the new strand; works only after replication (other processes use different repair systems); occurs in all organisms.

Methyl-directed mismatch repair

The E. coli MMR system that corrects errors by targeting the newly synthesized, unmethylated strand.

Right after replication the parent strand is methylated and the new strand is not, so the repair system can tell which strand is the daughter strand; once the new strand is methylated the strands can no longer be distinguished.

MutS

Scans DNA and binds the mismatched base in methyl-directed MMR.

MutL

Links MutS and MutH.

MutH

Cuts the unmethylated (new) strand at a nearby hemimethylated GATC site.

Final steps of methyl-directed MMR

An exonuclease removes bases from the cut until it passes the mismatch; DNA polymerase III resynthesizes the strand and ligase seals it.

Effect of proofreading + MMR on mutation rate

Each repair system reduces errors ~100x, so together ~10,000x; overall rate becomes ~1 error per billion nucleotides (e.g. E. coli polymerase ~400 mutations/replication in vitro vs ~0.04 in vivo, per lecture).

Sickle cell anemia

Caused by a missense mutation in the HBB gene, which encodes beta-globin (a subunit of hemoglobin); autosomal recessive.

Most commonly the 6th amino acid changes from glutamic acid (charged) to valine (hydrophobic), exposing a hydrophobic patch on beta-globin.

At low oxygen the mutant hemoglobin sticks together and aggregates into fibers, making red blood cells sickle-shaped, which blocks small vessels and impairs oxygen delivery.

Sickle cell frequency and malaria

About 1/600 Black Americans have sickle cell anemia and 1/12 are carriers; in some parts of Africa a large fraction are carriers because carriers (heterozygotes) have better resistance to malaria (heterozygote advantage).

Phenylketonuria (PKU)

Caused by mutations (e.g. point mutations) in the PAH gene encoding phenylalanine hydroxylase; leads to excess phenylalanine and insufficient tyrosine; autosomal recessive.

Excess phenylalanine causes severe intellectual disability in newborns if untreated, but this is prevented with a special low-phenylalanine diet; low tyrosine reduces melanin (skin pigment) and thyroxine (growth/metabolism).

Phenylalanine hydroxylase

Enzyme that converts phenylalanine to tyrosine by adding a hydroxyl group.

Cystic fibrosis

Caused by mutations in the CFTR gene (a chloride channel), leading to thick sticky mucus that clogs airways and other ducts; autosomal recessive.

Mutant CFTR is misfolded, so it never reaches the plasma membrane; chloride ions are not secreted, water does not follow by osmosis, and mucus becomes dehydrated and sticky.

About 70% of cases are due to deletion of a phenylalanine codon (3-nucleotide in-frame deletion, delta F508).

Translocations diseases

Cause some lymphomas and leukemias.

Duplications diseases

Many oncogenes, fragile X syndrome, and Huntington's disease.

Inversions diseases

Connected to infertility.

Insertions diseases

Haemophilia A.

Deletion diseases

Duchenne muscular dystrophy.

Duchenne smile

Guillaume Duchenne (who described DMD) used electric shocks to study involuntary facial movements and found that genuine smiles use different muscles than fake smiles.

Permanence of mutations

A mutation becomes permanent (fixed) only after a second round of replication copies the altered base into both strands.

Mutations in transcription/translation are less critical because RNA and proteins are not heritable (DNA mutations are passed on).

Cytoskeleton

A network of protein filaments in the cytosol (and along the inner nuclear membrane as the lamina): microtubules, actin filaments, intermediate filaments.

Microtubule

Long hollow cylinder of alpha- and beta-tubulin dimers, 25 nm in diameter, with 13 protofilaments.

Plus end (beta-tubulin exposed, fast growing) and minus end (alpha-tubulin exposed, usually anchored at the MTOC).

Guide intracellular transport (vesicles, organelle movement and positioning), separate chromosomes in mitosis, bend cilia and flagella (sweep fluid over membranes), pigment/color changes, and give cell structure.

Microtubule polymerization in vitro vs in vivo

In vitro polymerization occurs at both ends; in vivo, polymerization and depolymerization occur mainly at the plus end.

Alpha-tubulin

Faces the minus end; binds GTP but does not hydrolyze it.

Beta-tubulin

Faces the plus end; binds GTP and hydrolyzes it to GDP once the dimer is in the microtubule.

Gamma-tubulin

Forms a ring complex at the MTOC that acts as a template for nucleation, bypassing the rate-limiting step of nucleating a microtubule; caps the minus end.

Protofilament

A linear chain of tubulin dimers joined head to tail; 13 protofilaments associate side by side around a hollow core to form a microtubule.

Dynamic instability

Microtubules oscillate between growing and rapidly shortening (catastrophe and rescue).

GTP cap

Not a physical structure; a region of GTP-bound tubulin at the plus end that prevents protofilaments from peeling away. If GTP hydrolysis outpaces addition of new GTP-tubulin the cap is lost and the microtubule rapidly depolymerizes (catastrophe).

Colchicine and colcemid

Bind tubulin and prevent microtubule polymerization in all cells, blocking spindle formation and arresting cells in mitosis (stop growing/dividing).

Vincristine and vinblastine

Vinca alkaloids that prevent microtubule polymerization; used against rapidly dividing cancer cells.

Taxol (paclitaxel)

Stabilizes microtubules and prevents depolymerization; used against rapidly dividing cancer cells.

Microtubule-organizing center (MTOC)

A site in eukaryotic cells where microtubules grow (nucleate).

Centrosome

The major MTOC in animal cells; most plants and protists lack centrosomes/centrioles; the minus (pointed) end of microtubules is always anchored in the centrosome.

Centriole

One of a pair of perpendicular structures in a centrosome, made of 9 sets of triplet microtubules.

Triplet microtubules

A, B, and C tubules: A tubule is a complete 13-protofilament microtubule; B and C tubules are incomplete (10-11 protofilaments) and share walls.

Pericentriolar material (PCM)

An amorphous collection of proteins surrounding the centrioles that provides platforms (gamma-tubulin rings) to nucleate new microtubules.

Kinetochore

A multiprotein structure (motor, structural, regulatory proteins) assembled on the centromere of each chromatid; grabs spindle microtubules so sister chromatids move when microtubules polymerize/depolymerize.

Kinetochore microtubules

Spindle microtubules that extend from a centrosome and attach to a kinetochore.

Polar (interpolar) microtubules

Spindle microtubules that do not attach to kinetochores; they overlap with microtubules from the opposite pole and push the poles apart.

Astral microtubules

Microtubules that radiate from the centrosomes toward the cell periphery (cortex), positioning the spindle.

Motor proteins

Proteins that use ATP hydrolysis to move along cytoskeletal filaments.

Helps in movement of cargo along a filament, movement of the filament itself, or bending of the filament.

Motor protein head

Binds the filament and binds/hydrolyzes ATP (ATP -> ADP + Pi).

Motor protein hinge (neck)

When ATP is hydrolyzed the hinge bends, moving the head forward to where it rebinds the filament and pushes back (so the motor moves forward).

Motor protein tail

Attaches to cargo (other proteins or cellular molecules).

Dynein

Microtubule motor that moves toward the MINUS end; works as a pair with intertwined tails; also drives cilia/flagella bending.

Kinesin

Microtubule motor that moves toward the PLUS end; works as a pair with intertwined tails.

Flagella

Long projections (found singly or in pairs) that move the whole cell.

Cilia

Short projections that cover the cell surface; move cells or sweep fluid/particles across a stationary cell surface; shorter and more numerous than flagella.

Axoneme

Internal structure of cilia and flagella: microtubules, dynein, and linking proteins.

9+2 array

Axoneme arrangement in most eukaryotes: 9 outer doublet microtubules (complete A tubule + partial B tubule) surrounding 2 central single microtubules.

Dynein arms (axoneme)

Attached to the A tubule; use ATP to walk toward the minus (pointed) end along the B tubule of the adjacent doublet; linking proteins convert this sliding into bending of the axoneme.

Basal body

Source/anchor of microtubules of cilia and flagella; anchored on the cytoplasmic side of the plasma membrane; 9 triplets, structurally similar to a centriole.

Actin filament

Thinnest cytoskeletal fiber (7 nm); structurally highly conserved; polarized with plus (barbed, growing) and minus (pointed) ends.

Concentrated near the plasma membrane (cell cortex), where filaments are anchored to membrane proteins, providing cell shape and strength.

G-actin

Globular actin monomer with a binding site for ATP/ADP.

F-actin

Actin polymer: two strands of actin monomers twisted around each other in a helix.

Actin nucleation + polymerization/depolymerization

The slow, rate-limiting first step of polymer formation, where a few monomers (about 3) associate into a seed that can then elongate.

Polymerization is favored at the barbed (plus) end; depolymerization is favored at the pointed (minus) end.

ATP-actin adds to the barbed end, then hydrolyzes its ATP to ADP after incorporation (with a delay); the newer ATP-rich end is stable, while older ADP-actin at the pointed end dissociates more easily.

Treadmilling

Steady state where subunits add at the barbed end at the same rate they leave the pointed end, so filament length stays constant while subunits flow through.

Actin-binding proteins (ABPs)

Regulate the structure, assembly, and disassembly of the actin cytoskeleton so actin can be assembled for many functions.

G-actin binding protein complexes

Control G-actin polymerization by increasing or decreasing its rate (e.g. profilin promotes, thymosin sequesters).

Filament nucleating proteins

Start new filaments: Arp2/3 complex (branched networks) and formins (long straight filaments).

Filament severing proteins

Break F-actin into smaller pieces, increasing the number of ends and accelerating turnover (e.g. cofilin, gelsolin).

Filament bundling proteins

Cross-link actin filaments into parallel (or antiparallel) bundles.

Filament anchoring proteins

Link actin filaments to the plasma membrane or organelles.

Filament capping proteins

Bind filament ends to prevent subunit addition or removal; stabilize filament length.

Filament cross-linking proteins

Connect actin filaments into 3D networks (e.g. filamin).

Terminal web

Actin-rich cytoskeletal network just beneath the plasma membrane (e.g. in intestinal epithelial cells) that gives structural support.

Myosin

A molecular motor that converts chemical energy (ATP) into mechanical energy, generating force and movement.