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Study of inheritance, variation, and expression of genetic material in microorganisms
Includes:
DNA structure
Replication
Mutation
Gene transfer
Gene regulation
| Feature | Genome | Gene |
|---|---|---|
| Definition | Total genetic material | Functional unit of heredity |
| Composition | Entire DNA (chromosomal + plasmid) | Specific DNA sequence |
| Function | Encodes all information | Encodes protein/RNA |
| Size | Large (kb–Mb) | Small segment |
| Feature | Chromosomal DNA | Extrachromosomal DNA |
|---|---|---|
| Structure | Circular (usually) | Circular |
| Location | Nucleoid | Cytoplasm |
| Copy number | Single | Multiple |
| Function | Essential genes | Accessory genes (resistance, virulence) |
| Example | Bacterial chromosome | Plasmid |
Single circular double-stranded DNA
Located in nucleoid (no nuclear membrane)
No histones (except some archaea)
High gene density → minimal non-coding DNA
Operon organization present
Transcription and translation coupled
Presence of:
Plasmids
Transposons
Single copy of genome
Mutation effects are immediately expressed
No masking by second allele
Important in:
Antibiotic resistance
Rapid adaptation
Genome size expressed in kb (kilobase) or Mb (megabase)
Examples:
Mycoplasma → ~0.6 Mb
E. coli → ~4.6 Mb
Clinical correlation:
Small genome → host dependence
Large genome → metabolic versatility
% of guanine + cytosine bases
Significance:
High GC → greater thermal stability
Used in classification and taxonomy
Influences:
DNA melting temperature
Gene expression patterns
Separation of DNA strands by:
Heat
Alkali
Causes:
Loss of double helix
Hyperchromic effect (increase in absorbance at 260 nm)
Rejoining of complementary strands
Depends on:
Temperature
Ionic conditions
Sequence complementarity
Cot = DNA concentration × time
Measures rate of DNA renaturation
Interpretation:
Repetitive DNA → fast reassociation
Unique DNA → slow reassociation
Use:
Genome complexity analysis
Flow of genetic information:
Continue with Google
DNA → RNA → Protein
DNA → RNA = Transcription
RNA → Protein = Translation
| Feature | Prokaryotes | Eukaryotes |
|---|---|---|
| Nucleus | Absent | Present |
| Chromosome | Single, circular | Multiple, linear |
| Histones | Absent | Present |
| Introns | Absent | Present |
| Gene organization | Operon | Individual genes |
| Transcription & translation | Coupled | Separate |
| mRNA | Polycistronic | Monocistronic |
DNA
↓ (Transcription)
mRNA
↓ (Translation)
Protein
Double-stranded DNA
↓ Heat
Single strands
↑ Absorbance (260 nm)
[Circular Chromosome]
+
[Plasmids]
+
[Transposons / IS elements]
Bacteria are haploid → mutations directly expressed
Operon system is a key feature of prokaryotes
GC content acts as a taxonomy and stability marker
Cot curve helps in genome complexity assessment
Central dogma forms the foundation of molecular biology
| Feature | Prokaryotic Genome | Eukaryotic Genome |
|---|---|---|
| Nucleus | Absent | Present |
| Chromosome | Single, circular | Multiple, linear |
| Histones | Absent (except archaea) | Present |
| Introns | Absent | Present |
| Gene density | High | Low |
| Repetitive DNA | Minimal | Abundant |
| Operon system | Present | Absent |
| Transcription & translation | Coupled | Separate |
| Feature | Chromosome | Plasmid |
|---|---|---|
| Definition | Main genetic material | Extra-chromosomal DNA |
| Size | Large | Small |
| Number | Usually single | Multiple copies |
| Essential genes | Present | Usually absent |
| Replication | Controlled | Independent |
| Function | Vital cellular functions | Resistance, virulence |
| Feature | DNA | RNA |
|---|---|---|
| Sugar | Deoxyribose | Ribose |
| Bases | A, T, G, C | A, U, G, C |
| Structure | Double-stranded | Single-stranded |
| Stability | More stable | Less stable |
| Function | Genetic storage | Protein synthesis |
| Location | Nucleus/nucleoid | Cytoplasm |
| Organism | Genome Size | Complexity |
|---|---|---|
| Viruses | Very small | Simple |
| Mycoplasma | ~0.6 Mb | Minimal functions |
| Bacteria (e.g., E. coli) | ~4–5 Mb | Moderate |
| Eukaryotes | Large (Gb) | Highly complex |
| Humans | ~3 Gb | Very high |
DNA
↓ (Transcription)
mRNA
↓ (Translation)
Protein
______________________
/ \
/ \
| Circular DNA (dsDNA) |
| |
\ /
\______________________/
Located in nucleoid
No nuclear membrane
Double-stranded DNA
↓ Heat / Alkali
Separation of strands
↓
Single-stranded DNA
↑
Increase in absorbance (260 nm)
(Hyperchromic effect)
A gene is a segment of DNA that codes for a functional product
Protein
RNA (rRNA, tRNA)
Smallest unit of heredity and function
Cistron
Functional unit of gene
Codes for a single polypeptide
Operon
Group of genes transcribed together under one promoter
Produces polycistronic mRNA
Regulon
Group of genes or operons regulated by same regulatory protein
Located at different sites in genome
Proposed by Jacob and Monod
Basic unit of gene regulation in prokaryotes
Allows:
Coordinated gene expression
Efficient metabolic control
Promoter
Binding site for RNA polymerase
Initiates transcription
Operator
Regulatory site
Binding of repressor protein
Structural Genes
Code for enzymes/proteins
Regulator Gene
Produces repressor protein
May be located outside operon
| Feature | Monocistronic | Polycistronic |
|---|---|---|
| Number of genes | One gene | Multiple genes |
| mRNA product | Single protein | Multiple proteins |
| Found in | Eukaryotes | Prokaryotes |
| Regulation | Individual | Coordinated |
Constitutive genes
Expressed continuously
Required for basic functions
Inducible genes
Expressed only when needed
Activated by substrate/inducer
Genetic element that can exist:
Independently (plasmid form)
Integrated into chromosome
Example:
F factor
Small DNA segments capable of transposition
Contain:
Transposase gene
Inverted repeat sequences
Do not carry additional genes
Simple transposons
Only transposition genes
Composite transposons
Carry additional genes (e.g., antibiotic resistance)
Flanked by IS elements
| Feature | Operon | Regulon |
|---|---|---|
| Structure | Cluster of genes | Scattered genes |
| Control | Single promoter | Multiple promoters |
| Regulation | One regulator | Common regulator |
| Example | Lac operon | SOS regulon |
| Feature | Structural Genes | Regulatory Genes |
|---|---|---|
| Function | Code for proteins/enzymes | Control gene expression |
| Location | Within operon | May be outside operon |
| Product | Functional proteins | Repressor/activator proteins |
| Feature | IS Elements | Transposons |
|---|---|---|
| Size | Small | Larger |
| Genes | Only transposase | Additional genes present |
| Function | Movement only | Movement + extra functions |
| Example | IS1 | Tn3 |
Regulator gene → Repressor protein
↓
Promoter → Operator → Structural genes → mRNA → Protein
Promoter → Operator → Gene 1 → Gene 2 → Gene 3
↓
Single mRNA (polycistronic)
[IS] — Resistance gene — [IS]
Transposase enzyme mediates movement
Each daughter DNA contains:
One parental strand
One newly synthesized strand
Proven by Meselson and Stahl experiment
Ensures genetic fidelity
Specific site where replication begins
In bacteria:
Single origin (OriC)
Rich in AT sequences → easier strand separation
Replication proceeds in both directions from OriC
Forms two replication forks
Increases efficiency
Y-shaped structure where DNA unwinds and replicates
Site of:
Strand separation
New DNA synthesis
DNA Polymerase
Synthesizes new DNA
Works in 5’ → 3’ direction
Has proofreading activity (3’ → 5’ exonuclease)
Helicase
Unwinds DNA helix
Primase
Synthesizes RNA primer
DNA Ligase
Joins DNA fragments
Topoisomerase (Gyrase)
Relieves supercoiling
Leading strand
Continuous synthesis
Same direction as replication fork
Lagging strand
Discontinuous synthesis
Opposite direction
Forms Okazaki fragments
Short DNA fragments formed on lagging strand
Later joined by DNA ligase
Essential for discontinuous synthesis
Occurs in circular DNA (bacteria)
Produces theta-shaped intermediate
Seen in:
Plasmids
Bacteriophages
Produces multiple copies rapidly
| Feature | Prokaryotes | Eukaryotes |
|---|---|---|
| Origin | Single | Multiple |
| Speed | Fast | Slow |
| DNA polymerase types | Few | Many |
| Okazaki fragments | Long | Short |
| Location | Cytoplasm | Nucleus |
| Polymerase | Function |
|---|---|
| DNA Pol I | Primer removal and repair |
| DNA Pol II | DNA repair |
| DNA Pol III | Main replication enzyme |
| Feature | Leading Strand | Lagging Strand |
|---|---|---|
| Synthesis | Continuous | Discontinuous |
| Direction | Same as fork | Opposite |
| Primer | Single | Multiple |
| Fragments | Absent | Okazaki fragments present |
Helicase
↓
5' -----------→ 3' (Leading strand)
3' ←----------- 5' (Lagging strand)
Okazaki fragments
Lagging strand:
Primer → Fragment → Primer → Fragment → Primer → Fragment
↓
Joined by ligase
Circular DNA
↓
Bubble formation
↓
Theta-shaped structure
↓
Two daughter circles
Circular DNA
↓ Nick
Single strand elongation
↓
Displacement of old strand
↓
Synthesis of multiple copies
Permanent heritable change in DNA sequence
Leads to:
Altered protein
Loss or gain of function
Change in a single nucleotide base
Types:
Silent mutation
No change in amino acid
Missense mutation
Change in amino acid
Nonsense mutation
Formation of stop codon → truncated protein
Insertion or deletion of bases
Alters reading frame
Produces abnormal protein
Mutation expressed only under specific conditions
Example:
Temperature-sensitive mutants
Spontaneous mutation
Occurs naturally
Due to replication errors
Induced mutation
Caused by mutagens
UV radiation → thymine dimers
Ionizing radiation → DNA breaks
Base analogs
Alkylating agents
Intercalating agents
Mutation that restores original phenotype
Types:
True reversion
Suppressor mutation
Genes that increase mutation rate
Affect DNA repair mechanisms
Repair of UV-induced damage
Enzyme: photolyase
Removal of damaged DNA
Replacement with correct sequence
Emergency repair system
Error-prone
Activated during severe damage
Structural alteration of genome
Includes:
Insertion
Deletion
Inversion
Movement of DNA segments within genome
Mediated by transposons
Enzyme: transposase
Detects mutagenic potential of chemicals
Principle:
Uses Salmonella mutant strain
Measures reversion to normal growth
Application:
Screening carcinogens
| Type | Description | Effect |
|---|---|---|
| Silent | No amino acid change | No effect |
| Missense | Amino acid change | Altered protein |
| Nonsense | Stop codon formed | Truncated protein |
| Frameshift | Reading frame altered | Severe dysfunction |
| Mechanism | Type of Damage | Enzyme |
|---|---|---|
| Photoreactivation | UV damage | Photolyase |
| Excision repair | Damaged bases | Endonuclease |
| SOS repair | Severe damage | Multiple enzymes |
| Mutagen | Type | Effect |
|---|---|---|
| UV rays | Physical | Thymine dimers |
| X-rays | Physical | DNA breaks |
| Base analogs | Chemical | Wrong base pairing |
| Alkylating agents | Chemical | DNA modification |
Point mutation:
ATG → ATA (single base change)
Frameshift mutation:
ATG-CGA-TTA
↓ deletion
ATG-GAT-TA...
DNA damage
↓
Recognition
↓
Repair mechanism:
→ Photoreactivation
→ Excision repair
→ SOS repair
DNA segment
↓
Cut by transposase
↓
Inserted into new site
Mutant bacteria (cannot grow)
↓ + Chemical
Reversion mutation occurs
↓
Growth observed → Mutagen present
Synthesis of RNA from DNA template
Occurs in cytoplasm (prokaryotes)
Direction: 5’ → 3’
Core enzyme synthesizes RNA
Requires sigma factor for initiation
Recognizes promoter region
Initiates transcription
Dissociates after initiation
Rho-dependent termination
Requires rho protein
Terminates transcription
Rho-independent termination
Hairpin loop formation
No rho factor needed
Synthesis of protein from mRNA
Occurs on ribosomes
Made of:
30S subunit
50S subunit
Forms 70S ribosome
Ribosome binding site on mRNA
Aligns mRNA for correct initiation
Triplet code (codon)
Properties:
Degenerate
Non-overlapping
Universal
Unambiguous
Control of protein synthesis at:
Transcription level
Translation level
Induction
Gene expression turned ON by inducer
Repression
Gene expression turned OFF by corepressor
Example of inducible system
Components:
Promoter
Operator
Structural genes (lacZ, lacY, lacA)
Mechanism:
No lactose → repressor active → gene OFF
Lactose present → repressor inactivated → gene ON
Example of repressible system
Mechanism:
Low tryptophan → gene ON
High tryptophan → repression → gene OFF
Glucose inhibits utilization of other sugars
Mediated by:
cAMP
CAP protein
Low glucose → high cAMP → gene activation
High glucose → low cAMP → repression
Regulation at transcription level
Seen in trp operon
Depends on tryptophan levels
mRNA stability
mRNA degradation
Ribosome binding control
Protein modifications after synthesis
Examples:
Phosphorylation
Cleavage
| Feature | Induction | Repression |
|---|---|---|
| Gene state | OFF → ON | ON → OFF |
| Effector | Inducer | Corepressor |
| Example | Lac operon | Trp operon |
| Feature | Lac Operon | Trp Operon |
|---|---|---|
| Type | Inducible | Repressible |
| Default state | OFF | ON |
| Effector | Lactose | Tryptophan |
| Function | Lactose metabolism | Tryptophan synthesis |
| Property | Description |
|---|---|
| Triplet | 3 bases per codon |
| Degenerate | Multiple codons for same amino acid |
| Universal | Same in most organisms |
| Non-overlapping | Codons read separately |
| Unambiguous | One codon → one amino acid |
DNA template
↓
RNA polymerase binds promoter
↓
RNA synthesis (mRNA)
↓
Termination
mRNA
↓
Ribosome binding
↓
tRNA brings amino acids
↓
Polypeptide chain formation
No lactose:
Repressor binds operator → Gene OFF
With lactose:
Repressor inactive → Gene ON
Low tryptophan:
Gene ON
High tryptophan:
Repressor active → Gene OFF
Technique of combining DNA from different sources
Produces recombinant DNA (rDNA)
Basis of:
Gene cloning
Biotechnology applications
Enzymes that cut DNA at specific sequences
Also called restriction endonucleases
Sticky ends
Staggered cuts
Produce overhangs
Facilitate easy joining
Blunt ends
Straight cuts
No overhangs
Enzyme that joins DNA fragments
Forms phosphodiester bonds
Enzyme that converts:
RNA → DNA (cDNA)
Found in retroviruses
Complementary DNA synthesized from mRNA
Represents expressed genes only
No introns
DNA molecules used to carry foreign DNA into host cell
Plasmids
Circular DNA
Commonly used
Bacteriophages
Viral vectors
Cosmids
Hybrid of plasmid + phage
Genomic library
Contains entire genome
Includes introns
cDNA library
Contains only expressed genes
No introns
Isolation of DNA
Cutting with restriction enzymes
Insertion into vector
Transformation into host
Selection of clones
Expression of gene
Inserted gene is:
Transcribed
Translated
Produces desired protein
Production of:
Insulin
Vaccines
Hormones
Gene therapy
Diagnostic probes
DNA fingerprinting
| Feature | Plasmids | Bacteriophages | Cosmids |
|---|---|---|---|
| Size of DNA insert | Small | Moderate | Large |
| Nature | Circular DNA | Virus | Hybrid |
| Efficiency | Moderate | High | High |
| Use | Routine cloning | Gene transfer | Large fragments |
| Type | Feature |
|---|---|
| Type I | Cut away from site |
| Type II | Cut at specific site |
| Type III | Cut near recognition site |
| Feature | Genomic Library | cDNA Library |
|---|---|---|
| Source | Total DNA | mRNA |
| Introns | Present | Absent |
| Genes | All genes | Expressed genes only |
| Use | Genome study | Protein expression |
DNA isolation
↓
Restriction enzyme cutting
↓
Insertion into vector
↓
Transformation into host
↓
Cloning and expression
Plasmid (cut open)
+
Foreign DNA fragment
↓
Joined by DNA ligase
↓
Recombinant plasmid
Genes used to identify cells that have taken up vector DNA
Common markers:
Antibiotic resistance genes
Example: Ampicillin resistance
Identify cells containing desired recombinant DNA
Methods:
Colony screening
Hybridization probes
Blue-white screening
Based on lacZ gene activity
Mechanism:
Intact lacZ → β-galactosidase produced → blue colonies
Disrupted lacZ (recombinant) → no enzyme → white colonies
👉 White colonies = desired clones
Technique to separate DNA fragments based on size
Uses:
Agarose gel
Electric field
Principle:
DNA is negatively charged → moves towards anode
Smaller fragments move faster
Determination of exact nucleotide sequence
Common method:
Sanger sequencing
Identifies locations of restriction enzyme sites
Helps in:
DNA analysis
Construct verification
Identification based on DNA pattern differences
Uses:
Variable number tandem repeats (VNTRs)
Applications:
Forensic identification
Paternity testing
Detection of DNA fragments using radioactive labeling
Produces visible bands on film
| Feature | Selection | Screening |
|---|---|---|
| Purpose | Identify transformed cells | Identify recombinant clones |
| Basis | Survival (antibiotic resistance) | Detection of insert |
| Result | Growth or no growth | Color/marker change |
| Example | Ampicillin resistance | Blue-white screening |
| Feature | Description |
|---|---|
| Medium | Agarose gel |
| Charge of DNA | Negative |
| Movement | Towards anode |
| Separation | Based on size |
| Visualization | Ethidium bromide / UV light |
Well → DNA loaded
↓
Electric field applied
↓
Small fragments → move faster
Large fragments → move slower
↓
Bands formed
Top (large fragments)
│ █
│ █
│ █
│ █
Bottom (small fragments)
Sample DNA
↓
Fragmentation
↓
Gel electrophoresis
↓
Band pattern (unique)
Technique used to introduce a specific, targeted change in DNA sequence
Allows precise alteration of gene structure
Based on use of synthetic oligonucleotide (primer) containing desired mutation
This primer:
Binds to target DNA
Introduces mutation during replication
Most common method
Steps:
Design synthetic primer with desired mutation
Primer binds to template DNA
DNA polymerase extends strand
New DNA contains mutation
Mutant DNA is amplified
Study of gene function
Identification of active sites in proteins
Development of:
Vaccines
Therapeutic proteins
Protein engineering
| Feature | Random Mutagenesis | Site-Directed Mutagenesis |
|---|---|---|
| Specificity | Non-specific | Highly specific |
| Control | Low | High |
| Mutation site | Anywhere | Predefined location |
| Method | Mutagens (UV, chemicals) | Synthetic primers |
| Use | Screening studies | Functional analysis |
Template DNA
↓
Mutant primer binds (with mismatch)
↓
DNA polymerase extends strand
↓
New DNA contains mutation
↓
Amplification of mutant DNA
Technique based on complementary base pairing between nucleic acid strands
DNA or RNA strands with complementary sequences bind (hybridize)
Applications:
Detection of specific genes
Identification of pathogens
Short single-stranded DNA/RNA fragments
Labeled with:
Radioactive markers
Fluorescent dyes
Bind to complementary target sequences
Detects specific DNA sequences
Steps:
DNA extraction
Restriction digestion
Gel electrophoresis
Transfer to membrane
Hybridization with labeled probe
Detects RNA (gene expression)
Same principle as Southern blot
Used to identify bacterial colonies with desired DNA
Colonies transferred to membrane
Probe binds to target sequence
Detects nucleic acids within cells or tissues
Maintains structural localization
Uses fluorescent probes
Allows visualization under microscope
Applications:
Chromosomal abnormalities
Pathogen detection
| Feature | Southern Blot | Northern Blot | Western Blot | FISH |
|---|---|---|---|---|
| Detects | DNA | RNA | Protein | DNA/RNA |
| Technique | Hybridization | Hybridization | Antibody-based | Fluorescent probe |
| Sample | DNA fragments | RNA | Proteins | Cells/tissues |
| Output | Bands | Bands | Bands | Fluorescent signals |
| Type | Description |
|---|---|
| Radioactive probes | High sensitivity |
| Fluorescent probes | Safe, visual detection |
| Enzyme-labeled probes | Colorimetric detection |
DNA/RNA sample
↓
Gel electrophoresis
↓
Transfer to membrane
↓
Probe hybridization
↓
Detection of bands
Single-stranded probe
+
Target DNA sequence
↓
Complementary binding
↓
Signal detection
Technique to amplify specific DNA sequences
Produces millions of copies
Denaturation (95°C)
Annealing (50–65°C)
Extension (72°C)
Uses two sets of primers
Increases:
Specificity
Sensitivity
Amplifies multiple DNA targets simultaneously
Uses multiple primer sets
Measures DNA amplification in real time
Uses fluorescent signals
Applications:
Viral load detection
Gene expression
Uses dideoxynucleotides (ddNTPs)
Causes chain termination
Produces fragments of different lengths
Sequence determined by fragment size
Genome editing tool
Uses:
Guide RNA (gRNA)
Cas enzyme
Mechanism:
gRNA directs Cas to target DNA
Cas enzyme cuts DNA
DNA repaired → gene edited
Alteration of DNA sequence
Methods:
CRISPR
TALENs
Zinc finger nucleases
Introduction of functional gene to treat disease
Types:
Somatic gene therapy
Germline gene therapy
Diagnosis of infections
Genetic disease detection
Cancer research
Vaccine development
Forensic analysis
| Feature | PCR | RT-PCR |
|---|---|---|
| Template | DNA | RNA |
| Enzyme | DNA polymerase | Reverse transcriptase + DNA polymerase |
| Purpose | DNA amplification | RNA detection |
| Application | Gene detection | Viral detection |
| Type | Feature |
|---|---|
| Conventional PCR | Standard amplification |
| Nested PCR | Increased specificity |
| Multiplex PCR | Multiple targets |
| Real-time PCR | Quantitative detection |
| Feature | CRISPR | Traditional Methods |
|---|---|---|
| Precision | High | Moderate |
| Efficiency | High | Lower |
| Complexity | Simple | Complex |
| Cost | Lower | Higher |
Denaturation (95°C)
↓
Annealing (50–65°C)
↓
Extension (72°C)
↓
Repeated cycles → amplification
Guide RNA binds target DNA
↓
Cas enzyme cuts DNA
↓
DNA repair
↓
Gene modification
Bacteria produce enzymes that inactivate antibiotics
Example:
β-lactamase → destroys β-lactam ring
Seen in:
Staphylococcus, E. coli
Active transport systems that pump antibiotic out of cell
Decreases intracellular drug concentration
Seen in:
Tetracycline resistance
Alteration of antibiotic target site
Prevents drug binding
Examples:
Altered PBPs → β-lactam resistance
Ribosomal changes → macrolide resistance
Resistance genes carried on R plasmids
Can transfer between bacteria
Responsible for:
Multi-drug resistance
Resistance genes carried on transposons
Can move between:
Plasmid ↔ chromosome
Enhances spread of resistance
Occurs via:
Conjugation (most important)
Transformation
Transduction
Leads to:
Rapid dissemination
Hospital-acquired infections
| Mechanism | Description | Example |
|---|---|---|
| Enzyme production | Drug inactivation | β-lactamase |
| Efflux pumps | Drug removal | Tetracycline resistance |
| Target modification | Altered binding site | MRSA (PBP change) |
| Feature | Genetic (Acquired) | Chromosomal (Intrinsic) |
|---|---|---|
| Origin | Mutation or gene transfer | Natural |
| Transfer | Transferable | Non-transferable |
| Speed | Rapid spread | Slow |
| Example | Plasmid resistance | Cell wall impermeability |
Bacteria
↓
Acquisition of resistance gene
↓
Expression of resistance mechanism
↓
Survival in presence of antibiotic
↓
Spread to other bacteria
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