10  Molecular Genetics

10.1 Learning Objectives

By the end of this chapter, you should be able to:

  1. Describe the evidence establishing DNA as the genetic material
  2. Explain the structure of DNA and how it enables replication and information storage
  3. Compare different types of mutations and their molecular consequences
  4. Describe DNA repair mechanisms and their importance
  5. Explain how genes are organized in prokaryotic and eukaryotic genomes
  6. Analyze the relationship between genotype and phenotype at the molecular level
  7. Describe techniques for analyzing DNA and genes
  8. Apply molecular genetics principles to understand genetic disorders

10.2 Introduction

Molecular genetics bridges classical genetics with biochemistry, providing a physical and chemical understanding of heredity. This chapter explores the molecular basis of genetic phenomena, from the structure of DNA to the mechanisms of mutation and repair. We will examine how genetic information is encoded in DNA molecules, how this information can change through mutation, and how cells maintain genomic integrity. Understanding molecular genetics provides the foundation for modern biotechnology, genetic engineering, and personalized medicine.

10.3 The Genetic Material

10.3.1 Historical Background

Early 20th century: Chromosomes known to carry genetic information, but composition debated

Candidates: Proteins (complex, diverse) vs. nucleic acids (simple, repetitive)

10.3.2 Griffith’s Transformation Experiment (1928)

Organism: Streptococcus pneumoniae

Strains: Smooth (virulent, encapsulated) and rough (avirulent, unencapsulated)

Key experiment: Heat-killed smooth + live rough → live smooth

Conclusion: “Transforming principle” from dead cells could change living cells

10.3.3 Avery, MacLeod, and McCarty (1944)

Question: What is the transforming principle?

Approach: Purify components from smooth bacteria, test transformation

Treatments: Proteases (destroy proteins), RNase (destroy RNA), DNase (destroy DNA)

Result: Only DNase destroyed transforming activity

Conclusion: DNA is the transforming principle (genetic material)

10.3.4 Hershey-Chase Experiment (1952)

Organism: Bacteriophage T2 (infects E. coli)

Labeling: ³²P (DNA) vs. ³⁵S (protein)

Experiment: Infect bacteria, blend to remove phage coats, centrifuge

Result: ³²P (DNA) inside bacteria, ³⁵S (protein) outside

Conclusion: DNA enters bacteria, directs phage reproduction (DNA is genetic material)

10.3.5 Chargaff’s Rules (1950)

Observations:

  1. DNA composition varies between species
  2. Within species: A = T, G = C (A+T ≠ G+C)

Significance: Provided clues to DNA structure

10.3.6 Watson, Crick, Franklin, and Wilkins (1953)

X-ray diffraction: Rosalind Franklin’s Photo 51 showed helical structure

Model building: Watson and Crick proposed double helix

Key features: Complementary base pairing, antiparallel strands, right-handed helix

10.4 DNA Structure

10.4.1 Chemical Components

Nucleotides: Building blocks of DNA

  • Deoxyribose sugar: 5-carbon sugar (2’-deoxy)
  • Phosphate group: Links nucleotides
  • Nitrogenous bases: Purines (A, G) and pyrimidines (C, T)

Nucleoside: Base + sugar (no phosphate)

Nucleotide: Base + sugar + phosphate(s)

10.4.2 Double Helix Structure

Key features:

  1. Two antiparallel strands: 5’→3’ and 3’→5’
  2. Right-handed helix: B-DNA is most common form
  3. Base pairing: A-T (2 hydrogen bonds), G-C (3 hydrogen bonds)
  4. Major and minor grooves: Sites for protein binding
  5. Diameter: 2 nm
  6. Helical repeat: ~10 base pairs per turn (3.4 nm)

Stabilizing forces:

  1. Hydrogen bonds: Between complementary bases
  2. Base stacking: Hydrophobic interactions between stacked bases
  3. Electrostatic interactions: Negative phosphate repulsion minimized by cations

10.4.3 DNA Conformations

B-DNA: Most common, right-handed, physiological conditions

A-DNA: Right-handed, shorter and wider, dehydrated conditions

Z-DNA: Left-handed, alternating purine-pyrimidine sequences

Triple helix: H-DNA, formed in polypurine-polypyrimidine regions

10.4.4 Chromosome Organization

Prokaryotes: Circular DNA, supercoiled, nucleoid-associated proteins

Eukaryotes: Linear chromosomes, histone proteins, nucleosomes, higher-order structures

Chromatin:

  • Euchromatin: Less condensed, transcriptionally active
  • Heterochromatin: Highly condensed, transcriptionally inactive

10.5 DNA Replication (Detailed)

10.5.1 Semiconservative Replication

Meselson-Stahl Experiment (1958):

  • Method: Density gradient centrifugation with ¹⁵N (heavy) and ¹⁴N (light)
  • Prediction: Conservative (all heavy or all light) vs. semiconservative (hybrid)
  • Result: After one generation: all hybrid; after two: half hybrid, half light
  • Conclusion: Semiconservative replication

10.5.2 Replication Machinery

Initiation complex:

  • Origin recognition complex (ORC): Binds origin in eukaryotes
  • Helicase: Unwinds DNA (MCM in eukaryotes)
  • Single-strand binding proteins (SSBs): Stabilize single strands
  • Topoisomerases: Relieve supercoiling (DNA gyrase in bacteria)

Priming:

  • Primase: Synthesizes RNA primer (10-12 nucleotides)
  • Primosome: Complex containing primase and helicase

Elongation:

  • DNA polymerase III: Main replicase in bacteria (ε and δ in eukaryotes)
  • Sliding clamp: PCNA (eukaryotes) or β-clamp (bacteria)
  • Clamp loader: RFC (eukaryotes) or γ-complex (bacteria)

Termination:

  • Bacteria: Specific termination sites, Tus protein
  • Eukaryotes: Telomeres, telomerase in stem/germ cells

10.5.3 Leading vs. Lagging Strand

Leading strand: Continuous synthesis 5’→3’

Lagging strand: Discontinuous synthesis (Okazaki fragments)

  • Size: 100-200 nucleotides in eukaryotes, 1000-2000 in bacteria
  • Processing: RNA primer removal, gap filling, ligation

10.5.4 Fidelity Mechanisms

  1. Base pairing specificity: Hydrogen bonding geometry
  2. 3’→5’ exonuclease proofreading: DNA polymerase activity
  3. Mismatch repair: Post-replication correction
  4. Error rate: ~10⁻⁹ per base (1 error per billion bases)

10.5.5 Eukaryotic Features

Multiple origins: 30,000-50,000 in human genome

Replication timing: Euchromatin replicates early, heterochromatin late

Licensing: Pre-replication complex ensures single replication per cycle

Telomere replication: Telomerase adds repeats to chromosome ends

10.6 Mutations

10.6.1 Types of Mutations

Point mutations (single base changes):

  • Transition: Purine to purine (A↔︎G) or pyrimidine to pyrimidine (C↔︎T)
  • Transversion: Purine to pyrimidine or vice versa

Functional consequences:

  • Silent mutation: Same amino acid (synonymous)

  • Missense mutation: Different amino acid

    • Conservative: Similar properties
    • Non-conservative: Different properties

  • Nonsense mutation: Stop codon created

  • Frameshift mutation: Insertion/deletion not multiple of 3

Chromosomal mutations:

  • Deletion: Loss of segment
  • Duplication: Repeat of segment
  • Inversion: Segment reversed
  • Translocation: Segment moved to different chromosome

10.6.2 Causes of Mutations

Spontaneous:

  • Replication errors: Polymerase mistakes
  • Tautomeric shifts: Rare base forms mispair
  • Depurination: Loss of purine base (~10,000/cell/day)
  • Deamination: C → U (~100/cell/day)

Induced:

  • Chemical mutagens:

    • Base analogs: 5-bromouracil (T analog)
    • Alkylating agents: EMS, adds alkyl groups
    • Intercalating agents: Ethidium bromide, between bases

  • Radiation:

    • UV light: Pyrimidine dimers (TT, CT, CC)
    • Ionizing radiation: Single- and double-strand breaks

10.6.3 Mutation Rates

Variation across organisms:

  • RNA viruses: 10⁻³ to 10⁻⁵ per base per replication
  • DNA viruses: 10⁻⁶ to 10⁻⁸
  • Bacteria: ~10⁻⁹ to 10⁻¹⁰
  • Eukaryotes: ~10⁻⁹ to 10⁻¹¹

Factors affecting rate:

  • Genome size
  • Generation time
  • DNA repair efficiency
  • Environmental exposure

10.7 DNA Repair Mechanisms

10.7.1 Direct Reversal

Photolyase: Light-dependent repair of pyrimidine dimers

O⁶-methylguanine methyltransferase (MGMT): Removes methyl groups from guanine

10.7.2 Base Excision Repair (BER)

For: Small, non-helix-distorting base lesions

Steps:

  1. DNA glycosylase removes damaged base
  2. AP endonuclease nicks backbone
  3. DNA polymerase fills gap
  4. DNA ligase seals nick

Examples: Uracil (from deaminated cytosine), oxidized bases

10.7.3 Nucleotide Excision Repair (NER)

For: Bulky, helix-distorting lesions

Steps:

  1. Recognition of damage
  2. Excision of oligonucleotide (24-32 bases)
  3. DNA synthesis to fill gap
  4. Ligation

Types:

  • Global genomic NER: Throughout genome
  • Transcription-coupled NER: Active genes

Diseases: Xeroderma pigmentosum (NER defect → skin cancer)

10.7.4 Mismatch Repair (MMR)

For: Replication errors (base mismatches, small insertions/deletions)

Recognition: MutS protein binds mismatch

Key feature: Distinguishes template strand from new strand (methylation in bacteria, nicks in eukaryotes)

Disease: Hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome)

10.7.5 Double-Strand Break Repair

Non-homologous end joining (NHEJ):

  • Direct ligation of broken ends
  • Error-prone (loss/gain of nucleotides)
  • Major pathway in mammalian cells

Homologous recombination (HR):

  • Uses sister chromatid as template
  • Error-free
  • Requires homologous sequence
  • Major pathway in yeast

10.7.6 Translesion Synthesis

Purpose: Bypass lesions that block replication

Mechanism: Specialized DNA polymerases (error-prone)

Trade-off: Cell survival vs. mutation

10.8 Gene Organization

10.8.1 Prokaryotic Genes

Operons: Clustered genes with related functions

  • Polycistronic mRNA: Single transcript encoding multiple proteins
  • Examples: lac operon, trp operon

Gene density: High (~1 gene per kb)

Non-coding DNA: Minimal (regulatory sequences, origins)

10.8.2 Eukaryotic Genes

Exon-intron structure:

  • Exons: Coding sequences
  • Introns: Intervening non-coding sequences
  • Alternative splicing: Different combinations of exons

Gene families: Related genes from duplication events

  • Examples: Globin genes, histone genes

Non-coding DNA:

  • Regulatory sequences: Promoters, enhancers, silencers
  • Repetitive DNA: Tandem repeats, transposable elements
  • Pseudogenes: Non-functional copies of genes

10.8.3 Genome Size Paradox (C-value paradox)

Observation: Genome size doesn’t correlate with organismal complexity

Examples:

  • Human: 3.2 Gb
  • Lungfish: 130 Gb
  • Paris japonica (plant): 150 Gb

Explanation: Non-coding DNA varies widely

10.9 Genotype to Phenotype

10.9.1 Biochemical Pathways

One gene-one enzyme hypothesis (Beadle and Tatum, 1941):

  • Each gene encodes one enzyme
  • Modified to: One gene-one polypeptide

Metabolic pathways: Mutations block steps

Example: Phenylketonuria (PKU)

  • Gene: Phenylalanine hydroxylase
  • Block: Phenylalanine → tyrosine
  • Result: Phenylalanine accumulation, mental retardation

10.9.2 Protein Structure and Function

Sickle cell anemia:

  • Mutation: β-globin Glu6Val
  • Effect: Hydrophobic patch causes polymerization
  • Phenotype: Sickled cells, anemia, pain crises

CFTR in cystic fibrosis:

  • Mutation: ΔF508 (deletion of phenylalanine)
  • Effect: Misfolding, defective chloride channel
  • Phenotype: Thick mucus, lung infections

10.9.3 Genetic Heterogeneity

Locus heterogeneity: Mutations in different genes cause same phenotype

Example: Retinitis pigmentosa (mutations in >60 genes)

Allelic heterogeneity: Different mutations in same gene cause same phenotype

Example: Cystic fibrosis (>2000 CFTR mutations)

10.9.4 Penetrance and Expressivity

Penetrance: Proportion of individuals with genotype who show phenotype

  • Complete: All show phenotype
  • Incomplete: Some show phenotype

Expressivity: Degree of phenotype expression

Example: Neurofibromatosis type 1 (variable expression)

10.10 Molecular Genetic Techniques

10.10.1 DNA Sequencing

Sanger sequencing (1977): Dideoxy chain termination

Next-generation sequencing (NGS): Massively parallel, high throughput

Third-generation sequencing: Single-molecule, long reads

10.10.2 Polymerase Chain Reaction (PCR)

Inventor: Kary Mullis (1983)

Components: Template DNA, primers, nucleotides, Taq polymerase

Cycles: Denaturation, annealing, extension

Amplification: 2ⁿ copies after n cycles

Applications: Cloning, diagnostics, forensics, ancient DNA

10.10.3 Gel Electrophoresis

Separates: DNA fragments by size

Agarose gels: Larger fragments (0.1-50 kb)

Polyacrylamide gels: Smaller fragments, higher resolution

10.10.4 Southern, Northern, Western Blotting

Southern: DNA detection (Edwin Southern, 1975)

Northern: RNA detection

Western: Protein detection

10.10.5 Genetic Engineering

Recombinant DNA technology: Combining DNA from different sources

Restriction enzymes: Cut DNA at specific sequences

DNA ligase: Joins DNA fragments

Vectors: Plasmids, viruses, artificial chromosomes

Applications: Gene cloning, protein production, transgenic organisms

10.11 Chapter Summary

10.11.1 Key Concepts

  1. DNA is genetic material: Evidence from transformation, phage, and biochemical experiments
  2. DNA structure: Double helix with complementary base pairing enables replication and information storage
  3. DNA replication: Semiconservative, requires multiple enzymes, high fidelity
  4. Mutations: Changes in DNA sequence, spontaneous and induced, various types
  5. DNA repair: Multiple mechanisms maintain genomic integrity
  6. Gene organization: Differs between prokaryotes and eukaryotes
  7. Genotype-phenotype relationship: Through biochemical pathways and protein function
  8. Molecular techniques: Enable analysis and manipulation of DNA

10.11.2 Evidence for DNA as Genetic Material

Experiment Organism Key Finding Year
Griffith S. pneumoniae Transforming principle 1928
Avery et al. S. pneumoniae DNA is transforming principle 1944
Hershey-Chase T2 phage DNA enters bacteria 1952
Chargaff Various A=T, G=C 1950
Watson-Crick - Double helix structure 1953

10.11.3 DNA Repair Pathways

Pathway Damage Type Key Enzymes Diseases if Defective
BER Small base lesions Glycosylases, AP endonuclease -
NER Bulky lesions XPA-XPG proteins Xeroderma pigmentosum
MMR Mismatches MutS, MutL HNPCC (Lynch syndrome)
NHEJ Double-strand breaks Ku, DNA-PKcs, XRCC4 Radiation sensitivity
HR Double-strand breaks Rad51, BRCA1/2 Breast/ovarian cancer

10.11.4 Mutation Types and Effects

Mutation Type Molecular Change Possible Effect
Silent Same amino acid None
Missense Different amino acid Variable (beneficial to deleterious)
Nonsense Stop codon Truncated protein, often nonfunctional
Frameshift Insertion/deletion Completely different protein sequence
Splice site Alters splicing Altered mRNA, often nonfunctional

10.12 Review Questions

10.12.1 Level 1: Recall and Understanding

  1. Summarize the evidence that DNA is the genetic material.
  2. Describe the structure of the DNA double helix.
  3. What does “semiconservative replication” mean?
  4. List three types of DNA repair mechanisms.
  5. Distinguish between exons and introns.

10.12.2 Level 2: Application and Analysis

  1. If a DNA molecule is 20% adenine, what are the percentages of thymine, cytosine, and guanine?
  2. Explain how a point mutation in a gene could have no effect, a small effect, or a large effect on the protein product.
  3. Why are mismatch repair systems more effective immediately after DNA replication?
  4. Compare the organization of genes in prokaryotes and eukaryotes.
  5. How does the one gene-one enzyme hypothesis explain inborn errors of metabolism?

10.12.3 Level 3: Synthesis and Evaluation

  1. Design an experiment to determine whether a new chemical is mutagenic.
  2. Evaluate the statement: “DNA repair systems have evolved to balance fidelity with adaptability.”
  3. How does our understanding of molecular genetics explain why some genetic diseases show variable expression?
  4. Propose how knowledge of DNA repair mechanisms could lead to new cancer treatments.

10.13 Key Terms

  • Transformation: Uptake and expression of foreign DNA
  • Double helix: Two antiparallel polynucleotide chains wound around each other
  • Complementary base pairing: A with T, G with C
  • Semiconservative replication: Each new DNA molecule contains one old and one new strand
  • Mutation: Change in DNA sequence
  • Point mutation: Change in single base pair
  • Frameshift mutation: Insertion or deletion altering reading frame
  • DNA repair: Correction of DNA damage
  • Exon: Coding region of gene
  • Intron: Non-coding intervening sequence
  • Operon: Cluster of prokaryotic genes with related functions
  • Polymerase chain reaction (PCR): Amplification of specific DNA sequences
  • Restriction enzyme: Enzyme cutting DNA at specific sequences
  • Recombinant DNA: DNA combined from different sources

10.14 Further Reading

10.14.1 Books

  1. Watson, J. D., et al. (2014). Molecular Biology of the Gene (7th ed.). Pearson.
  2. Lewin, B., et al. (2018). Genes XII. Jones & Bartlett.
  3. Alberts, B., et al. (2022). Molecular Biology of the Cell (7th ed.). W. W. Norton.

10.14.2 Scientific Articles

  1. Watson, J. D., & Crick, F. H. C. (1953). Molecular structure of nucleic acids: A structure for deoxyribose nucleic acid. Nature, 171(4356), 737-738.
  2. Meselson, M., & Stahl, F. W. (1958). The replication of DNA in Escherichia coli. PNAS, 44(7), 671-682.
  3. Avery, O. T., MacLeod, C. M., & McCarty, M. (1944). Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Journal of Experimental Medicine, 79(2), 137-158.

10.14.3 Online Resources

  1. DNA Learning Center: https://dnalc.cshl.edu
  2. NCBI Molecular Biology Resources: https://www.ncbi.nlm.nih.gov/guide/molecular-biology/
  3. Protein Data Bank: https://www.rcsb.org

10.15 Quantitative Problems

  1. DNA Composition:
    1. If a DNA molecule contains 18% cytosine, what are the percentages of the other bases?
    2. If a DNA molecule is 1,000 base pairs long and has 30% adenine, how many hydrogen bonds hold the two strands together?
    3. Calculate the length (in μm) of this DNA molecule if each base pair extends 0.34 nm.
  2. Mutation Rates: The human genome is 3.2 × 10⁹ bp. Mutation rate is 1 × 10⁻⁸ per base per generation.
    1. How many new mutations per genome per generation?
    2. If 2% of the genome is coding, how many affect protein-coding regions?
    3. If 90% of these are neutral, how many potentially deleterious mutations occur per generation?
  3. PCR Amplification: Starting with 1 DNA molecule, after 30 PCR cycles:
    1. How many copies are theoretically produced?
    2. If efficiency is 90% per cycle, how many copies are actually produced?
    3. If each amplicon is 500 bp, what is the total mass of DNA (in ng) if each bp averages 660 Da?

10.16 Case Study: BRCA Genes and DNA Repair

Background: BRCA1 and BRCA2 genes are involved in DNA repair, particularly homologous recombination.

Questions:

  1. What types of DNA damage do BRCA proteins help repair?
  2. How do mutations in BRCA genes increase cancer risk?
  3. Why are breast and ovarian tissues particularly susceptible?
  4. How does understanding the molecular function of BRCA genes inform cancer treatment strategies?

Data for analysis:

  • BRCA1: 1863 amino acids, multiple domains
  • BRCA2: 3418 amino acids, binds RAD51
  • Mutation carriers: 45-65% lifetime breast cancer risk (vs. 12% general)
  • Treatment implications: PARP inhibitors for BRCA-deficient cancers

Next Chapter: Regulation of Gene Expression