Chapter 14 and chapter 10

Chapter 14 - Molecular Genetics

Part 1: Learning Objectives

1. Summarize the experiments and/or ideas that contributed to our understanding of molecular genetics by the following researchers:

   • Meischer

   • Levene

   • Griffith

   • Avery, MacLeod, and McCarthy

   • Hershey and Chase

   • Chargaff

   • Wilkins & Franklin

   • Watson & Crick

   • Meselson and Stahl

Page 3: DNA… What Is It?

• Definition:

  • DNA stands for Deoxyribonucleic Acid.

• Composition:

  • Formed from nucleotides held together by covalent bonds.

  • Contains 2 polynucleotides held together by hydrogen bonds.

  • Exhibits a double-stranded helical structure.

• Significance:

  • Serves as the molecule containing genetic instructions for all living organisms.

  • DNA encodes genetic information in its unique chemical language.

  • It is reproduced in all cells of the body.

Page 4: DNA History Timeline

• Gregor Mendel: 1866

• Fredrick Meischer: 1869

• Fredrick Griffith: 1928

• Avery, MacLeod & McCarthy: 1944

• Erwin Chargaff: 1950

• Wilkins & Franklin: 1952

• Watson & Crick: 1958

• Phoebus Levene: 1920

Page 5: Finding DNA

• Friedrich Miescher (1869):

  • Isolated phosphate-rich acidic compounds (later termed nuclein) from the nuclei of leucocytes in hospital bandages.

  • The first to show a specific molecule in the nucleus that is not a protein.

  • Later used sperm cells from salmon to confirm:

  • This nucleus compound contained Carbon, Nitrogen, and Hydrogen.

  • It was rich in Phosphorous and lacked Sulfur (typically found in proteins).

Page 6: Chemical Nature of DNA

• Phoebus Levene (1920s):

  • Isolated the sugars of DNA and RNA as well as the four different nucleotides in DNA.

  • Determined:

  • The composition of nucleotides and how they link together in chains.

  • Established the Tetranucleotide Hypothesis, positing four nucleotides (A, C, G, T) occurred in roughly equal ratios.

Page 7: Determining the Transforming Agent

• Frederick Griffith (1928):

  • Studied Streptococcus pneumoniae and worked with two strains:

  • Pathogenic strain (smooth, “S”)

  • Nonpathogenic strain (rough, “R”)

  • Used an in-vivo model (mice) resulted in the conclusion that living ‘R’ bacteria had been transformed into pathogenic ‘S’ bacteria through an unknown, heritable substance from the dead S cells.

Page 8: DNA as the Heritable Substance

• Avery, MacLeod, & McCarthy:

  • Utilized an in-vitro model (test tubes) with the bacteria studied by Griffith.

  • Determined that DNA was the material transmitted from one generation to the next:

  • Conclusions:

    1. Transformation cannot occur unless DNA is present.

    2. DNA is the transforming substance.

Page 9: Base Composition in DNA

• Erwin Chargaff:

  • Analyzed the base composition of DNA in various organisms, resulting in two major findings known as Chargaff's rules:

  1. The composition of DNA varies from one species to another.

  2. For each species, the percentage of A (Adenine) is approximately equal to T (Thymine), and the percentage of G (Guanine) is approximately equal to C (Cytosine):

     • A = T and G = C

  • His findings provided evidence of molecular diversity among species.

Page 10: DNA as the Genetic Material

• Alfred Hershey and Martha Chase:

  • Geneticists studying bacteriophages (viruses that infect bacteria) performed experiments using the known phage T2.

Page 11: The Bacteriophage

• Definition:

  • Viruses that specifically infect bacteria; the name means “devour bacteria.”

• Significance in Research:

  • Widely used tools in molecular genetics.

  • Most are double-stranded DNA (dsDNA) viruses; estimates suggest there are around $10^{31}$ (ten nonillion) bacteriophages globally, with a single average teaspoon of seawater containing five times as many phages as individuals in Rio de Janeiro.

Page 12: Imaging DNA

• Rosalind Franklin & Maurice Wilkins:

  • Employed X-ray crystallography to analyze the molecular structure of DNA, providing significant evidence of DNA's helical structure.

  • Concluded that DNA consists of two antiparallel sugar-phosphate backbones with nitrogenous bases paired in the interior.

Page 13: Elucidating the Double Helix

• James Watson & Francis Crick:

  • Developed a double-helical model for DNA with key features:

  • Composed of two antiparallel polynucleotide strands.

  • Nitrogenous bases paired internally: A with T (two hydrogen bonds) and C with G (three hydrogen bonds).

  • Their studies revealed specificity in nucleotide pairing, influenced by molecular structure and the number of hydrogen bonds formed.

  • Key features of DNA structure (noted coordinates): $0.34 ext{ nm}$ per base pair, with a complete turn of the helix measuring $3.4 ext{ nm}$.

Page 14: How Does DNA Replicate?

• Models of DNA Replication:

  • Conservative replication: The original parent DNA double strand remains intact while two new double helix strands are synthesized.

  • Semi-conservative replication: The two strands of the parent double helix separate, each acting as a template for a new complementary strand synthesis.

  • Dispersive replication: Both original parent strands are fragmented into pieces, which are then incorporated into newly synthesized strands.

Page 15: Elucidating How DNA Replicated

• Meselson and Stahl:

  • Conducted experiments demonstrating that DNA replication is semi-conservative:

  • Each of the two new daughter DNA molecules contains one strand from the parent and one newly synthesized strand.

  • Used model organism E. coli, initially growing the bacteria in heavy nitrogen ($^{15}N$), then switching to lighter nitrogen ($^{14}N$) for subsequent growth.

  • Resulted in DNA sedimenting halfway between $^{15}N$ and $^{14}N$ levels after one round of division (indicating fifty percent $^{14}N$).

Page 17: Learning Objectives

1. Describe the structure of double-stranded DNA.

2. Calculate the percentage of nucleotides if given the % of one nucleotide in double-stranded DNA.

3. Describe how DNA and histones form nucleosomes and how chromatin is organized into higher-ordered structures.

4. Distinguish between euchromatin and heterochromatin.

5. Define the various structures of a chromosome.

Page 18: The Nucleotide

• Composition of a Nucleotide:

  • Consists of:

  • A phosphate group.

  • A nucleoside (sugar + nitrogenous base).

• Base Pair Combinations:

  • Paired as follows:

  • Adenine with Thymine (A with T)

  • Cytosine with Guanine (C with G)

  • The pairing occurs due to structural compatibility and involves different amounts of hydrogen bonding:

  • A with T = 2 hydrogen bonds

  • C with G = 3 hydrogen bonds

• Base Groups:

  • Pyrimidines (C, U, T; sharp: C U T)

  • Purines (A, G; pure: A G)

Page 19: DNA Double Helix

• Structure:

  • Made up of two polynucleotide strands, which are repeating units of nucleotides.

  • The strands run in antiparallel directions (one strand 3’ to 5’, the other 5’ to 3’).

Page 20: DNA Directionality

• Orientation of Polynucleotides:

  • DNA polynucleotides are oriented antiparallel due to the phosphodiester bond formation, which relies on specific carbons on the DNA sugar-phosphate backbone.

  • 3' and 5' ends:

  • 3’ (3 prime): 3rd carbon on the sugar with a hydroxyl group attached.

  • 5’ (5 prime): 5th carbon attached to the phosphate group.

Page 21: The Chromosome

• Definition:

  • A chromosome is made of DNA and proteins, including genes and regulatory elements alongside regions of unknown purpose.

• Gene Capacity:

  • Each chromosome contains hundreds to thousands of genes, with humans possessing 23 pairs (46 chromosomes total).

• **Types of Chromosomes:

  • Nuclear chromosomes (autosomal & sex chromosomes).

  • Mitochondrial DNA, inherited solely from the mother.

Page 22: Karyotype and Chromosomal Structure

• Chromosome Count Across Species:

  • Each species has a specific number of chromosomes:

  • Humans: 46 total (23 homologous pairs).

  • Example: Chinook salmon have 68 chromosomes (34 homologous pairs).

• Karyotype:

  • An image displaying an individual’s complete collection of chromosomes, exemplifying homologous pairs.

Page 23: The Anatomy of Chromosomes

• Key Structures:

  • Centromere:

  • Joining point for replicated chromosomes; condensed and constricted part, separating ‘p’ and ‘q’ regions.

  • Telomere:

  • Repeating sequences at the ends of chromosomes (e.g., in humans, sequences of CCCTAA/TTAGGG).

  • Sister Chromatid:

  • Represents 3 billion base pairs packed together, with identical copies seen in a cell.

Page 24: Telomeres

• Function of Telomeres:

  • They prevent erosion of genes located near ends of DNA molecules, maintaining structural integrity.

  • Active enzymes (telomerase) can extend telomeres, positively impacting cellular aging.

Page 25: Homologous Chromosomes and DNA Inheritance

• Diploids (2n):

  • Humans inherit one copy of each chromosome from both parents (23 from mom and 23 from dad equals a total of 46 chromosomes).

• Homologous Chromosomes:

  • Similar but not necessarily identical within each set or pair.

Page 26: Important Distinction

• Unduplicated vs. Duplicated Homologous Chromosomes:

  • A pair of unduplicated homologous chromosomes (one from mom, one from dad) duplicates before mitosis, becoming sister chromatids.

Page 27: Preparing for Cell Division

• Overview of Chromosome Preparation:

  • DNA is replicated, and chromosomes condense, where sister chromatids are held together at centromeres.

Page 28: Eukaryotic Cell Organization

• DNA in Prokaryotic vs. Eukaryotic Cells:

  • Prokaryotic DNA: Single circular chromosome, housed in a nucleoid region, passed to new daughter cells by binary fission.

  • Eukaryotic DNA: Has multiple linear chromosomes, typically includes heterologous sex chromosomes, and is housed in the nucleus.

Page 29: DNA Packaging and Organization

• Size Comparisons and Packaging:

  • E. coli has approximately 4 million base pairs in its genome—unpacked, this measures about 1mm or 500 times the cell's length.

  • For humans, chromosome 21 has approximately 48 million base pairs, which would be roughly 1.5 inches (40mm) long when unpacked.

  • The total human genome spans approximately 3 billion base pairs, stretching about 6 feet packed in 46 chromosomes within somatic cells, or 3 feet in sperm or egg DNA.

Page 30: Chromosome Packaging Levels

• Structural Hierarchy:

  • DNA is packaged in levels of increasing density:

  • DNA strand → Nucleosome → 30 nm fiber → looped domains → chromatid.

• Chromatin Organization Levels:

  • 10-nm fiber: histones + DNA

  • 30-nm fiber: nucleosome interactions

  • 300-nm fiber: looped domains

  • 700-nm fiber: chromatid

Page 31: DNA Packaging - The 2 and 10

• 2nm fiber: Width of the double helix in an unpacked state.

• 10nm fiber: DNA wraps around histone proteins (forming nucleosomes), resembling “beads on a string” linked by linker DNA.

• Histones:

  • Proteins providing structural support and regulatory sites, key to all eukaryotes.

• Chemical Modifications of Histones:

  • Changes in chromatin organization due to modifications affecting gene expression.

Page 32: DNA Packaging - 30nm and 300nm

• 30nm Fiber:

  • 30-nm fibers form through nucleosome interactions causing further coiling and folding.

• 300nm Fiber:

  • 30-nm fibers form looped domains attached to proteins.

Page 34: DNA Packaging - 700nm

• 700nm Fiber:

  • The coiling of looped domains occurs, only seen in metaphase (highly condensed).

Page 35: DNA Structure Visualization

• Overview of DNA elements:

  • The DNA double helix diameter is 2nm.

  • The nucleosome diameter is 10nm, described as “beads on a string.”

  • Chromatids (700 nm) are composed of looped domains and the filamenting structure during metaphase.

Page 36: A Note on Chromatin

• Chromatin Density:

  • Most chromatin is loosely packed during interphase and condenses before mitosis.

  • Euchromatin: Loosely packed chromatin, generally expressed DNA.

  • Heterochromatin: Densely packed chromatin typically not expressed.

Page 37: Learning Objectives

1. Describe the differences between somatic cells and gametes.

2. Compare/Contrast cell division in prokaryotes & eukaryotes.

3. Recall the Cell Theory and explain why cells undergo division.

Page 38: Cell Division Overview

• Definition:

  • The process by which a single cell splits into two new daughter cells.Goals of Cell Division:

  1. Genetically identical daughter cells.

  2. Genetically different daughter cells, if applicable.

• Cell Division Steps:

  1. Replicate DNA.

  2. Separate DNA.

  3. Divide the cytoplasm.

  4. Separate into daughter cells.

• Reasons for Cell Division:

  1. Growth & Development.

  2. Maintenance of tissues.

  3. Cell & Tissue Repair.

  4. Reproduction.

Page 39: Why Divide - Growth & Development

• Growth Dynamics:

  • Organism growth results from an increase in cell number, not size (particularly important in embryos and childhood).

  • Not all cells grow in the same manner; fat and muscle cells can change size based on factors like health or age (e.g., uterus size post-menopause).

Page 40: Why Divide - Cell & Tissue Repair

• Wound Healing Phases:

  • Hemostasis (clotting), Inflammation, Proliferation, and Remodeling must occur in proper sequence and timing for effective healing.

Page 41: Why Divide - Maintenance

• Cell and Tissue Renewal:

  • Replacement of damaged tissues occurs differently than wound repair, as scar tissue is not the same as regenerated tissue (e.g., in hair, nails, and organs).

Page 42: Why Divide - Reproduction

• Purpose of Cell Division:

  • Necessary for reproducing organisms via both sexual and asexual means (e.g., mitosis and meiosis).

Page 43: Preparing to Divide

• DNA Replication Requirement:

  • Cells must replicate DNA to ensure each new cell possesses a complete set of genetic material.

  • Prokaryotic Cell Division: Primarily conducted via binary fission and cytokinesis.

  • Eukaryotic Cell Division: Accomplished by mitosis and cytokinesis.

Page 44: Prokaryotic Cell Division - Binary Fission

• Steps of Binary Fission:

  1. Circular DNA replication.

  2. Replicated strands attach to the plasma membrane.

  3. Elongation of the plasma membrane separates DNA.

  4. Inward growth of the membrane at the cell's center, leading to cell division.

  5. Cyokinesis: separation of cytoplasm.

  • FtsZ Protein: The prokaryotic equivalent of tubulin that facilitates binary fission.

Page 45: Learning Objectives for the Cell Cycle

1. Explain the different phases of the cell cycle.

2. Describe the stages of mitosis.

3. Define functions of mitosis and cytokinesis.

4. Discuss roles of specific enzymes during replication:

   • Helicase

   • Single-strand binding protein

   • Topoisomerase

   • Primase

   • DNA polymerase

   • DNA ligase

5. Investigate replication differences between the leading and lagging strands.

Page 46: The Cell Cycle

• Definition:

  • An ordered sequence of events in a cell’s life.

  • Two Major Phases:

  • Interphase: Time for normal growth and preparation for division (includes G1, S, G2 phases).

  • Mitotic Phase: The replicated DNA and cytoplasm are divided, and the cell divides (mitosis and cytokinesis).

Page 47: Interphase Details

• Characteristics of Interphase:

  • Longest phase of the cell cycle (~90% of the cell’s life, approximately 21.5 hours for human cells).

  • Activities include growth, metabolic activity, DNA replication (S phase), and preparation for mitosis (G2).

  • G0 Phase: A resting phase that can be temporary or permanent depending on cell type and environmental conditions.

Page 48: Interphase G1 Phase

• G1 Phase:

  • Primary growth stage, where the cell doubles in size, duplicates organelles, and synthesizes proteins necessary for the S phase.

Page 49: G0 Resting Phase

• Overview of G0 Phase:

  • Occupied by non-dividing cells (some remain in this permanent state; e.g., cardiac or nerve cells).

  • Other cells can enter and exit based on signals (e.g., liver cells).

Page 50: S Phase (DNA Replication)

• S Phase Details:

  • Formation of identical DNA copies (sister chromatids) joined at the centromere.

  • Centrosomes also begin producing mitotic spindles to facilitate movement during cell division.

Page 51: DNA Replication Overview

• Mechanism:

  • The complementary nature of DNA strands allows each strand to serve as a template for new strand synthesis.

  • Parent DNA separates into two single strands for the replication process.

Page 52: DNA Replication Origins

• Beginning of Replication:

  • Occur at special sites called Origins of Replication:

  • Prokaryotes have one origin; Eukaryotes have many (thousands per chromosome).

Page 53: Proteins in DNA Replication

• Key Proteins Involved in Replication:

  • Helicase: Unwinds the double helix.

  • Single-strand Binding Protein (SSB): Stabilizes single-stranded DNA.

  • Topoisomerase: Relieves torsion stress ahead of the replication fork.

  • Primase: Synthesizes an RNA primer at the start site.

  • DNA Polymerases I & III: Involved in DNA strand synthesis.

  • DNA Ligase: Joins Okazaki fragments on the lagging strand.

Page 54: DNA Replication Challenges

• Challenge 1: Torsion

  • The stable double helix must be opened for replication, introducing topological stress (mitigated by topoisomerases).

• Challenge 2: Antiparallel Elongation

  • DNA polymerases can only extend nucleotides to the 3’ end.

• Challenge 3: Priming DNA Synthesis:

  • An RNA primer is necessary for DNA synthesis, where each Okazaki fragment on the lagging strand requires a separate primer.

Page 55: The Replication Fork and Bubble

• Replication Dynamics:

  • The replication fork is the region where elongation occurs; DNA polymerase adds nucleotides to the new growing strand at the fork.

Page 56: DNA Lagging Strand Synthesis

• Mechanism for Lagging Strand Formation:

  • Involves making multiple Okazaki fragments, each initiated by an RNA primer, synthesized by primase and subsequently extended by DNA polymerase III.

Page 57: Summary of DNA Replication Proteins

Page 62: Interphase G2 Phase

• G2 Phase:

• Organelles reproduce, and energy, proteins, lipids, and ribosomes are synthesized in preparation for daughter cells.

• DNA is checked for damage and completeness before mitotic division.

Page 63: The Mitotic Phase

• Definition:

  • Consists of two main steps:

  1. Karyokinesis: Division of the nucleus (also referred to as mitosis).

  2. Cytokinesis: Separation of the cytoplasm, resulting in two daughter cells.

• Outcome:

  • Leads to two genetically identical daughter cells with the same chromosome number as the original parent cell.

Page 64: Mitosis Steps

• Stages of Mitosis:

  1. Prophase

  2. Prometaphase

  3. Metaphase

  4. Anaphase

  5. Telophase

Page 65: Mitosis - Prophase

• Prophase Characteristics:

  • Lasts about 23 minutes in humans.

• Key Steps:

  1. Breakdown of the nuclear envelope.

  2. Dispersal of membranous organelles.

  3. Disappearance of the nucleolus.

  4. Centrosomes migrate to poles.

  5. Microtubule spindle formation.

  6. Sister chromatids coil tighter, aided by condensin proteins.

Page 66: Mitosis - Prometaphase

• Prometaphase Details:

  • Sister chromatids acquire kinetochores at the centromere, connecting to spindle microtubules.

  • The mitotic spindle apparatus arises from the centromere, assisting in chromosome movement.

Page 67: Mitosis - Metaphase

• Metaphase Characteristics:

  • Lasts around 30 minutes in humans.

• Key Steps:

  1. Chromosomes align along the metaphase plate.

  2. Cohesion proteins keep sister chromatids together.

  3. The spindle's attachment is scrutinized before concluding this critical stage.

Page 68: Mitosis - Anaphase

• Anaphase Dynamics:

  • Lasts about 10 minutes in humans.

• Key Events:

  1. Cohesin proteins degrade, allowing chromatids to separate.

  2. Separated chromatids are pulled to opposite poles.

  3. The cell elongates to facilitate separation of the poles.

Page 69: Mitosis - Telophase

• Characteristics of Telophase:

  • Lasts about 14 minutes in humans.

• Key Steps:

  1. Chromosomes reach opposite poles, and decondensation begins.

  2. Spindles disassemble into tubulin for cellular components.

  3. Nuclear envelopes form around chromosome sets, leading to the formation of nuclei in daughter cells.

Page 70: Cytokinesis Overview

• Definition:

  • The division of the cytoplasm, typically occurring alongside telophase and lasting about 4 minutes in humans.

• Animal Cells:

  • Formation of a cleavage furrow through cytoplasmic constriction.

• Plant Cells:

  • Formation of a cell plate (inner cell wall section), beginning at the cell's center and expanding outward.

Part 5: Learning Objectives

1. Summarize methods to minimize mutations in the genome.

2. Define the end replication problem and explain eukaryotic solutions.

3. Describe purposes of telomeres and telomerase.

4. Discuss consequences of unfixed errors.

Page 71: DNA Replication & Repair Overview

• Replication Characteristics:

  • Rapid but requires high accuracy, important due to large numbers of base pairs involved.

• Repair Mechanisms:

  • Occur concurrently with replication, utilizing enzymes responsible for both functions (e.g., DNA polymerases).

Page 72: Uncorrected Errors and Mutations

• Error Rates in DNA Replication:

  • While DNA replication has a high accuracy rate, uncorrected errors lead to mutations affecting protein sequences.

• Types of Mutations:

  • Point mutations

  • Frameshift mutations

  • Chromosome mutations.

Page 73: DNA Proofreading & Repair Mechanisms

• DNA Polymerases:

  • Proofread new DNA strands, replacing incorrect nucleotides, leading to an error rate of approximately $1 imes 10^{-10}$ per replication.

• Mismatch Repair:

  • Repair enzymes correct base-pairing errors, including AP endonucleases responsible for NER (nucleotide excision repair).

Page 74: Environmental Damage Effects

• Sources of DNA Damage:

  • Environmental elements (e.g., UV light) can disrupt DNA structure, leading to mutations like thymine dimers (covalently bonded thymines).

• Repair Mechanisms:

  • Cells excise and replace these mutations during normal cellular processing, ensuring integrity.

Page 75: Nucleotide Excision Repair Process

• Repair Method:

  • Enzymes cut and replace mismatches detected post-replication through nuclease action, followed by replacement by DNA polymerase and sealing by DNA ligase.

Page 76: The End Replication Conundrum

• Eukaryotic DNA Replication Issue:

  • Ends of linear chromosomal DNA shorten with each replication due to the absence of RNA primers initially present.

• Circular DNA in Prokaryotes:

  • Prokaryotes do not face this challenge due to the nature of their chromosomal DNA.

Page 77: Telomerase Functionality

• Purpose of Telomerase:

  • Enzymatic involvement in telomere replication, acting primarily in certain cell stages and types (e.g., germ cells, lymphocytes, epithelial cells).

Page 78: Risks of Shortened Telomeres

• Consequences of Shortened Telomeres:

  • If germ cell chromosomes shorten after each cycle, vital genes could be lost in the produced gametes (eggs/sperm).

Page 79: Elongation Polymerase Chain Reaction (PCR)

• Overview of PCR:

  • A laboratory technique amplifying specific DNA sequences exponentially in short periods, allowing extensive research on particular genomic sections.

• Stages of PCR:

  1. Denaturation

  2. Annealing

  3. Elongation (replication).

Page 80: Utilization of Thermocyclers

• Role of Thermocyclers:

  • Automatic temperature adjustments facilitate the PCR process in laboratory settings.

Page 81: The PCR Exponential Growth

• Exponential Growth Rate:

  • Demonstrated as 20 = 1, 21 = 2, 22 = 4, 230 = >1 ext{ Gbp}

• PCR Process:

  • Exponential “growth” allows for extensive amplification of DNA segments, aiding visual analysis via gel electrophoresis in lab settings.