DNA Biology and Technology chapter 22

DNA and RNA Structure and Function

  • Deoxyribonucleic Acid (DNA): The genetic material of the cell.

    • Genes: Short segments of DNA that contain instructions for a specific trait.
    • Chromosomes: Structures into which genes are organized. In eukaryotic cells, DNA is primarily located in the nucleus, though a small amount is found in the mitochondria.
    • Primary Functions of DNA:
      • Replication: Allows DNA to be transmitted to the next generation.
      • Information Storage: Holds the blueprints for cellular activity.
      • Genetic Variability: Undergoes changes (mutations) to provide variability within a population.

  • The Structure of DNA:

    • Double Helix: DNA is composed of two strands that spiral about each other.
    • Nucleotides: Each strand is a series of nucleotides, each consisting of three subunits:
      • Phosphate group.
      • Pentose Sugar: Specifically deoxyribose.
      • Nitrogen-containing Base:
        • Purines (Two Rings): Adenine [A] and Guanine [G].
        • Pyrimidines (One Ring): Cytosine [C] and Thymine [T].
    • Backbone and Bonding:
      • The phosphate and sugar molecules form the backbone, while the bases project to one side.
      • Complementary Base Pair Rules: Bases are joined by hydrogen bonds.
        • A pairs with T through two hydrogen bonds.
        • G pairs with C through three hydrogen bonds.
    • Antiparallel Orientation: The two strands run in opposite directions. One side ends with a 5′5' carbon and the other with a 3′3' carbon, determined by the position of carbon molecules on the deoxyribose sugar.

DNA Replication

  • Definition: The process of copying DNA, which occurs during the S phase of interphase.
  • Mechanism: Semiconservative replication, meaning each new double helix consists of one original (template) strand and one newly synthesized daughter strand.
  • Steps and Enzymes:
    • DNA Helicase: Unwinds and "unzips" the DNA by breaking the hydrogen bonds between the nitrogenous bases.
    • DNA Polymerase: Adds new DNA nucleotides following complementary base pairing rules.
    • Directionality:
      • Leading Strand: Synthesized continuously, following the helicase enzyme.
      • Lagging Strand: Synthesized in short segments called Okazaki fragments.
    • DNA Ligase: Seals any breaks in the sugar-phosphate backbone, joining segments together.
    • Outcome: Two identical double-helix molecules, each matching the original DNA molecule.
  • Mutations: Errors in replication that result in a sequence of bases different from the parental strand.
    • Repair enzymes typically fix these, but if they fail, a mutation occurs.
    • Mutations are not always negative; they introduce genetic variability by producing new alleles that alter the phenotype.

RNA Structure and Function

  • Ribonucleic Acid (RNA): Composed of nucleotides containing the sugar ribose.
    • RNA Nucleotides: Adenine (A), Uracil (U) (which replaces Thymine), Cytosine (C), and Guanine (G).
    • Pairing: A pairs with U; C pairs with G.
    • Structure: Typically single-stranded.
  • Major Types of RNA:
    • Messenger RNA (mRNA): Produced in the nucleus using DNA as a template; carries genetic information to the ribosomes in the cytoplasm for protein synthesis.
    • Ribosomal RNA (rRNA): Produced in the nucleolus using DNA. It joins with proteins to form large and small subunits of ribosomes. These subunits combine in the cytoplasm to form the ribosome where proteins are manufactured.
    • Transfer RNA (tRNA): Produced in the nucleus. It transfers specific amino acids to the ribosomes. There are 20 different types of amino acids and a corresponding tRNA for each.
  • Small RNAs:
    • Small nuclear RNAs (snRNAs): Involved in splicing mRNA before it leaves the nucleus.
    • Small nucleolar RNAs (snoRNAs): Modify ribosomal RNAs within the nucleolus.
    • MicroRNAs (miRNAs): Attach to mRNAs in the cytoplasm to prevent unnecessary translation.
    • Small interfering RNAs (siRNAs): Bind to mRNAs and mark them for degradation.

Gene Expression: Transcription and Translation

  • Overview: DNA acts as a template for RNA, which then acts as a template for protein manufacture.

  • Proteins: Composed of 20 different amino acids. The number, order, and sequence of amino acids determine the protein's shape, structure, and function.

    • Functions: Structural components, enzymes, neurotransmitters, antibodies, and hormones.

  • The Genetic Code:

    • Codon: A three-base sequence in mRNA representing a specific amino acid.
    • Total Codons: 64 total possible codons.
      • 61 code for specific amino acids.
      • Start Codon: AUG (codes for methionine).
      • Stop Codons: Three codons signal the termination of the polypeptide.
    • Redundancy: Most amino acids have more than one codon, which protects against harmful mutations.

  • Transcription: The first step of gene expression occurring in the nucleus.

    • Mechanism: A segment of DNA serves as a template. RNA polymerase adds RNA nucleotides using complementary base pairing (A−UA-U and C−GC-G).
    • mRNA Processing: Primary mRNA is modified into mature mRNA before leaving the nucleus.
      • Introns: Non-coding segments that interrupt genes; these are removed.
      • Exons: Portions of the gene that are expressed into protein; these are joined together.
      • Modifications: A cap (altered guanine nucleotide) is added to the 5′5' end, and a poly-A tail (multiple adenosine nucleotides) is added to the 3′3' end.
      • Spliceosome: A complex of RNA and protein that performs splicing. The RNA part acts as an enzyme, known as a ribozyme.
      • Alternate Splicing: Cells can use different combinations of exons from a single gene to produce different proteins.

  • Translation: The second step of gene expression occurring at the ribosome.

    • Components:
      • Translation Complex: Small and large ribosomal subunits bound to mRNA.
      • Ribosome Sites: A (Amino acid), P (Peptide), and E (Exit) sites.
      • tRNA and Anticodons: tRNA has an amino acid binding site and an anticodon (three-base sequence complementary to an mRNA codon).
    • Steps of Translation:
      • 1. Initiation: mRNA binds to the small ribosomal subunit, and the initiator tRNA (pairing with AUG) enters the P site. The large subunit then joins.
      • 2. Elongation: The polypeptide chain lengthens. New tRNA arrives at the A site, the peptide chain is transferred to the new amino acid via a peptide bond, and the ribosome moves forward. Spent tRNA exits via the E site.
      • 3. Termination: Occurs when a stop codon reaches the A site. A release factor protein binds to the stop codon, cleaving the polypeptide from the last tRNA. The ribosomal subunits then dissociate.
    • Polyribosome: Multiple ribosomes moving along a single mRNA simultaneously to synthesize several copies of the same polypeptide.

Regulation of Gene Expression

  • Mechanisms of Control:
    • Pretranscriptional: In the nucleus, DNA must uncoil and protective modifications must be removed to allow enzyme access.
    • Transcriptional: Involves transcription factors (DNA-binding proteins) that determine which genes are transcribed and at what rate.
    • Posttranscriptional: Occurs in the nucleus during mRNA processing/splicing.
    • Translational: Occurs in the cytoplasm; involves miRNAs and siRNAs that inhibit translation or destroy mRNA.
    • Posttranslational: Occurs in the cytoplasm after the protein has been synthesized.

DNA Technology and Biotechnology

  • Biotechnology: Use of natural biological systems to create products or achieve human-desired ends.

  • Genetic Engineering: Modification of genomes to improve characteristics or create products.

  • DNA Sequencing: Determining the order of nucleotides in DNA.

    • Allows identification of disease-linked alleles.
    • Aids forensic biology and evolutionary history studies.

  • Polymerase Chain Reaction (PCR): Used to make many copies of a DNA segment.

    • 1. Denaturation: DNA is heated to approximately 95∘C95^{\circ}C to become single-stranded.
    • 2. Annealing: DNA is cooled to approximately 55∘C55^{\circ}C to allow primers to attach.
    • 3. Extension: Heated to 72∘C72^{\circ}C for Taq DNA polymerase to add complementary bases.

  • DNA Fingerprinting (Profiling):

    • Uses PCR to amplify fragments and gel electrophoresis to separate them by size.
    • Used in crime scene investigation, identifying remains, and testing for genetic disorders.

  • Cloning:

    • Gene Cloning: Producing many copies of a single gene.
    • Recombinant DNA (rDNA): Contains DNA from more than one source.
    • Vectors: Used to introduce foreign genes into host cells. Plasmids (small rings of bacterial DNA) are common vectors.
    • Process:
      1. Restriction Enzyme: Cleaves both human and plasmid DNA at specific sites (forming a gap).
      2. DNA Ligase: Seals the foreign gene into the plasmid.
      3. Bacterial cells take up the recombinant plasmid and replicate it.
    • Expression in Bacteria: Human genes used in bacteria must lack introns and include bacterial regulatory regions.
    • Reverse Transcriptase: Enzyme used to create complementary DNA (cDNA) from mRNA; cDNA does not contain introns.

  • Genome Editing (CRISPR):

    • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats): Targets specific DNA sequences for removal or replacement.
    • Cas9 Enzyme: Acts as molecular scissors governed by a guide RNA molecule.
    • Applications: Treating sickle-cell disease and cancer (via ex vivo editing of bone marrow or T cells), developing tests for viruses like SARS-CoV-2, and potentially editing donor sperm for IVF therapy.

Genetically Modified Organisms (GMOs)

  • Transgenic Organisms: Organisms with a foreign gene inserted into their genome.
  • Transgenic Bacteria: Grown in bioreactors.
    • Examples: Oil-eating bacteria (engineered with "suicide" genes), frost-resistant (frost-minus) bacteria for strawberries, and corn-colonizing bacteria producing insect toxins.
  • Transgenic Plants:
    • Resistance: Corn, potatoes, soybeans, and cotton resistant to pests or herbicides.
    • Shelf Life: Knocking out the browning gene in apples.
    • Climate Adaptation: Leaves engineered to lose less water and absorb more CO2CO_2.
    • Biopharming: Plants producing hormones, clotting factors, and antibodies.
  • Transgenic Animals:
    • Food Source: Salmon engineered with growth hormones to grow faster (and sterile).
    • Pest Control: Mosquitoes that carry proteins killing their offspring (Zika control).
    • Gene Pharming: Transgenic farm animals producing pharmaceuticals in their milk (e.g., antibiotics, vaccines).
    • Models: Mouse models for human diseases like cystic fibrosis or cancer.
    • Xenotransplantation: Using animal organs for human transplantation.

Genomics, Proteomics, and Bioinformatics

  • Genomics: The study of genomes.
    • Human Genome Project (HGP): Completed in 2003. Revealed 3 billion bases and just under 20,000 functional genes. Humans are 99.9% identical at the base level.
    • Findings: Less than 2% of the genome codes for proteins; 98% produces regulatory RNA.
    • Functional Genomics: Studies how genes function and the role of noncoding (intergenic) DNA.
    • Comparative Genomics: Compares human genomes to other species (e.g., humans are 95–98% similar to chimps and 85% similar to mice).
  • Proteomics: The study of the structure, function, and interaction of cellular proteins. Computer modeling of 3D shapes is crucial.
  • Bioinformatics: The use of computers to analyze genomic data and identify significant patterns.

Gene Therapy

  • Definition: Insertion of genetic material into cells to treat a disorder.
  • Ex Vivo Gene Therapy: Treatment happens outside the body.
    • Stem cells are removed, modified using a viral vector (like a retrovirus) carrying the normal gene, and returned to the patient.
    • Used for: SCID, Hemophilia A, Alzheimer's, Parkinson's, Crohn's.
  • In Vivo Gene Therapy: Therapeutic DNA is injected directly into the body.
    • Often uses a viral vector sprayed or injected into the target tissue (e.g., respiratory tract for cystic fibrosis patients lacking a chloride transporter gene).
    • Used for: Cardiovascular diseases, endocrine disorders, Huntington's disease.