Genetics Exam III Study Guide: Bacterial Genetics, Translation, Mutation & Repair, Gene Regulation, and Recombinant DNA Technology

Chapter 6: Genetic Analysis in Bacteria & Bacteriophages

Bacterial Growth Curve

The bacterial growth curve describes the distinct phases of population growth in a closed system.

• Lag Phase: Period of adaptation; cells are metabolically active but not dividing.

• Log (Exponential) Phase: Rapid cell division and population growth; cells are most susceptible to antibiotics.

• Stationary Phase: Growth rate slows as resources are depleted; cell death balances cell division.

Quantitative Assays: Serial Dilutions and Colony Counts

Serial dilution and colony counting are used to estimate the original density of bacteria in a sample.

• Serial Dilution: Stepwise dilution of a bacterial culture to reduce concentration for counting.

• Colony Count: Number of colonies multiplied by the dilution factor gives the original density.

Formula:

$\text{Original density (CFU/mL)} = \frac{\text{Number of colonies}}{\text{Dilution factor} \times \text{Volume plated (mL)}}$

Bacterial Conjugation: F-, F+, Hfr, F’ Strains

• F-: Recipient cell lacking the F plasmid.

• F+: Donor cell containing the F plasmid; can transfer F plasmid to F-.

• Hfr (High frequency recombination): F plasmid integrated into the bacterial chromosome; transfers chromosomal genes to F-.

• F’: F plasmid excised from chromosome with some bacterial genes; can transfer these genes to F-.

Mating Outcomes: F+ x F- yields two F+ cells; Hfr x F- transfers chromosomal genes but recipient remains F-; F’ x F- can result in partial diploids (merozygotes).

Genetic Recombination in Prokaryotes

• Conjugation: Direct transfer of DNA via cell-to-cell contact.

• Transformation: Uptake of free DNA from the environment.

• Transduction: Transfer of DNA by bacteriophages.

Experimental Evidence: Classic experiments (e.g., Lederberg & Tatum) demonstrated these processes by showing genetic exchange and recombination in bacteria.

Time-Mapping Using Hfr Strains

Hfr strains transfer genes in a linear fashion; interrupted mating experiments allow mapping of gene order and distance based on time of entry.

Bacteriophage Life Cycles and Plaque Assays

• Lytic Cycle: Phage replicates and lyses host cell.

• Lysogenic Cycle: Phage DNA integrates into host genome (prophage); can later enter lytic cycle.

• Plaque Assay: Used to quantify phage particles; clear zones (plaques) indicate lysis of bacteria.

Chapter 14: Translation & Proteins

Charging tRNA

• Enzyme: Aminoacyl-tRNA synthetase attaches amino acid to tRNA.

• Energy Source: ATP is required for the charging process.

• Steps: Amino acid is activated by ATP, then transferred to tRNA.

Translation: Initiation, Elongation, Termination

• Initiation: Assembly of ribosome, mRNA, initiator tRNA, and initiation factors.

• Elongation: Sequential addition of amino acids; elongation factors assist.

• Termination: Stop codon recognized; release factors promote polypeptide release.

Peptide Bond Formation

• Peptide Bond: Covalent bond between amino group of one amino acid and carboxyl group of another; catalyzed by ribosome.

• Significance: Provides stability and directionality to proteins.

Reading Direction and Polypeptide Termini

• mRNA Reading: Ribosomes read mRNA 5’ to 3’.

• Polypeptide: Synthesized from N-terminus (amino) to C-terminus (carboxyl).

Translation: Eukaryotes vs. Bacteria

• Location: Eukaryotes—cytoplasm; Bacteria—cytoplasm (coupled with transcription).

• Initiation: Eukaryotes use 5’ cap; bacteria use Shine-Dalgarno sequence.

• Ribosome Size: Eukaryotes (80S), Bacteria (70S).

Gene-Protein Relationship Evolution

• One Gene-One Enzyme Hypothesis: Beadle and Tatum’s experiments with Neurospora mutants showed each gene encodes a specific enzyme.

• Modern View: One gene can encode multiple polypeptides via alternative splicing.

Amino Acid Structure and Groups

• General Structure: Central carbon, amino group, carboxyl group, hydrogen, R group (side chain).

• Groups: Nonpolar, polar, acidic, basic.

Post-Translational Modifications

• Phosphorylation, glycosylation, methylation, acetylation, etc., alter protein function and localization.

Protein Structure Levels

• Primary: Amino acid sequence.

• Secondary: Alpha helices and beta sheets (hydrogen bonding).

• Tertiary: 3D folding of a single polypeptide.

• Quaternary: Association of multiple polypeptides.

Protein Examples and Functions

• Enzymes: Catalyze biochemical reactions (e.g., DNA polymerase).

• Structural Proteins: Provide support (e.g., collagen).

• Transport Proteins: Move molecules (e.g., hemoglobin).

Chapter 15: Gene Mutation & DNA Repair

Types of Mutation

• Point Mutation: Single nucleotide change.

• Frameshift Mutation: Insertion or deletion alters reading frame.

• Transition: Purine to purine or pyrimidine to pyrimidine substitution.

• Transversion: Purine to pyrimidine or vice versa.

Mutation Detection and Rates

• Mutations detected via phenotypic screening, replica plating, or molecular methods.

• Mutation Rate Calculation:

$\text{Mutation rate} = \frac{\text{Number of mutations}}{\text{Total number of cells or generations}}$

Mutagens and Their Effects

• UV Light: Causes thymine dimers, leading to errors in replication.

• Base Analogs: Incorporated into DNA, causing mispairing.

• Other Mutagens: Chemicals, radiation, etc.

DNA Repair Mechanisms

• Photoreactivation Repair: Photolyase enzyme uses light energy to break thymine dimers.

• Excision Repair: Damaged DNA is removed and replaced; includes base and nucleotide excision repair.

• Recombination Repair: Uses undamaged DNA as a template to repair gaps.

Mutations and Human Disorders

• Examples: Sickle cell anemia (point mutation in HBB gene), cystic fibrosis (deletion in CFTR gene).

Chapter 16: Gene Regulation in Prokaryotes

Operons and Their Components

• Operon: Cluster of genes under control of a single promoter and regulatory elements.

• Components: Promoter, operator, structural genes, regulatory gene.

Operon Regulation

• Inducible Operon: Usually off; turned on by inducer (e.g., lac operon).

• Repressible Operon: Usually on; turned off by corepressor (e.g., trp operon).

• Negative Control: Repressor protein inhibits transcription.

• Positive Control: Activator protein enhances transcription.

lac and trp Operon Examples

• lac Operon: Inducible; lactose induces expression of genes for lactose metabolism.

• trp Operon: Repressible; tryptophan represses genes for its own synthesis.

Mutations in Gene Regulation

• Constitutive Mutations: Genes expressed continuously due to defective regulation.

• Merozygote Analysis: Partial diploids used to study dominance and regulatory mechanisms.

• Attenuation: Regulation of gene expression by premature termination of transcription (e.g., trp operon).

Chapter 20: Introduction to Recombinant DNA Technology (RDT)

Goals and Definitions

• Goals: Clone, analyze, and manipulate genes for research, medicine, and biotechnology.

• Definition: RDT involves combining DNA from different sources to create recombinant molecules.

Tools and History

• Tools: Restriction enzymes, DNA ligase, vectors, PCR, etc.

• History: Developed in the 1970s; Cohen and Boyer pioneered gene cloning.

General Steps in RDT

1. Isolate DNA of interest.

2. Cut DNA with restriction enzymes.

3. Ligate DNA into vector.

4. Transform host cells.

5. Screen for recombinant clones.

Vectors

Vector Type	Insert Size	Host	Features
Plasmid	Up to ~10 kb	Bacteria	Easy to manipulate, antibiotic resistance
Bacteriophage	Up to ~20 kb	Bacteria	High efficiency, larger inserts
BAC (Bacterial Artificial Chromosome)	Up to ~300 kb	Bacteria	Large inserts, stable maintenance
YAC (Yeast Artificial Chromosome)	Up to ~2 Mb	Yeast	Very large inserts, eukaryotic system

DNA Library Construction and Screening

• Genomic Library: Collection of clones representing entire genome.

• cDNA Library: Clones of expressed genes (from mRNA via reverse transcription).

• Screening: Use of probes to identify clones with gene of interest.

Techniques for DNA Analysis

• PCR (Polymerase Chain Reaction): Amplifies specific DNA sequences.

• RT-PCR: Reverse transcription PCR; amplifies cDNA from RNA.

• qPCR: Quantitative PCR; measures DNA amplification in real time.

• Restriction Mapping: Determines locations of restriction sites in DNA.

• Southern Blot: Detects specific DNA sequences.

• Northern Blot: Detects specific RNA sequences.

• Western Blot: Detects specific proteins.

• FISH (Fluorescence In Situ Hybridization): Visualizes DNA sequences on chromosomes.

DNA Sequencing

• Principle: Determining the order of nucleotides in DNA.

• Methods: Sanger sequencing (chain termination), next-generation sequencing.

• Recent Progress: High-throughput, massively parallel sequencing technologies.

Knockout and Knock-in Animals

• Knockout Mice: Gene inactivated to study function; created via homologous recombination in embryonic stem cells.

• Knock-in Animals: Specific gene inserted or modified to study effects.

Additional info: For all topics, understanding experimental design and the rationale behind techniques is crucial for application in genetics research and biotechnology.