Bacterial Genetics Quiz

Course Objectives

• Define essential terms: genotype, phenotype, mutation, vertical/horizontal gene transfer, wild type.
• Discuss causes of mutations: spontaneous vs. induced.
• Describe DNA repair mechanisms including SOS repair.
• Explain auxotroph and prototroph concepts and the Ames Test for carcinogen screening.
• Cover the mechanisms of horizontal gene transfer: transformation, transduction, and conjugation.

Genetic Change in Bacteria

• Genotype: Sequence of nucleotides; Phenotype: Observable traits.
• Mutation: Change in nucleotide sequence leading to vertical gene transfer.
• Horizontal gene transfer: DNA movement between organisms.
• Wild Type: Typical phenotype derived from natural strains.

Spontaneous Mutation

• Random occurrences caused by normal cellular processes, with a mutation rate of 10^-4 to 10^-12.
• Reversion: Some mutations can revert to the original state.
• Base Substitution: Most common mutation type involving point mutations (silent, missense, nonsense mutations).
• Deletion/Addition of Nucleotides: Impact varies based on context; can lead to frameshift mutations which may result in nonfunctional proteins (knockout mutations).
• Transposons: Mobile genetic elements that can inactivate genes and carry transcriptional terminators.

Induced Mutations

• Result from external influences (mutagens).
• Chemical Mutagens: Include:
  • Nucleobase modifiers: Increase incorrect nucleotide incorporation.
  • Base analogs: Similar to nucleotides, such as 5-bromouracil for thymine.
  • Intercalating agents: Cause frameshift mutations, e.g., ethidium bromide.
• Radiation:
  • Ultraviolet light: Induces thymine dimers, distorting DNA.
  • X-rays: Cause strand breaks and changes in nucleobases.

Repair of Damaged DNA

• Error Correction:
  • Proofreading by DNA polymerase improves fidelity, though not perfect.
  • Mismatch repair involves enzymes that cut and repair the DNA backbone.
• Thymine Dimer Repair: Methods include photoreactivation and excision repair.
• Modified Nucleobase Repair: Specific repair pathways exist.
• SOS Repair: Activated under extensive DNA damage, allowing synthesis without proofreading, increasing errors.

Ames Test

• Tests for carcinogenic potential by measuring mutation rates in histidine-auxotroph Salmonella.
• Auxotroph: Mutants needing a growth factor; Prototroph: Does not require growth factors.
• Most carcinogens also act as mutagens.

Horizontal Gene Transfer

• Transformation: Bacteria uptake DNA from the environment.
• Transduction: DNA transfer mediated by bacteriophages.
• Conjugation: Direct DNA transfer between bacterial cells through physical contact.

Biotechnology and Recombinant DNA

• Focus on key technologies such as restriction enzymes and gel electrophoresis.
• Applications of genetically engineered organisms in various industries.

Restriction Enzymes

• Recognize specific nucleotide sequences and cut DNA to create restriction fragments.
• Produce cohesive ends (sticky ends) that facilitate annealing for recombinant DNA formation.

Gel Electrophoresis

• Technique to separate DNA fragments based on size using an electric current.

Genetically Engineered Bacteria

• Used for DNA cloning and producing valuable proteins such as insulin and vaccines.
• "Shotgun cloning" allows random DNA sample cloning for research.

Genetically Engineered Eukaryotes

• Transgenic organisms: Engineered to express desirable traits, e.g., pest resistance in crops.
• Biotech applications involve enriched nutritional value and novel vaccine production.

Concerns Regarding Genetic Engineering

• Emphasis on safety testing for new technologies, with ethical considerations regarding human DNA sequencing.
• GM organisms: Debates over allergens, environmental impacts, and public perception of GM food safety.

Polymerase Chain Reaction (PCR)

• Enables rapid amplification of DNA copies for analysis in hours.
• Operates in three temperature-controlled steps: denaturation, primer annealing, and DNA synthesis.

Probe Technologies

• DNA Probes: Locate specific sequences through hybridization with complementary DNA.
• Colony Blotting: Detects colonies with desired DNA sequences.
• FISH: Uses fluorescent probes for visualizing nucleotide sequences in fixed cells.
• DNA Microarrays: Analyze gene expression by isolating mRNA and converting it to cDNA.