Prokaryotic and Eukaryotic Cells, DNA Structure, and Gene Expression

Comparison of Prokaryotic and Eukaryotic Cells

  • Taxonomy and Classification:

    • Prokaryotic Cells: Comprise True bacteria (eubacteria) and Archaebacteria.

    • Eukaryotic Cells: Comprise Protists, fungi, plant, and animal cells.

  • Size Comparisons:

    • Prokaryotic cells are generally smaller, ranging from 100 nm100\,\text{nm} to 10 μm10\,\mu\text{m}.

    • Eukaryotic cells are larger, ranging from 10−100 μm10-100\,\mu\text{m}.

  • Structural Differences:

    • Prokaryotic Cells: Characterized by the absence of a nucleus; DNA is located directly in the cytoplasm within a nucleoid region. They contain no membrane-bound organelles.

    • Eukaryotic Cells: Characterized by DNA enclosed within a membrane-bound nucleus. They contain many specialized organelles.

Structure and Features of the Prokaryotic Cell

  • Anatomical Components:

    • Pili: Hair-like appendages on the surface.

    • Ribosomes: Sites of protein synthesis.

    • Nucleoid region: Contains the bacterial chromosome.

    • Capsule: An outer smooth coat found in certain virulent strains.

    • Cell wall: Provides structural support and protection.

    • Plasma membrane: Inner boundary regulating substance entry/exit.

    • Flagella: Tail-like structures used for locomotion.

History of DNA as the Inherited Genetic Material

  • Friedrich Miescher (1869):

    • The Swiss biologist identified a cellular substance from the nucleus which he called "nuclein."

    • Observations: Nuclein could not be broken down by proteases.

    • Questions & Discussion: Based on the fact that proteases (enzymes that break down protein) did not degrade nuclein, it was concluded it was not a protein.

    • Renaming: Nuclein was found to have acidic properties and was later renamed Nucleic Acid. The two types are DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid).

  • Frederick Griffith (1928):

    • Streptococcus pneumoniae Strains:

      • S cells (Smooth strain): Virulent/pathogenic; surrounded by a smooth capsule coat that protects them from the host's immune system.

      • R cells (Rough strain): Harmless/non-pathogenic; lack a capsule.

    • Experimental Procedure:

      • (a) Injected living S strain: Mouse dies.

      • (b) Injected living R strain: Mouse remains healthy.

      • (c) Injected heat-killed S strain: Mouse remains healthy.

      • (d) Injected a mixture of heat-killed S strain and living R strain: Mouse dies.

    • Results: Living S cells were recovered from the blood of mice that died from the mixture (treatment d), indicating that the harmless R cells had been transformed into virulent S cells.

  • Transformation:

    • Definition: The process by which bacteria take in DNA from their surroundings.

    • Mechanism in Griffith's Experiment: Heat treatment lysed the S cells and their capsules, releasing DNA. The living R cells took up this S cell DNA, which transformed their properties, making them virulent.

    • Applications: Molecular biologists use transformation routinely to introduce specific genes into bacteria for cloning purposes.

  • Avery, MacLeod, and McCarty (1944):

    • Provided definitive evidence that DNA is the inherited genetic material.

    • Method: Divided a culture of S cell extract into three treatments: Proteases (degrade protein), RNases (degrade RNA), and DNases (degrade DNA).

    • Findings: Only the extract treated with DNase failed to transform living R cells into S cells. This proved that DNA was the "transforming factor" originally observed by Griffith.

Molecular Structure of DNA

  • Nucleotide Building Blocks: DNA is a polynucleotide made of repeating units called nucleotides. Each contains:

    1. Pentose (5-carbon) sugar: Deoxyribose.

    2. Phosphate group: Attached to the 5′5' carbon of the sugar.

    3. Nitrogenous base: Attached to the 1′1' carbon.

  • Nitrogenous Bases:

    • Purines (Double-ring structure): Adenine (AA) and Guanine (GG).

    • Pyrimidines (Single-ring structure): Thymine (TT) (DNA only), Cytosine (CC), and Uracil (UU) (RNA only).

  • Comparison of Sugars: Deoxyribose (found in DNA) lacks an oxygen atom on the 2′2' carbon (HH group), whereas Ribose (found in RNA) has a hydroxyl group (OHOH) on the 2′2' carbon.

  • Discovery of the Double Helix:

    • Rosalind Franklin and Maurice Wilkins (1951): Provided X-ray crystallography data.

      • h=3.4 A˚h = 3.4\,\text{\AA}: Distance between individual bases.

      • p=34 A˚p = 34\,\text{\AA}: Distance required for one complete turn of the helix.

    • James Watson and Francis Crick (1953): Used data from Franklin, Wilkins, and Chargaff's rules of base pairing to develop the wire model of DNA.

    • Publication: "The Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid" in the journal Nature on April 25, 1953.

  • Chemical Bonding and Polarity:

    • Phosphodiester bonds: Covalent bonds that hold nucleotides together in a single strand; they connect the phosphate group of an incoming nucleotide to the hydroxyl group on the 3′3' carbon of the existing oligonucleotide.

    • Hydrogen bonds: Hold the two strands together. AA pairs with TT (2 bonds), and GG pairs with CC (3 bonds).

    • Antiparallel Polarity: The two strands run in opposite directions; one is 5′5' to 3′3', the other is 3′3' to 5′5'. Polarity refers to the numbering of carbons on the deoxyribose sugar.

Chromosomes and Genomic Organization

  • Chromatin vs. Chromosomes:

    • Chromatin: Strings of DNA wrapped around DNA-binding proteins called histones. This is the state of DNA when the cell is NOT dividing.

    • Chromosomes: Tightly coiled arrangements of DNA and proteins formed during cell division.

  • Human Chromosome Sets:

    • Somatic Cells: Diploid (2n2n); contain 46 total chromosomes arranged in 23 homologous pairs (one maternal, one paternal).

    • Autosomes: Chromosome pairs 1 through 22.

    • Sex Chromosomes: Pair 23 (XX and YY).

    • Gametes: Sex cells (sperm/egg); contain a single set of 23 chromosomes (nn, haploid).

  • Chromosome Anatomy:

    • Chromatid: One copy of a newly replicated chromosome.

    • Sister Chromatids: Exact replicas joined at a centromere.

    • Centromere: The region of intertwined DNA/protein that joins sister chromatids and delineates the chromosome into two arms: the p-arm (short) and q-arm (long).

    • Kinetochore: Proteins located at the centromere that attach chromosomes to microtubules for movement during division.

    • Telomere: Highly conserved repetitive nucleotide sequences at the ends of arms. They attach chromosomes to the nuclear envelope and protect against gene loss during division. Telomeres shorten with age and cancer progression.

  • Karyotypes: A visual representation used to study chromosome number and structure. G-bands are used for identification.

    • Questions & Discussion: A karyotype for Trisomy 21 (Down Syndrome) would show three chromosomes at the 21st position instead of two.

  • Genomes and Genes:

    • Genome: All the DNA in an organism's cell. The human (haploid) genome is 3.2 billion base pairs3.2\,\text{billion base pairs}.

    • Gene: A sequence of nucleotides (average length 1,000−4,0001,000-4,000) providing instructions to synthesize proteins or RNA (e.g., tRNA). Humans have approximately 20,00020,000 protein-coding genes.

    • Trait: A specific characteristic influenced by genes.

    • Genomics: The study of genomes.

The Cell Cycle and DNA Replication

  • Cell Cycle Phases:

    1. Interphase: Includes G1G_1 (growth), SS (DNA replication), and G2G_2 (preparation for division).

    2. M-phase: Mitosis (division of the nucleus) and Cytokinesis (division of the cytoplasm).

  • Replication Mechanisms:

    • Semiconservative Replication: Each resulting DNA molecule contains one original parental strand and one newly synthesized strand.

    • Step 1: Unwinding: Helicase breaks hydrogen bonds to "unzip" the double helix. Single-strand binding (SSB) proteins prevent strands from re-annealing at the origins of replication.

    • Step 2: Priming: DNA Primase (an RNA polymerase) synthesizes short RNA primers (10−1510-15 nucleotides) to provide a binding site for DNA Polymerase.

    • Step 3: Synthesis: DNA Polymerase scans the template in the 3′3' to 5′5' direction and synthesizes new DNA in the 5′5' to 3′3' direction. It integrates dNTPs, releasing pyrophosphate and hydrogen.

    • Leading vs. Lagging Strands:

      • Leading strand: Synthesized continuously toward the replication fork.

      • Lagging strand: Synthesized discontinuously in short Okazaki fragments away from the fork.

    • Step 4: Ligation: DNA Polymerase replaces RNA primers with DNA. DNA Ligase forms covalent bonds to join the fragments.

Protein Synthesis: Transcription and mRNA Processing

  • The Central Dogma: Information flows from DNA →\rightarrow RNA →\rightarrow Protein.

  • Transcription Process:

    • Occurs in the nucleus. RNA Polymerase binds to a promoter (containing TATA and CAAT boxes) aided by Transcription Factors (TF).

    • The template strand is read 3′3' to 5′5'; the RNA transcript is synthesized 5′5' to 3′3'.

    • Enhancers: Regulatory DNA sequences that bind activators to speed up transcription.

    • Termination: RNA Pol reaches a termination sequence, often forming a hairpin loop (GC-rich) followed by a string of uracils in the RNA, causing release.

  • mRNA Processing (Post-transcriptional Modifications):

    1. RNA Splicing: Removal of non-coding introns and joining of protein-coding exons. Alternative splicing allows one gene to produce multiple different proteins.

    2. 5′5' Cap: A methylated guanine base added to the 5′5' end for ribosome recognition.

    3. 3′3' PolyA Tail: Addition of 100−300100-300 adenine nucleotides to protect mRNA from degradation and increase stability.

  • Types of RNA:

    1. mRNA: Carries genetic code from nucleus to cytoplasm.

    2. tRNA: Transports amino acids to ribosomes.

    3. rRNA: Structural component of ribosomes.

The Genetic Code and Protein Translation

  • Genetic Code Properties: Universal and degenerate (64 codons for only 20 amino acids).

    • Codon: A three-nucleotide unit of mRNA.

    • Start Codon: AUGAUG (codes for Methionine, Met).

    • Stop Codons: UGAUGA, UAAUAA, UAGUAG (do not code for amino acids; signal termination).

  • Ribosome Structure: Aggregates of rRNA and protein consisting of a small (18S18S) and large (28S28S) subunit. Sites include:

    • A (Aminoacyl) site: Where "charged" aminoacyl tRNA enters.

    • P (Peptidyl) site: Where the growing peptide chain is held.

    • E (Exit) site: Where empty tRNAs leave.

  • Stages of Translation:

    1. Initiation: Recruitment of the ribosome to the 5′5' Cap of mRNA. The small subunit scans for the start codon (AUGAUG), and the initiator tRNA binds.

    2. Elongation: Stepwise addition of amino acids. Peptidyl transferase catalyzes the peptide bond between amino acids. Translocation shifts the ribosome along the mRNA.

    3. Termination: Occurs when a stop codon reaches the A site. Releasing factor proteins interact with the stop codon to release the polypeptide and dissociate the ribosome.

Regulation of Gene Expression

  • Gene Expression: The process of producing mRNA (and protein) from a gene. Genes can be upregulated (turned on) or downregulated (silenced).

  • Eukaryotic Regulation:

    • Transcriptional Control: Use of promoters, enhancers, and activators.

    • Steroid Hormone Example: Testosterone (lipid soluble) diffuses through the membrane, binds a receptor in the nucleus, and the complex acts as an activator binding to an Androgen Response Element (5′−TGTTCT−3′5'-TGTTCT-3') to stimulate mRNA synthesis.

  • Bacterial Regulation (Operons):

    • Operon: A cluster of related genes controlled by a single promoter and an operator.

    • lac Operon Players:

      • LacI: Codes for the repressor protein.

      • LacZ: Codes for β\beta-Galactosidase (breaks lactose into glucose and galactose).

      • LacY: Codes for permease (transports lactose into the cell).

      • LacA: Codes for transacetylase (protective function).

    • Mechanism: Lactose acts as an inducer by binding to the repressor, changing its shape so it falls off the operator. Glucose allows the repressor to stay bound, preventing transcription.

  • RNA Interference (RNAi):

    • siRNA (small interfering RNA): Derived from long dsRNA in the cytoplasm (≈21\approx 21 nt); silences specific target mRNA by cleaving it.

    • miRNA (micro RNA): Begins in the nucleus (≈21\approx 21 nt); can regulate many genes by inhibiting translation or causing mRNA degradation.

Mutations and Genetic Diversity

  • Mutation: A change in the DNA nucleotide sequence.

  • Causes: Spontaneous (replication errors) or induced (X-rays, UV).

  • Types of Mutations:

    • Point Mutations: Transition vs. Transversion.

    • Silent: No change in amino acid.

    • Missense: Changes one amino acid.

    • Nonsense: Creates a premature stop codon.

    • Frameshift: Insertion or deletion that shifts the reading frame.

  • Inheritance:

    • Inherited (Germline): Passed via gametes; present in all cells of offspring.

    • Acquired (Somatic): In somatic cells; not passed to offspring; can lead to cancer.

  • SNPs (Single Nucleotide Polymorphisms): Account for most genetic variation (0.1%0.1\% difference between humans, or 3.2 million3.2\,\text{million} differences).

Epigenetics and DNA Extraction Techniques

  • Epigenome: Modifications in chromatin structure that do not change the DNA sequence. Influenced by diet and environment.

  • Major Processes:

    • DNA Methylation: Represses gene transcription.

    • Histone Acetylation: Increases levels of gene transcription.

    • Non-coding RNAs: Mechanism for gene silencing.

  • Phenol:Chloroform Extraction:

    • Cell lysate is mixed with phenol:chloroform.

    • Phase Separation: Two phases form. DNA/RNA partitions to the upper aqueous layer. Denatured/coagulated proteins partition to the lower organic phase or the interface.

    • Clean-up: The aqueous layer is pipetted off, and ethanol is used to precipitate the DNA.