Genetics, Viruses, and Laboratory Techniques in Microbiology: Study Notes

Genetic Information Flow and Nucleic Acid Structure

The Central Dogma of Molecular Biology

The central dogma describes the directional flow of genetic information in cells, outlining how genetic instructions are used to synthesize proteins.

• DNA stores genetic information.

• Transcription: DNA is transcribed into RNA.

• Translation: RNA is translated into protein.

• Memory aid: DNA makes RNA, RNA makes protein.

Structural Features of DNA and RNA

• DNA: Double-stranded, contains deoxyribose sugar, bases are A, T, C, G.

• RNA: Single-stranded, contains ribose sugar, bases are A, U, C, G (uracil replaces thymine).

• Both DNA and RNA nucleotides have a phosphate group, sugar, and nitrogenous base.

DNA Replication

Key Enzymes in DNA Replication

• Gyrase: Relieves supercoiling/twisting of DNA.

• Helicase: Unwinds the DNA double helix.

• DNA Polymerase III: Adds new nucleotides to the growing DNA strand.

• DNA Polymerase I: Removes RNA primers and replaces them with DNA.

• Primase: Synthesizes RNA primers to initiate DNA synthesis.

• Ligase: Joins Okazaki fragments on the lagging strand.

Directionality and Mechanism of Replication

• DNA synthesis occurs in the 5’ → 3’ direction.

• DNA strands are anti-parallel.

• Replication begins at the origin of replication and proceeds at the replication fork.

• Leading strand: Synthesized continuously.

• Lagging strand: Synthesized discontinuously, forming Okazaki fragments.

• Replication is semi-conservative: each new DNA molecule contains one old and one new strand.

Gene Expression: Transcription and Translation

Transcription in Prokaryotes

• Transcription: DNA is transcribed to messenger RNA (mRNA) by RNA polymerase.

Translation in Prokaryotes

• Translation: mRNA is translated into protein.

• mRNA: Carries genetic code from DNA.

• rRNA: Structural and catalytic component of ribosomes.

• tRNA: Brings amino acids to the ribosome; contains anticodons that pair with mRNA codons.

• Codon: 3-base sequence on mRNA.

• Anticodon: Complementary 3-base sequence on tRNA.

Protein Synthesis: Prokaryotes vs. Eukaryotes

• Prokaryotes: Transcription and translation occur simultaneously in the cytoplasm.

• Eukaryotes: Transcription occurs in the nucleus; translation occurs in the cytoplasm.

• Introns: Noncoding regions, removed during RNA processing (eukaryotes).

• Exons: Coding regions, remain in mature mRNA.

Using the Codon Table

• Use the mRNA codon table to determine amino acids.

• Example: AUG codes for methionine (start codon).

Gene Regulation and Operons

Operon Structure and Function

• Operon: A cluster of genes under control of a single promoter and operator.

• Parts: Promoter, Operator, Structural genes, Repressor.

• Inducible operon: Usually OFF, turned ON when needed (e.g., lac operon).

• Repressible operon: Usually ON, turned OFF when product is abundant (e.g., trp operon).

• Advantage: Efficient gene regulation and energy conservation.

Reverse Transcription and Mutations

Reverse Transcription

• Reverse transcription: Synthesis of DNA from an RNA template.

• Example: HIV (a retrovirus) uses this process.

Mutations and Evolution

• Mutations: Changes in DNA sequence; source of genetic variation and evolution.

• Base analogs: Mimic normal bases, cause incorrect pairing.

• UV light: Causes thymine dimers, leading to mutations.

Types of Mutations

• Deletion: Removal of a base.

• Insertion: Addition of a base.

• Frameshift: Reading frame changes (unless insertion/deletion is in multiples of 3).

• Silent: No change in amino acid.

• Nonsense: Creates a stop codon.

• Missense: Changes one amino acid.

Minimizing Mutation Impact

• If insertion/deletion is in multiples of 3, the reading frame is preserved, minimizing impact.

Ames Test and DNA Repair

• Ames test: Detects mutagenic/carcinogenic substances.

• Excision repair: Damaged DNA is removed and replaced with correct bases.

Genetic Recombination and Horizontal Gene Transfer

Recombination

• Recombination: Exchange of genetic material between DNA molecules.

Horizontal Gene Transfer Mechanisms

• Conjugation: DNA transfer between bacteria via pili; increases genetic diversity.

• Transformation: Uptake of naked DNA from environment (demonstrated by Griffith’s experiment with S. pneumoniae).

• Transduction: Bacteriophages transfer bacterial DNA between cells (generalized transduction).

Plasmids

• Plasmids: Small, circular DNA molecules separate from the chromosome.

• Often carry antibiotic resistance genes and virulence factors.

Viruses and Prions

General Characteristics of Viruses

• Acellular entities.

• Contain either DNA or RNA genome.

• Surrounded by a capsid (protein coat).

• Some have a lipid envelope.

Viral Evolution and Classification

• RNA viruses evolve faster due to higher mutation rates (RNA polymerases lack proofreading).

• Classification criteria:

  • Nucleic acid type (DNA or RNA)

  • Capsid shape

  • Envelope presence

  • Host type

Antigenic Shift and Drift in Influenza

• Antigenic drift: Small, gradual mutations.

• Antigenic shift: Major genetic reassortment.

• Both contribute to influenza virus evolution and outbreaks.

Bacteriophage Replication Cycles

• Lytic cycle: Immediate replication and cell lysis.

• Lysogenic cycle: Viral DNA integrates into host genome.

Phage Conversion

• Phage conversion: Phage DNA adds new traits to bacteria, often increasing virulence.

Animal Virus Replication Steps

• Attachment → Entry → Uncoating → Replication → Assembly → Release

Virus Culturing Methods

• Cell culture

• Embryonated eggs

• Live animals

Oncogenic Viruses

• Oncogenic viruses: Can cause cancer in hosts.

• Examples:

  • HPV → cervical cancer

  • HBV → liver cancer

Antiviral Drugs

• Block viral replication.

• Examples:

  • Acyclovir

  • Oseltamivir

Prions

• Prions: Misfolded infectious proteins.

• Diseases:

  • Creutzfeldt-Jakob disease

  • Mad cow disease

• Transmitted by ingestion, contaminated instruments, or inherited mutations.

Laboratory Techniques: Staining and Microscopy

Simple Staining

• Positive simple stain: Stains the cell, background remains clear.

• Negative simple stain: Stains the background, cell remains clear.

• Basic stain: Positively charged dye binds to negatively charged cell.

• Acidic stain: Negatively charged dye stains the background.

• Simple staining uses one dye.

• Reveals cell shape, size, and arrangement (e.g., cocci, bacilli, chains).

Differential and Structural Staining

• Differential stains use more than one dye to distinguish cell types or structures.

• Can differentiate Gram-positive vs. Gram-negative bacteria and cell wall differences.

• Structural stains highlight capsules, spores, and flagella.

• Common structural stains: Endospore stain, Capsule stain, Flagella stain.

Acid-Fast Stain

• Steps: Carbol fuchsin → Heat → Acid-alcohol → Methylene blue.

• Works due to mycolic acid in cell wall of acid-fast bacteria.

• Clinical use: Diagnosing Mycobacterium infections (e.g., tuberculosis).

• Interpretation:

  • Red/pink cells = acid-fast positive

  • Blue cells = acid-fast negative

Endospore Stain

• Green spores = endospore positive

• Pink/red vegetative cells = normal cells

• Genera with endospores: Bacillus, Clostridium

Gram Stain

• Differentiates bacteria based on cell wall structure (peptidoglycan thickness).

• Steps: Crystal violet → Iodine → Decolorizer (alcohol/acetone-alcohol) → Safranin.

• Gram-positive: Thick peptidoglycan, retains crystal violet, appears purple.

• Gram-negative: Thin peptidoglycan + outer membrane, loses crystal violet, takes safranin, appears pink.

Smear Preparation and Heat Fixing

• Steps: Place sample on slide → Spread thinly → Air dry → Heat fix.

• Purpose of heat fixing: Kills bacteria, adheres cells to slide, prevents washing off during staining.

Bacterial Morphology and Arrangements

• Coccus: Round

• Bacillus: Rod-shaped

• Spirillum: Spiral

• Arrangements:

  • Diplo-: Pairs

  • Strepto-: Chains

  • Staphylo-: Clusters

  • Tetrad: Groups of 4

  • Palisade: Side-by-side rods

Summary Table: Types of Mutations

Type	Description	Effect
Deletion	Base removed	May cause frameshift
Insertion	Base added	May cause frameshift
Frameshift	Reading frame altered	Usually severe effect
Silent	Base change, no amino acid change	No effect on protein
Nonsense	Base change creates stop codon	Premature termination
Missense	Base change alters amino acid	May affect protein function

Summary Table: Staining Techniques

Stain Type	Purpose	Key Features
Simple Stain	Visualize cell shape/arrangement	One dye; cell or background stained
Gram Stain	Differentiate Gram+/- bacteria	Crystal violet, iodine, decolorizer, safranin
Acid-Fast Stain	Identify acid-fast bacteria	Carbol fuchsin, acid-alcohol, methylene blue
Endospore Stain	Detect endospores	Green spores, pink/red cells
Capsule Stain	Visualize capsules	Negative staining technique