Gene Cloning Tools: cDNA and PCR

• Key tools in gene cloning include preparing cDNA, cloning vectors, enzymes, PCR, and host cells.

cDNA Preparation

• mRNA is extracted from a sample, then reverse transcriptase produces a single-stranded DNA molecule complimentary to the mRNA.
• DNA polymerase synthesizes a second DNA strand, creating a double-stranded cDNA molecule.
• cDNA contains only coding sequences (exons) of a gene, unlike DNA, which contains both coding and non-coding sequences.
• mRNA is needed for cDNA preparation.
• cDNA starts as single-stranded, then is converted to double-stranded using DNA polymerase.

Polymerase Chain Reaction (PCR)

• Rapidly produces millions to billions of copies of a specific cDNA.
• Amplifies cDNA for storage in cDNA libraries or for gene expression studies.

Features of cDNA

• Contains only coding sequences (exons)
• Used in gene cloning and other research experiments.

DNA Vectors

• DNA carriers that transport desired DNA fragments into a host cell for replication and/or expression
• Features include origin of replication, restriction sites, and a selectable marker.
• Plasmids are the most important vectors as they can infect bacteria and other cells and organisms.
• Phages can only infect bacteria not humans
• Retroviruses are mostly used for research for genetic problems

Restriction Enzymes

• Recognize and cut DNA at specific sequences called restriction sites
• Produce fragments with blunt ends (cut at the same position) or sticky ends (overhangs).
• In bacteria, restriction enzymes serve as a defense mechanism against invading viruses (bacteriophages).
• Blunt ends are palindromic.
• Sticky ends can easily pair with a complementary sequence.
• Over 600 different restriction endonucleases are commercially available, with EcoR1 being a common example.
• DNA fragments resulting from restriction enzyme digestion are called restriction fragments.

Function of Vectors and DNA Ligase

• A vector is a DNA molecule (often plasmid or virus) used to carry a particular DNA segment into a host cell for cloning or recombinant DNA.
• Restriction enzymes cut the vector, creating sticky ends for DNA fragment insertion.
• DNA ligase catalyzes the formation of covalent phosphodiester linkages between sticky ends, sealing gaps between nucleotides.
• After ligation, the complete plasmid can be transformed into bacterial cells for propagation.

Plasmids and Antibiotic Resistance

• Plasmids carrying antibiotic resistance genes allow easy detection of plasmid-containing bacteria on selective media.
• Ensures only cells with the desired genetic modification will survive.

Role of Restriction Endonucleases in Nature

• Prevent replication of bacteriophages' DNA by cutting it into many pieces.
• Named for their ability to restrict, or limit the number of strains of bacteriophages that can infect a bacterium.

Bacteria as Hosts for Recombinant DNA

• Bacteria and yeast are widely used in molecular cloning due to their small genome size and rapid reproduction time.
• Both can carry and amplify foreign DNA as circular DNA plasmids that replicate independently.
• Bacteria are easy to culture, grow fast, and produce high yields of recombinant protein.

DNA Recombination Products

• Antibiotics
• Blood clotting factors VII and IX
• Hormones (insulin, Erythropoietin)
• Growth Factors
• Interleukins
• Interferons
• Metabolic Enzymes
• Vaccines
• Monoclonal antibodies

Gene Cloning in Bacteria

• Double-stranded recombinant plasmid DNA is introduced into a bacterial cell.
• Cell culture produces hundreds of millions of new bacteria.

Transgenic Organisms

• Transgenic animals are animals that have had a foreign gene inserted into their DNA through DNA recombination techniques.
• Ethical considerations are involved in altering the genetic makeup of animals and plants.

DNA Probes

• Radiolabeled or fluorescently labeled single-stranded sequences of DNA or RNA complementary to a target gene

Hybridization Techniques

• Southern Blotting: study DNA
• Northern Blotting: study RNA
• Western Blotting: study proteins

Southern Blotting

• Detection and quantification of a specific DNA sequence in DNA samples.
• Purified DNA is digested with restriction enzymes, separated by electrophoresis, and transferred to a nitrocellulose membrane.
• The membrane is exposed to a DNA probe labeled with a radioactive, fluorescent, or chemical tag.
• Allows visualization of DNA fragments containing complementary sequences to the DNA probe.

Northern Blotting

• Used to study gene expression by detection of RNA (or isolated mRNA) in a sample.
• Electrophoresis is used to separate RNA samples by size and detection with a hybridization probe complementary to part of or the entire target sequence.

Western Blotting

• Used to detect specific proteins in a sample of cell homogenate or extract.
• Involves sample extraction, electrophoresis, membrane transfer, labeled probe incubation, and target detection.

Agarose Gel Electrophoresis

• Agarose and buffer solution is poured into a plastic tray with a comb on one end.
• DNA samples colored with a tracking dye are pipetted into the wells.
• The tray is placed in a chamber that generates electrical current through the gel.
• DNA has a negative charge and moves towards the positive electrode.
• Smaller DNA molecules travel faster through the gel.
• One well contains a DNA ladder for size comparison.

Bacterial Pathogenicity

• Pathogenicity: the ability of a microorganism to cause disease in a host.
• Directly related to bacterial morphology and lifestyle.
• Morphology: G+ vs G- Bacteria; Cocci vs Bacilli
• Lifestyle: Extracellular vs intracellular bacteria; Aerobic Metabolism vs anaerobic metabolism
• Pathogenicity: a qualitative term indicating whether the microorganism can cause disease.

Important Definitions for Pathogenicity

• Pathogen: a microbe that can cause disease.
• Host: the organism being infected.
• Pathogenicity: the ability to cause disease.
• Opportunistic Pathogen: a microbe that infects an immunocompromised host.
• Reservoir: the natural home for a pathogen. May be animate (human or animal) or inanimate (fomite – water, soil, food).
• Carrier: hosts that carry pathogens and show no obvious signs and symptoms of infection.
• Zoonosis: an infection that primarily occurs within animal populations but can be spread to humans.
• Nosocomial Infection: a hospital-acquired infection.
• Mode of transmission: the mechanism by which a pathogen is picked up by a host.
• Bacteremia: bacteria in the bloodstream, which can quickly spread.
• Septicemia: outcome of systemic bacteremia which can result in septic shock and death

Modes of Transmission

• Direct contact: Host to host contact (kissing or sexual intercourse)
• Vertical transmission: Mother to infant transfer of a pathogen, either before or immediately after birth
• Droplet transmission: the infected host expels pathogens in tiny droplets by coughing or sneezing, which are then inhaled by a host nearby
• Indirect contact involves transfer of infectious agent through some type of intermediary, such as a contaminated object or person. The pathogen might be deposited on an inanimate object called a fomite which is then used by another person
• Fecal-oral transmission: pathogens are transmitted from an infected person’s feces to the mouth of another person.

Virulence Factors

• Microbial structures that contribute to virulence.
• Genes coding for virulence factors are found on the pathogen's chromosome or plasmid DNA, called pathogenicity islands.
• Pathogenicity islands facilitate the sharing of virulence factors between bacteria due to horizontal gene transfer leading to the development of a new pathogen over time.
• Virulent: describes a pathogen that is relatively toxic or capable of causing relatively more harm to the health of its host compared with other pathogens.

Pathogenicity Island

• A collection of genes on a chromosome and/or plasmid that helps the bacterium to cause disease
• Encodes virulence factors and antibiotic resistance enzymes.
• Transferred through horizontal gene transfer (conjugation), transduction, and transformation

Examples of Virulence Factors

• Adherence Factors (capsule, pilus, flagellum) which provides resistance to host phagocytosis

Invasive Enzymes

• Collagenase: allows the pathogen to spread by breaking down the collagen found in connective tissue.
• Leucocidins: destroy the host’s white blood cells, decreasing resistance.
• Hemolysins: lyse the host’s red blood cells.

Bacterial Toxins

• Exotoxins: released/secreted outside of the cell

  • AB toxins (ALWAYS PRODUSE GRAM POSITIVE)
  • Superantigens
  • Proteins
  • Highly immunogenic
  • Symptoms: cytotoxin, enterotoxin & neurotoxin
  • G+ diseases: such as botulism, diphtheria, and tetanus

• Endotoxins

  • Lipopolysaccharides (LPS)
  • Within the cell wall of the bacteria ALWAYS PRODUCE GRAM NEGATIVE Weakly immunogenic
  • Symptoms: fever, diarrhea, and vomiting
  • A complex of lipids and polysaccharides
  • G- Disease: Septicemia, septic shock

Lipopolysaccharide (LPS)

• O-antigen, Core Polysaccharide, Lipid A which is attached to the core polysaccharide, each of these have an important role in the pathogenicity of Gram-negative bacteria.
• LPS is released during bacterial division or when the bacteria are destroyed.
• LPS causes toxic effects, including fever, multi-organ failure, septic shock, and sepsis

Superantigens

• G+ exotoxins that can cause a massive immune system dysfunction.
• Trigger the release of pro-inflammatory cytokines leading to uncontrolled inflammation.
• Produced mainly by Staphylococcus aureus and Streptococcus pyogenes.

Cytolytic Exotoxins

• Cytoplasmic membrane damaging toxins (MDTs) that cause pores/cytolysis.
• An example is hemolytic exotoxins

AB Exotoxins

• G+ exotoxins where the A segment is the active part that damages cells, while the B segment is. the adhesion/attachment part to the cell surface.
• An example is the tetanus toxin produced by the bacterium Clostridium tetani.
• B attaches to the target cell, A segment can enter the cell and cause damage
• The A subunit will cause a decrease in neurotransmitter release in neurons, resulting in spastic paralysis of the host
• B portion of botulinum toxin (produced by Clostridium botulinum) binds to acetylcholine receptors on the cardiac SA node cell surface, allowing the A portion to enter the neuron and block signal transduction at cardiac neuromuscular junctions causing myocytes weakness and/or paralysis

Viruses & Viral Infections

• Viruses are acellular, infectious nucleic acids, and obligate intracellular pathogens.
• Consist of a nucleic acid (DNA OR RNA) never both and is surrounded by a protein coat called a capsid.
• Some viruses have an envelope (lipoprotein) in addition to a capsid.
• They lack ribosomes & mitochondria.

Virion

• Complete, infectious unit of a virus
• Consists of a core of RNA or DNA enclosed in a coat (capsid and/or envelope).

Bacteriophages

• Viruses that infect and replicate ONLY in bacterial cells, do not infect human
• An important part of the human gut microbiome that attack pathogenic bacteria

Baltimore Classification of Viral Genomes

• Group I: DS DNA
• Group II: SS DNA
• Group III: DS RNA
• Group VI: +SSRNA
• Group V: - SSNA
• Group VI: SS RNA with RTase
• Group VII: DS DNA with RTase

Examples of Viruses by Baltimore Classification

• Group I: Double-stranded DNA viruses (adenoviruses, Herpesvirus)
• Group II: Single-Stranded DNA viruses (Parvovirus)
• Group III: Double-stranded RNA viruses (Reoviruses)
• Group IV: Positive-sense-single-stranded RNA viruses (Picornavirus)
• Group V: Negative-sense single-stranded RNA viruses (orthomyxoviruses)
• Group VI: Single-stranded RNA viruses with DNA intermediate (Retroviruses)
• Group VII: Double-stranded DNA viruses with an RNA intermediate (Hepadnaviruses)

Viral Replication Steps

• Attachment: attach to the cell
• Penetration: needs to get into the cell, usually via endocytosis, virus gets inside the host
• Uncoating: Needs to uncoat capsid and envelope because nucleic acid needs to be exposed
• NA replication: nucleic acid replication
• Transcription
• Translation
• Assembly: put them together to make a complete virus/virium
• Release: New viruses getting released from the host cell to infect other cells

Immunology

• Study of the immune system's ability to distinguish between self and non-self,
• Produce appropriate immune responses against non-self
• Keep the immunological memory of non-self for later attacks

The Immune Response

• The self’s way of defending against nonself based on recognition

Immune System Components

• Immune organs
• Immune cells
• Immune proteins

Physiologic and Pathological Responses of the Immune System

• Physiologic responses:
  • Proper non-self recognition
  • Effective immune responses
  • Complete elimination
  • Long-lasting remembrance
• Pathological Responses
  • Hyperactivity: Autoimmunity, Hypersensitivity
  • Hypoactivity: Immune deficiencies, Malignancies, Infections

Primary vs. Secondary Immune Organs

• Primary Immune Organs: Bone marrow (B cells originate here) and Thymus (T cells differentiate here)
• Secondary Immune Organs: Lymph node (T and B lymphocytes present)

Non-Self Classifications

• Obligatory intracellular
  • Mycobacterium tuberculosis
• Obligatory extracellular
  • Staphylococcus aureus
• Facultative intracellular
  • Listeria monocytogenes
• Opportunistic pathogens
  • Pseudomonas aeruginosa
• Transformed (mutated) pathogens
  • Methicillin-resistant staphylococcus aureus
• Infected and transformed cells: Liver cell infected with Hepatitis A virus and Cancer cells

Criteria of Nonself

• Antigenic Determinants (Epitopes): Markers on nonself used by the immune system to identify non-self
• Collection of markers on nonself that will be used by immune system to identity non self
• Antigenic determinants 2 groups: antigen and immunogen

Antigen vs. Immunogen

• Antigen Provokes weak or NO IRS, Can react with IRs products, Produces weak or NO memory
• Immunogen Can causes immunity, Provokes strong IRs, Can react with IRs products, Produces long lasting memory

Specific vs. Non-Specific Immune Cells

• Specific immune cells
  • B lymphocytes : From bone marrow, After 1st exposure can save memory, during 2nd exposure they can act quickly to fight against it
  • T lymphocytes: From thymus, After 1st exposure can save memory, during 2nd exposure they can act quickly to fight against it
• Nonspecific Immune cells:
  • Phagocytes & AG (antigen) presenting cells (APC) Does not save memory
  • Natural Killer Cells (NKC) Do not save memory

Specific Immune Cells

• The Key Feature: Immune memory, The ability to recognize and respond more effectively to pathogens that it has encountered before
• B-Lymphocytes (from bone marrow)
• T-Lymphocytes (from thymus)

Humoral (Antibody-Mediated) Immune Responses (HIR)

• B cell → activation → Plasma cell: differentiated form of B-lymphocytes
• Plasma cells make custom antibodies against antigens

Cell-Mediated Immune Responses (CMIR)

• T cell → activation → Th cells or Ts cells or Tc cells:
• T lymphocytes can become
  • T helper cells: Turn on immune responses, turn on immune responses
  • T suppressor cells: Turn off immune responses, turn off immune responses
  • T cytotoxic cells: Cytotoxic to: cancer cells pathogens infected cells Foreign transplant cells

Main Types of T Cells

• Cytotoxic T cells (CD8+): directly kill infected or cancerous cells by releasing toxic substances.
• Helper T cells (CD4+): turn on immune response (IRS)
  • Helper T1 and Helper T2.
• Suppressor T cells: Turn off IRS CD = cell differentiation marker

Non-Specific Immune Cells

• First line of immune defense, but no immune memory
• They respond in a general way to any pathogen
  • Ag presenting cells (APC): responsible to present antigen to the cells that turn on the immune response (T helper cells) = receive antigen and become active against it.
  • Phagocytes: When they see pathogen, they eat the pathogen.
  • Natural killer cells (NKC) Cytotoxic to: cancer cells, pathogen infected cells, foreign transplant cells

Types of Non-Specific INnune Cells

• Neutrophil → phagocytosis & antigen presentation: most important cells in 1st line of defense
• Basophil → Inflammation & Allergies
• Eosinophil → Anti-multicellular parasites
• Monocyte → when they migrate in our tissue they can become: dendritic cell, macrophage Macrophage same as monocyte = phagocytosis and antigen presentation
• NKC → Anti-viral & Anti-tumor cells

Non-Specific Immune Responses (Innate IRs)

• Phagocytosis/Pinocytosis
• Antigen Presentation
• NKC- mediated cytotoxicity
• Innate Lymphoid cells
• Complement-mediated cell lysis
• Cytokine-mediated responses

Phagocytes vs Pinocytosis

• Phagocytosis = eating
• Pinocytosis = drinking

Pattern Recognition Receptor

• Pattern recognition Receptor: Pathogen, Phagosome, Phagolysosome, Degradation, Degraded Pathogen (Ags)

Antigen Presenting Cells (APCs)

• Dendritic cells →Th-lymphocytes: In tissues
• Monocytes → Th-lymphocyte: In blood
• Macrophages → Th-lymphocytes: In tissues
• B-lymphocytes → Th-lymphocytes: Bone marrow
• Th-lymphocytes → Tc-lymphocytes: Thymus

Self/Nonself Recognition

• Via Major Histocompatibility complex (MHC), most important protein in immune response, only found in nucleated cells of humans and animals
• Types of MHC: MHC-I & MHC-II
• MHC-I is self-marker found on the surface of all nucleated cells -MHC-II is communication marker found on the surface of immune cells,if one is carrying that communicating marker (MHC-I) you can communicate via MHC-II
• MHC-I protein on nucleated cells interacts exclusively with CD-8 protein on cytotoxic T-lymphocytes

MHC-I Self

• Cytotoxic T cells (CD8+)
• No antigen presented = no reaction, if antigen is present = reaction -CD8 protein is on cytotoxic T cells.

Immune Cell Communication

• MHC- II on professional immune cells (APCs, B- lymphocytes & T-Lymphocytes) interacts exclusively with CD- 4 protein on helper T- Lymphocytes
• Helper T, has CD4 protein CD 4 protein interacts exclusively with MHC 2

How Th1 and Th2 are created

• Antigen presenting cell (APC) triggers T cell differentiation aided by cytokine: interleukin-1 secreted by APC
• Interaction between MHC- II on antigen presenting cells and CD-4 protein on Th will activate Th to differentiate into Th-1 & Th-2

Humoral Immune Responses

• Immune responses triggered by B-lymphocytes and antibodies
• Antibody-dependent immune responses (Ab-dependent IRS)
• Th2 triggers HIRs aided by interleukin-2 secreted by Th2: Interleukin-2 presents antigen, once presented it turns into a plasma cell which make antibodies to destroy the antigen.
• B-lymphocytes will become activated (B → PC) against nonself

Important Proteins

• IL-1 = made by antigen presenting cells (APCs), Stimulates stronger interaction between APCs and Th cells.
• IL-2 = made by T helper cells (Th), Stimulates stronger interaction between B lymphocytes and Th cells

Cell-Mediated (CM) Immune Responses

• Triggered by Th cells,Th-1 triggers CMIRs aided by Interleukin-2 secreted by Th1
• Cytotoxic T-lymphocyte will differentiate into effector cytotoxic cells to produce:
  • Perforin: forms a pore for delivery of granzyme
  • Granzymes: induce cell death in infected cells and/or cancer cells
• With perforin forming a pore, granzymes can be injected into that cell and explode that infected/cancer cell

Immune Proteins

• Specific (Adaptive), comes with memory
  • Immunoglobulins (lg) or Antibodies (Ab) Globulins = proteins
• Non-specific (innate) -Cytokines Interleukin 1 Interleukin 2 Interferons
  • Complement proteins -Immune regulatory proteins

Immunity

• Specific immune protein
• Antibodies (Ab)
• Immunoglobulin (Ig)

5 Classes of Immunoglobulin

• Igm: largest/shortest lifetime during primary immune responses
• Ig g: Longest lifetime -produced during secondary Immune responses. IG G is the ONLY class of Ab which can pass the placenta
• Ig a: Secretions: Only antibody which can only be transmitted to the new born through breastfeeding
• Ig E→ Allergic reactions
• Ig d→ we don't know what this antibody does Ig M and Ig G measured in blood circulation ,Ig M recently infected Ig G is from a while ago Each class of immunoglobulin is responsiblePentamere: 5 subunits of

Antibody Structure

• Antibody structure is Y shaped -2 light chains = light chains have fewer amino acids, 2 heavy chains = more amino acids -Antigen binding portion: Pathogen binding site, 1 LC, 1 HC Difference in these pathogen binding site -specific immunity Pathogen binding site - also called antibody specificity portion Each antibody is specific for each antigen FC portion lower part of the Y -Made up of only 2 heavy chains-FC receptor
• Who has FC receptor? -Phagocytes: Can do phagocytosis without antibody But with the help of antibody, more active and is faster 2 form of phagocytosis Independent of antibody Dependent of antibody
• NKCs (Natural Killer Cells) -Basophils/Mast cells

FC receptor

• Phagocytes: Can do phagocytosis without antibody = is faster.
• NKCs (Natural Killer Cells): Can do cytotoxicity without antibody = cytotoxicity enhances more in antibodies
• Responsible for cell-mediated toxicity
• Basophils/Mast cells: Have allergens instead of antigens: Basophils in blood - Mast cells in connective tissue Allergens are antigens which cause allergic reactions.

Immune Response vs. Time

Following pathogen injection in a mouse:

• Week 0 → Week 1 : No immune response
• Week 1: Immune response starts
• Week 2: declines quick to 0 after week 2

1. Latent/Lag phase (first exposure): Time the immune system is using to recognize and identify that pathogen
2. Immune response phase :Immune system is making antibodies here
3. Decline phase :Level of antibodies measured, decreases until it gets back to 0 Following second exposure of pathogen

Following Second Exposure of Pathogen

When immune responses have declined back to 0 at Week 2

1. Latent phase is very short
2. Immune response phase is very long and stronger (higher level of antibodies)

• Decline phase is shorter and doesn’t reach 0
• 1st exposure -Immune response is not that strong-Immune system needs to take time to identify this new pathogen and make the antibodies to fight against it = takes time-IgM
• 2nd exposure- Alot faster- Alot stronger- Immune system recognizes the pathogen - can react faster and stronger- IgG