Chapter 13: Molecular Basis of Inheritance

The Search for the Genetic Material

Early research sought to identify the molecule responsible for heredity. DNA and protein were considered the main candidates, with proteins initially favored due to their complexity. However, experiments in the mid-20th century shifted the focus to DNA.

• Key Point 1: Proteins were thought to be the genetic material until the 1940s.

• Key Point 2: DNA's role was not well understood until pivotal experiments demonstrated its importance.

Evidence that DNA Can Transform Bacteria

Frederick Griffith's experiment in 1928 revealed that bacteria could acquire new traits through the uptake of external DNA, a process called transformation. This experiment used two strains of Streptococcus pneumoniae: a lethal smooth (S) strain and a nonlethal rough (R) strain.

• Transformation: The uptake of external DNA by a cell, resulting in a genotypic and phenotypic change.

• Example: Heat-killed S strain mixed with live R strain produced live S strain bacteria, demonstrating genetic material transfer.

Evidence that Viral DNA Can Program Cells

Hershey and Chase's 1952 experiment used bacteriophages (viruses that infect bacteria) to show that DNA, not protein, is the genetic material. Bacteriophages consist of a protein coat and a nucleic acid core.

• Bacteriophage: A virus that infects bacteria and replicates by hijacking bacterial machinery.

• Key Point: Only viral DNA enters the bacterial cell during infection, confirming DNA as the genetic material.

Hershey & Chase Experiment

Radioactive labeling of protein and DNA allowed tracking of viral components during infection. Radioactive sulfur labeled the protein coat, while radioactive phosphorus labeled the DNA. Only the DNA entered the cell, confirming its role in heredity.

DNA Structure and Composition

DNA is a polymer of nucleotides, each consisting of a nitrogenous base (adenine, thymine, guanine, cytosine), a pentose sugar (deoxyribose), and a phosphate group. The arrangement of these components forms the backbone and the base pairs of the DNA molecule.

• Nitrogenous Bases: Adenine (A), Thymine (T), Guanine (G), Cytosine (C).

• Phosphate Group: Provides structural support and polarity to the DNA strand.

Chargaff's Rules

Erwin Chargaff discovered that DNA base composition varies between species, but within a species, the percentage of A and T bases are roughly equal, as are G and C bases. This provided clues to the double helix structure.

Building a Structural Model of DNA

Rosalind Franklin's X-ray diffraction images (Photo 51) provided critical evidence for the helical structure of DNA. Watson and Crick used this data to describe DNA as a double helix with antiparallel strands and specific base pairing (A-T, C-G).

• Double Helix: Two strands of DNA run in opposite directions (antiparallel) and are held together by hydrogen bonds between complementary bases.

• Major and Minor Grooves: Structural features of the helix important for protein binding.

Detailed DNA Structure

The 5' end of each DNA strand has a free phosphate group, while the 3' end has a free hydroxyl group. The sugar-phosphate backbone is on the outside, and the nitrogenous bases are paired in the interior.

DNA Replication: Principles and Mechanisms

DNA replication is the process by which a cell copies its DNA before cell division. The two strands of DNA are complementary, allowing each to serve as a template for a new strand. Replication follows the semiconservative model, where each new DNA molecule contains one old and one new strand.

• Semiconservative Replication: Each daughter DNA molecule consists of one parental and one newly synthesized strand.

• Base Pairing: Ensures accurate copying of genetic information.

Meselson-Stahl Experiment

This experiment confirmed the semiconservative model by using isotopic labeling of DNA strands and tracking their distribution after replication.

Components and Steps of DNA Replication

DNA replication involves multiple enzymes and proteins that work together to ensure accurate and efficient copying of genetic material.

• Origin of Replication (ORI): Specific DNA sequences where replication begins. Prokaryotes have one ORI; eukaryotes have multiple.

• Replication Fork: Y-shaped region where DNA is unwound and replication occurs.

Unwinding the DNA: Proteins and Enzymes

• Helicase: Unwinds the DNA double helix.

• Single-Strand Binding Proteins (SSB): Stabilize separated strands.

• Topoisomerase: Relieves strain from supercoiling.

Primase and DNA Polymerase

Primase synthesizes short RNA primers to provide a starting point for DNA polymerase. DNA polymerase builds new DNA strands in the 5' to 3' direction, elongating from the free 3' hydroxyl group.

• DNA Polymerase: Main enzyme for DNA synthesis; requires a primer and template.

• Rate of Elongation: 500 nucleotides/second in bacteria, 50 nucleotides/second in humans.

Antiparallel Elongation: Leading and Lagging Strands

DNA replication occurs differently on the two strands due to their antiparallel orientation. The leading strand is synthesized continuously, while the lagging strand is synthesized in short fragments called Okazaki fragments.

• Leading Strand: Synthesized continuously in the direction of the replication fork.

• Lagging Strand: Synthesized discontinuously, opposite the fork, in Okazaki fragments.

• DNA Ligase: Joins Okazaki fragments to form a complete strand.

Steps of DNA Replication

1. Helicase unwinds DNA.

2. Topoisomerase relieves supercoiling.

3. SSBs stabilize single strands.

4. Primase adds RNA primers.

5. DNA Polymerase III elongates new strands.

6. DNA Polymerase I replaces RNA primers with DNA.

7. DNA Ligase joins Okazaki fragments.

Proofreading and Repairing DNA

DNA polymerases proofread newly synthesized DNA, correcting errors. Additional repair mechanisms, such as mismatch repair and nucleotide excision repair, further ensure DNA integrity.

• Proofreading: DNA polymerase removes incorrect nucleotides during replication.

• Mismatch Repair: Enzymes correct base-pair mismatches missed during proofreading.

• Nucleotide Excision Repair: Damaged DNA is cut out and replaced with correct bases.

Replicating the Ends of DNA Molecules: Telomeres and Telomerase

Linear DNA in eukaryotes poses a challenge for replication at the ends. Telomeres are repetitive, non-coding sequences that protect chromosome ends. Telomerase is an enzyme that extends telomeres in germ cells and some cancer cells.

• Telomeres: Repeating sequences (e.g., TTAAGGG) at chromosome ends, acting as a buffer zone.

• Telomerase: Enzyme that lengthens telomeres, preventing loss of essential genes.

DNA-Based Technology

Modern techniques allow manipulation and study of DNA for various purposes, including cloning, PCR, gel electrophoresis, and DNA fingerprinting.

• DNA Cloning: Producing many identical copies of a DNA sequence in a host cell.

• Polymerase Chain Reaction (PCR): Rapidly amplifies specific DNA sequences in a test tube.

• Gel Electrophoresis: Separates DNA fragments by size using an electric current.

• DNA Fingerprinting: Uses genetic markers to identify individuals.

Components of PCR

• Template DNA

• DNA primers

• Thermo-stable DNA polymerase

• Deoxyribonucleotides

Steps of PCR

1. Denaturation (high temperature)

2. Annealing (low temperature)

3. Extension (moderate temperature)

Gel Electrophoresis

• DNA fragments migrate from cathode (negative) to anode (positive).

• Larger fragments remain near the top; smaller fragments move toward the bottom.

DNA Fingerprinting

• Uses polymorphic genetic markers, such as single nucleotide polymorphisms (SNPs), to distinguish individuals.