The Molecular Basis of Inheritance

Concept 16.1: DNA is the Genetic Material

  • Early 20th century: Identifying the molecule of inheritance was a major challenge.

  • Question: Which large biological molecule is responsible for passing on heritable information? (DNA, protein, etc.)

Griffith's Experiment: Genetic Transformation

  • Different strains of bacterium:

    • S strain (smooth): pathogenic (causes disease)

      • Produces a polysaccharide coat.

    • R strain (rough): non-pathogenic

      • Lacks the polysaccharide coat.

  • Experiment:

    • Living S cells injected into mouse: Mouse dies.

    • Living R cells injected into mouse: Mouse healthy.

    • Heat-killed S cells injected into mouse: Mouse healthy.

    • Mixture of heat-killed S cells and living R cells injected into mouse: Mouse dies, living S cells are found in the mouse (contradicts original observation).

  • Conclusion:

    • Non-pathogenic (R) bacteria were converted into pathogenic (S) bacteria.

    • Genetic transformation occurred: heritable information transferred from heat-killed S strain to living R strain.

Avery, McCarty, and MacLeod Experiment

  • Objective: Identify the source of heritable information using cell-free extracts (contents of ruptured cells with no intact cells).

  • Experimental Design:

    • Isolate and purify components from S cells (lipids, RNA, DNA, proteins, etc.).

    • Test each component for its ability to transform R cells into S cells.

    • Treat each solution to destroy a different type of molecule.

  • Results:

    • If DNA is destroyed, no genetic transformation occurs.

    • Only when DNA is used can genetic transformation be stimulated.

  • Conclusion: DNA is the genetic material in cells and the source of heritable information in bacteria.

Hershey-Chase Experiment: Viral DNA Programs Cells

  • Viruses (bacteriophages) infect bacteria and inject heritable information for virus replication and protein production.

  • Two phases of viral infection:

    • Lysogenic: Viral DNA is integrated into host genome; does not destroy host immediately.

    • Lytic: Viral DNA replicates, new viruses are assembled, and the host cell is destroyed.

  • Objective: Determine whether protein or DNA is injected into bacteria during infection by using radioactive isotopes.

  • Radioactive Labels:

    • $^{35}S: Labels proteins (sulfur is present in proteins but not DNA).

    • $^{32}P: Labels DNA (phosphorus is present in DNA but not proteins).

  • Experiment:

    • Infect E. coli with radioactively labeled phages.

    • Separate phages from bacteria via centrifugation.

    • Measure radioactivity in the supernatant (containing phage coats) and the bacterial cells.

  • Results:

    • $^{32}P (DNA) was found inside the bacterial cells.

    • $^{35}S (protein) was found in the supernatant.

  • Conclusion: DNA, not protein, is the genetic material that enters E. coli during phage infection. The viral DNA takes over the host cell's machinery to produce more viruses (lytic cycle).

Chargaff's Rules: Base Composition of DNA

  • Base composition of DNA varies between species.

  • Species more closely related have more similar base compositions.

  • The number of A and T bases are equal, and the number of C and G bases are equal.

    • A=TA = T and C=GC = G

  • Ratios:

    • A+TG+C\frac{A+T}{G+C} varies between species.

Watson-Crick Model: Structural Model of DNA

  • Rosalind Franklin used X-ray crystallography to determine the molecular structure of DNA.

    • X-ray diffraction images showed a repeating pattern in DNA.

  • Key features of DNA structure:

    • DNA is a double helix with complementary base pairing (A with T, C with G).

    • The two strands are antiparallel (run in opposite directions).

    • Repeating nucleotides can bond to one another.

  • Dimensions:

    • Diameter: 2nm2 nm.

    • Distance between bases: 0.34nm0.34 nm.

    • One full turn: 3.4nm3.4 nm (10 base pairs).

  • Base pairing:

    • Adenine (A) pairs with Thymine (T) via 2 hydrogen bonds.

    • Guanine (G) pairs with Cytosine (C) via 3 hydrogen bonds.

  • Purine and Pyrimidine:

    • Purine + purine = too wide.

    • Pyrimidine + pyrimidine = too narrow.

    • Pyrimidine + purine = width consistent with X-ray data.

Concept 16.2: DNA Replication

  • The relationship between structure and function is evident in the double helix.

  • Watson and Crick noted that base pairing suggests a copying mechanism for genetic material.

The Basic Principle: Base Pairing to a Template Strand

  • Non-covalent hydrogen bonds between strands allow separation.

  • Each strand can function as a template for new strand formation.

  • The result is the creation of a new "daughter DNA".

Models of DNA Replication

  • Conservative model: Parental DNA maintains its original structure and serves as a template for a completely new DNA molecule.

  • Semiconservative model: Each new DNA molecule consists of one parental strand and one newly synthesized strand.

  • Dispersive model: Each strand of DNA contains a mixture of parental and newly synthesized segments.

Meselson and Stahl Experiment

  • Objective: Determine which model of DNA replication is correct.

  • Method:

    • Bacteria cultured in medium with $^{15}N$ (heavy isotope).

    • Bacteria transferred to medium with $^{14}N$ (lighter isotope).

    • DNA samples centrifuged after first and second replications.

  • Predictions:

    • Conservative: After one replication, there would be one completely heavy strand and one completely light strand.

    • Semiconservative: After one replication, there would be a hybrid strand with one heavy and one light strand.

    • Dispersive: After one replication, there would be a hybrid strand, but the original parental DNA gradually decreases with each replication.

  • Results:

    • After one replication, all DNA was of intermediate density (hybrid), thus disproving the conservative model.

    • After two replications, there were both light and intermediate density bands.

  • Conclusion: DNA replication is semiconservative.

Semiconservative Replication in Eukaryotes

  • Taylor, Woods, and Hughes experiment.

  • Dividing cells from root tips of a broad bean (eukaryotic).

  • Autoradiography showed that after one round of replication in the presence of radioactive isotopes, each sister chromatid contained radioactivity. After the second round, only one chromatid was radioactive, proving semiconservative replication in eukaryotes.

DNA Replication: Mechanisms and Enzymes

Origins of Replication

  • Prokaryotes: have only 1 origin of replication on their circular chromosomes.

  • Eukaryotes: have multiple origins of replication on their linear chromosomes.

Key Proteins Involved in DNA Replication

  • Helicase: unwinds the double helix by breaking hydrogen bonds.

  • Single-strand binding proteins: stabilize single-stranded DNA to prevent re-pairing.

  • Topoisomerase: relieves strain ahead of the replication fork by breaking, swiveling, and rejoining DNA strands.

  • Primase: synthesizes RNA primers to initiate DNA synthesis.

  • DNA polymerase: catalyzes the elongation of new DNA strands.

DNA Polymerase

  • DNA polymerases add nucleotides to the 3' end of a pre-existing strand.

  • Energy for polymerization comes from deoxyribonucleoside triphosphates (dNTPs, e.g., dATP, dTTP, dGTP, dCTP).

  • Polymerization is an anabolic process (building things up).

Replication Forks and Bidirectional Synthesis

  • DNA replication is bidirectional and proceeds from the origin of replication.

  • AT-rich regions are typical at origins of replication since they are less stable (only two hydrogen bonds).

Leading and Lagging Strands

  • Leading strand: synthesized continuously in the 5' to 3' direction.

  • Lagging strand: synthesized discontinuously in short fragments (Okazaki fragments) in the 5' to 3' direction.

  • Okazaki fragments: small DNA fragments synthesized on the lagging strand.

Steps in Lagging Strand Synthesis

  • Primase adds an RNA primer.

  • DNA polymerase synthesizes an Okazaki fragment until it reaches the previous primer.

  • DNA polymerase I replaces the RNA primer with DNA.

  • DNA ligase joins the Okazaki fragments.

DNA Replication Machine

  • The proteins involved in DNA replication form a large multi-enzyme complex, the DNA replication machine.

Telomeres and the End Replication Problem

  • Telomeres: special nucleotide sequences at the ends of linear chromosomes in eukaryotes.

  • The lagging strand cannot be fully replicated at the 5' end due to the need for a primer.

  • Without telomeres, chromosomes would shorten with each round of replication.

  • Telomeres postpone the erosion of genes at the ends of chromosomes.

  • Shortening of telomeres is associated with aging.

Telomerase

  • Telomerase: an enzyme that catalyzes the lengthening of telomeres in germ cells.

  • Telomerase contains an RNA template that it uses to extend the 3' end of the DNA, allowing the lagging strand to be completed.

  • Telomerase ensures that essential genes are not lost during DNA replication in germ cells.

From RNA to DNA

  • Telomerase goes from RNA to DNA (reverse transcriptase).

  • Maintains genome integrity.

Concept 16.3: Chromosome Structure

  • A chromosome consists of a DNA molecule packed together with proteins.

Prokaryotic Chromosomes

  • The bacterial chromosome is a double-stranded, circular DNA molecule associated with a small amount of protein.

  • In bacteria, the DNA is supercoiled and found in a region of the cell called the nucleoid.

Eukaryotic Chromosomes

  • Eukaryotic chromosomes have linear DNA molecules associated with a large amount of protein.

  • DNA is combined with proteins into a complex called chromatin.

  • Histones: proteins responsible for DNA packing.

  • Chromatin goes through changes in packing during the cell cycle.

  • Interphase chromosomes occupy specific regions of the nucleus.

Types of Chromatin

  • Euchromatin: loosely packed chromatin, actively transcribed.

  • Heterochromatin: tightly packed chromatin, generally not transcribed.

  • Facultative Heterochromatin: can convert from heterochromatin to euchromatin.

  • Constitutive Heterochromatin: always heterochromatic (e.g., repetitive DNA sequences).