Microbial Genetics and Recombinant DNA Technology: Structured Study Notes

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Genetics: Flow of Genetic Information

Central Dogma of Molecular Biology

The central dogma describes the flow of genetic information in cells: DNA stores instructions, RNA carries a copy, and proteins perform cellular functions. Understanding this flow is fundamental to genetics.

DNA → RNA → Protein: Information is transcribed from DNA to RNA, then translated into protein.
Replication: DNA is copied before cell division.
Transcription: DNA is used to synthesize RNA.
Translation: RNA is used to synthesize proteins.

Example: The gene for an enzyme is transcribed into mRNA, which is then translated into the enzyme protein.

Core Definitions

Gene: A region of DNA coding for a protein or functional RNA.
Genome: All DNA in a cell, including coding and non-coding regions.
Phenotype: Observable characteristics resulting from gene expression.

Prokaryotic vs Eukaryotic DNA

Structural Differences

Prokaryotes and eukaryotes differ in DNA structure and location, affecting replication and gene regulation.

Prokaryotes: Circular DNA, no nucleus, DNA in cytoplasm, often have plasmids.
Eukaryotes: Linear DNA, DNA inside nucleus, DNA wrapped around histones.

Example: Escherichia coli (prokaryote) vs. human cells (eukaryote).

DNA Packaging and Gene Control

Chromatin Structure

DNA wraps around histones to form chromatin, influencing gene accessibility and regulation.

Open chromatin: Loosely packed, genes accessible and active.
Closed chromatin: Tightly packed, genes inaccessible and inactive.

DNA Structure and Components

Nucleotide Composition

DNA is composed of nucleotides, each containing a phosphate group, deoxyribose sugar, and a nitrogenous base.

Backbone: Sugar-phosphate chain.
Bases: Adenine (A), Thymine (T), Guanine (G), Cytosine (C).
Base pairing: A-T (2 hydrogen bonds), G-C (3 hydrogen bonds).

Equation:

DNA Replication

Semi-Conservative Replication

DNA replication produces two molecules, each with one old and one new strand. Replication occurs in the 5' to 3' direction.

Leading strand: Built continuously toward replication fork.
Lagging strand: Built in Okazaki fragments away from fork, joined by DNA ligase.
Key enzymes: Helicase, Topoisomerase, Primase, DNA Polymerase III, DNA Polymerase I, DNA ligase.
Energy source: Phosphate bonds of incoming nucleotides.

Equation:

DNA Methylation

Regulation and Protection

Methylation adds a CH3 group to DNA, helping distinguish self from non-self and regulate gene expression.

Prokaryotes: Methylate adenine.
Eukaryotes: Methylate cytosine.
Functions: Protects DNA from restriction enzymes, regulates replication and gene expression.

Transcription (DNA to RNA)

RNA Synthesis

Transcription copies one DNA strand into RNA using RNA polymerase, which does not require a primer.

RNA polymerase: Unwinds DNA, copies one strand, uses uracil (U) instead of thymine (T).
Types of RNA: mRNA (message), rRNA (ribosome), tRNA (amino acids), RNA primer (replication).
Location: Prokaryotes (cytoplasm), Eukaryotes (nucleus).

Eukaryotic mRNA Processing

Splicing and Export

Eukaryotic mRNA contains introns (non-coding) and exons (coding). The spliceosome removes introns, joining exons for export.

Introns: Removed.
Exons: Joined together.
Processed mRNA: Leaves nucleus for translation.

Translation (mRNA to Protein)

Protein Synthesis

Translation converts mRNA codons into amino acids, forming proteins. The ribosome reads codons and tRNA delivers amino acids.

Codon: Three nucleotides coding for one amino acid (e.g., AUG = methionine/start).
Ribosome sites: A (accepts tRNA), P (holds polypeptide), E (exit).
Energy: GTP powers translation.

Equation:

Gene Regulation

Control of Gene Expression

Not all genes are active at all times. Some are always on, others respond to environmental conditions.

Constitutive genes: Always active (ribosomal proteins, membrane proteins, glycolysis enzymes).
Inducible genes: Turned on/off as needed.

Operons (Prokaryotes Only)

Operons are groups of genes controlled together under one promoter, allowing efficient response to environmental changes.

Lactose operon: Usually off, turns on when lactose is present.
Components: Promoter, operator, structural genes.

Mutation

Types and Effects

Mutation is a permanent change in DNA sequence, which can be harmful, neutral, or beneficial.

Types: Base substitution, insertion, deletion, frameshift, inversion, duplication, transposition.
Effects: Silent (no change), missense (one amino acid changes), frameshift (major disruption).

Horizontal Gene Transfer (Prokaryotes)

Mechanisms of Gene Acquisition

Bacteria can acquire genes from other bacteria, contributing to genetic diversity and antibiotic resistance.

Transformation: Uptake of DNA from environment (e.g., Griffith's experiment with Streptococcus pneumoniae).
Transduction: Virus transfers DNA between bacteria.
Conjugation: Direct transfer via cell-to-cell contact using F plasmid.

Transposons

Jumping Genes

Transposons are DNA segments that move within the genome, increasing genetic variation and sometimes carrying antibiotic resistance genes.

Found in: Prokaryotes and eukaryotes.
Function: Can move within or between chromosomes and plasmids.

Radiation as a Mutagen

DNA Damage by Radiation

Radiation can cause mutations by damaging DNA structure.

Gamma rays: High energy, deep penetration, cause double-strand breaks.
X-rays: Cause strand breaks and base changes.
UV radiation: Causes thymine dimers, distorting DNA and increasing mutation risk.

Phenotype vs Genotype

Definitions and Relationship

Genotype is the DNA sequence; phenotype is the observable trait resulting from gene expression.

Genotype: Genetic information.
Phenotype: Observable characteristics (e.g., eye color, enzyme function).

Equation:

DNA Polymerase vs RNA Polymerase

Comparison

Feature	DNA Polymerase	RNA Polymerase
Primer required?	Yes	No
Copies	Entire DNA	One gene
Nucleotides used	Deoxyribonucleotides	Ribonucleotides
Accuracy	High	Lower

Control of Translation

Regulatory Mechanisms

miRNA: Binds mRNA, prevents translation.
Riboswitch: Changes shape in response to environment, turns gene expression on/off.

Mutagens and DNA Repair

Mutagen Types

Radiation: Gamma rays, X-rays, UV.
Chemicals: Benzo[a]pyrene (frameshifts), aflatoxin (liver mutations).

DNA Repair Mechanisms

Mismatch Repair: Corrects replication errors using methylation to identify old strand.
Base Excision Repair: Removes damaged bases, fills gap.
Pyrimidine Dimer Repair: Light repair (photolyase), dark repair (excision).
SOS Repair: Emergency, error-prone repair under severe damage.

Selection and Ames Test

Positive and Negative Selection

Positive selection: Mutants survive under selective conditions (e.g., antibiotic resistance).
Negative selection: Mutants are removed if they cannot survive.
Ames Test: Assesses mutagenicity of chemicals.

Recombinant DNA Technology (Genetic Engineering)

Definition and Goals

Recombinant DNA technology manipulates genes for industrial, medical, and agricultural purposes.

Eliminate unwanted traits (e.g., remove disease genes).
Combine traits (e.g., engineered research animals).
Produce useful products (e.g., vaccines, hormones).

Tools of Recombinant DNA Technology

PCR: Amplifies DNA sequences.
Mutagens: Induce mutations.
Reverse transcriptase: Makes DNA from RNA.
Synthetic nucleic acids: Lab-made DNA/RNA.
Restriction enzymes: Cut DNA at specific sites.
Vectors: DNA carriers (plasmids, viruses).

Polymerase Chain Reaction (PCR)

PCR is a lab technique to amplify DNA, enabling applications in sequencing, pathogen detection, and genetic mapping.

Steps: Denaturation (heat), annealing (cool), extension (polymerase).
Each cycle: Doubles DNA amount.

Equation:

Restriction Enzymes

Restriction enzymes cut DNA at specific palindromic sequences, protecting bacteria from viral DNA.

Example: HindIII from Haemophilus influenzae.
Protection: Bacterial DNA is methylated to prevent self-cutting.

Reverse Transcriptase

Reverse transcriptase synthesizes DNA from RNA, useful for creating cDNA and studying gene expression.

Synthetic Nucleic Acids

Lab-made DNA/RNA sequences are used for gene design, probes, and engineering mutations.

Vectors

Vectors transfer genes into cells; must replicate in host and often carry selectable markers.

Common vectors: Plasmids, viruses.
Features: Origin of replication, antibiotic resistance marker.

Applications of Recombinant DNA Technology

Pharmaceutical and Therapeutic Applications

Protein synthesis: Bacteria/yeast produce insulin, interferon, growth hormones.
Benefits: Cheaper, safer, large-scale production.

Vaccines

Types: Live, inactivated, toxoids, subunit.
Recombinant vaccines: Safe production of antigens (e.g., hepatitis B surface antigen).

Genetic Screening

Detects: Pathogens, inherited conditions.
Allows: Early treatment and monitoring.

DNA Fingerprinting

Identifies: Individuals by unique DNA patterns.
Applications: Forensics, paternity, organism identification.

Gene Therapy

Replaces: Defective genes with normal copies.
Challenges: Immune reactions, delivery, long-term expression.

Medical Diagnosis

Detects: Pathogen DNA (e.g., hepatitis, HIV, TB, gonorrhea).
Benefits: Faster, more accurate diagnosis.

Agricultural Applications

Traits: Herbicide resistance, salt tolerance, freeze resistance, pest resistance, improved nutrition.
Example: Crops engineered to produce insecticidal proteins.

Plasmids

Definition and Types

Plasmids are small, circular DNA molecules in bacteria, carrying genes for traits like resistance, virulence, and conjugation.

Replicate independently of chromosome.
Not essential for basic survival.

Types of Plasmids

Type	Function	Example
Resistance (R) plasmids	Antibiotic/heavy metal resistance	E. coli with ampicillin resistance
Bacteriocin plasmids	Protein toxins against competitors	Gut bacteria
Fertility (F) plasmid	Conjugation, pili formation	F+ cells
Virulence plasmids	Pathogenic traits (toxins, adhesion)	E. coli causing diarrhea

Positive Selection

Identifying Mutants

Positive selection allows only mutants with a desired trait to survive under selective conditions (e.g., antibiotic resistance).

Step 1: Grow bacteria on normal media.
Step 2: Plate on selective media (e.g., with penicillin).
Result: Only resistant mutants survive.

DNA Repair Mechanisms

Maintaining Genetic Stability

Mismatch Repair: Corrects replication errors using methylation.
Base Excision Repair: Removes damaged bases, fills gap.
Pyrimidine Dimer Repair: Light repair (photolyase), dark repair (excision).
SOS Repair: Emergency, error-prone repair under severe damage.

Summary Table: DNA Repair Mechanisms

Repair Type	Damage Fixed	Key Enzymes	Accuracy
Mismatch Repair	Replication errors	Mismatch enzymes, DNA Polymerase I, DNA ligase	High
Base Excision Repair	Abnormal bases	Excision enzymes, DNA Polymerase I, DNA ligase	High
Photoreactivation (Light Repair)	Thymine dimers	DNA photolyase	High
Nucleotide Excision (Dark Repair)	Thymine dimers	Excision enzymes, DNA Polymerase I, DNA ligase	High
SOS Repair	Severe damage	Novel DNA polymerases	Low (error-prone)

Big Picture Understanding

Genetics: Information storage, copying, expression, regulation, and change.
Recombinant DNA: Uses natural mechanisms for gene manipulation.
Balance: Mutation (variation) and repair (stability) are essential for evolution and cell survival.