Class Notes

Exam Information and Southern Blot

• Exams are being graded and will hopefully be returned early next week.
• The class will be curved at the end of the semester after final exams.
• The class average usually ends up around a B or B-.
• Solutions to the exam will be distributed when the tests are returned.
• The key technique discussed this week is the Southern blot.

Southern Blot

• Named after Mr. Southern, the scientist who developed it.
• The first of the "blots".
• Used for separating DNA on a gel.
• Other blots:
  • Western blot: separates protein on a gel.
  • Northern blot: separates RNA on a gel.
  • Eastern blot: related to DNA modifications.
• Process:
  • Cut up DNA into pieces using a restriction enzyme.
  • Separate the fragments on a gel.
  • DNA is negatively charged, so it runs down the gel towards the positive end.
  • Smaller bands travel further, indicating smaller DNA fragments.
  • Probes are used to identify specific sequences within the fragments.
  • Complementary fragments to specific sequences (e.g., V or C sequence) are used to locate those sequences on the gel.

VDJ Recombination

• Southern blot was used to investigate VDJ recombination.
• Unique result: B cells from different individuals show different patterns of bands, unlike other genes.
• VDJ recombination allows for a large number of different antibodies to be made with a limited amount of genetic material.
• Building blocks: different V, D, and J options are combined to create antibody sequences.
• VDJC includes the constant region for the heavy chain.
• VDJ without C refers to the light chain.

DNA Visualization and Southern Blotting

• Southern blotting is a different way of visualizing DNA structure compared to crosses and progeny analysis.
• Crosses and progeny analysis: Used to determine what genes and alleles are present, and recombination mapping reveals chromosome structure.
• Southern blot provides even more information, but requires knowledge of how to label DNA.
• DNA probe: a short sequence of A, T, G, or C, complementary to the DNA target.
• Southern blot was developed in 1975.
• Southern blotting allows us to learn that DNA is restructured between immature and mature B cells.

Immune Response

• When we get sick, we go through an immune response.
• Graph:
  • Y-axis: Antibody concentration in blood (micrograms per milliliter).
  • X-axis: Time in days.
• Day 0: Exposure to antigen.
• Even before exposure, there is a low level of antibodies against the antigen.
• After about 6 days, antibody levels start to increase.
• Around 12 days, antibody levels reach a maximum and remain high for about a week.
• Antibodies prevent the virus from growing. They attach to targets, signaling for immune cells to destroy them.
• When antibody levels are high, you get well.
• After recovery, antibody levels drop but plateau at a lower level (around 10^{-1} micrograms per milliliter), which is still higher than the initial level.
• Secondary exposure: A faster and stronger antibody response occurs.
• The response is fast and strong enough that you don't get sick the second time.

Primary vs. Secondary Immune Response

• Primary response: The initial immune reaction to an antigen.
• Secondary response: A faster and stronger immune reaction to the same antigen upon re-exposure.
• Vaccination: Either prevents illness or results in a weaker response due to pre-existing immunity.

B Cell Development and Function

• Immature B cells undergo VDJ recombination to become mature B cells.
• Mature B cells express a specific sequence for encoding antibodies (IgM) on their surface.
• Immature B cells cannot express antibodies because they haven't formed the sequence yet.
• Before antigen exposure, mature B cells with antibodies attached to them are circulating.
• Antigen detection: When the antigen enters the body, some B cells latch onto it and recognize it.
• Class switching: Once an antigen is detected, B cells undergo class switching.
• This process is assisted by helper T cells.
• B cells start to secrete antibodies (IgG) into the bloodstream.
• These cells become plasma cells.
• The number of plasma cells increases significantly, leading to a rise in antibody levels.
• After recovery, the number of plasma cells decreases, but some remain as memory cells.
• Because the process of class switching and B cell amplification has already occurred, the response to a secondary exposure is much quicker.

Polyclonal vs. Monoclonal Antibodies

• Polyclonal antibodies:
  • For every antigen, there are multiple different antibodies that can bind to it.
  • Each antigen has different spots where an antibody can bind.
  • A natural immune response generates a polyclonal response.
  • Generated from more than one B cell.
• Monoclonal antibodies:
  • Produced from a single B cell clone.
  • Typically created when humans intentionally replicate an antibody.
  • Example: During COVID, antibodies that stick to the spike protein were isolated.
  • The B cell that produced that antibody was replicated in culture.
  • The antibodies were collected and delivered to patients.

Antibody Uses and Southern Blotting Relevance

• Monoclonal antibodies are used in laboratory settings for staining to identify specific proteins in cells.
• The Southern blot method is relevant in cancer research as a way to visualize DNA and identify cancer mutations.

Viruses: General Structure and Characteristics

• Viruses carry genetic material (genes) to replicate.
• Capsid: A protein or lipoprotein shell that protects and delivers the genetic material.
• Genetic material: Either DNA or RNA.
• Viruses are typically simple, containing as few as 10 proteins or up to a hundred.
• Viruses cannot replicate without a host.
• Host provides:
  • Energy (ATP) for viral replication.
  • Machinery to produce proteins and run reproduction.
• Viruses are not considered living because they cannot self-replicate.
• Viruses can evolve through mutation and heredity, similar to living organisms.

Types of Viruses

• Open structure: Rod-shaped.
• Closed structure: Ball or soccer ball-shaped.
• Enveloped: Has a lipid bilayer, like our cells.
• Non-enveloped: Lacks a lipid bilayer.

Common Viruses

• Influenza:
  • RNA virus (minus strand).
  • Enveloped.
• HIV:
  • Positive-strand RNA virus.
  • Spherical and enveloped.
• COVID:
  • Positive-strand RNA virus.
  • Helical and enveloped.

Enveloped vs. Non-Enveloped Viruses

• Enveloped viruses:
  • Have a lipid shell that can be disrupted by soap (surfactant).
  • Washing hands with soap is effective against enveloped viruses.
  • Easily break down when dried out.
• Non-enveloped viruses:
  • Can survive on surfaces for extended periods.
  • Example: Norovirus, which can persist on surfaces like those found on cruise ships.

Viral Life Cycle

1. Cell Entry
   • Endocytosis: The cell membrane engulfs the virus.
   • Fusion (for enveloped viruses): The viral membrane fuses with the cell membrane.
2. Synthesis
   • DNA Viruses:
     • Use existing cellular mechanisms to replicate viral genes (DNA to DNA).
     • Use existing mechanisms for DNA to RNA to protein, to make the viral proteins for the coat.
     • Then the viral protein and the viral genes assemble into new virus.
   • Positive-Sense RNA Viruses:
     • Can directly make proteins.
     • To replicate genes:
       • Use RNA-dependent RNA polymerase to make the negative strand (complement).
       • Then copy it again to make positive-strand RNA.
   • Minus-Strand RNA Viruses:
     • Copy to positive strand, RNA and then use same method described above.
   • Retroviruses (e.g., HIV):
     • Use reverse transcriptase to make DNA from RNA.
     • The DNA integrates into the host genome (lysogeny).
     • The virus can replicate along with the cell's DNA without immediately making new virus.
     • When the virus wants to make new virus:
       • DNA integrated into the host cell is used to make RNA and protein (viral protein).
       • Then new virus particles are assembled.
3. Release
   • Budding (for enveloped viruses): Viral proteins go to the cell membrane and bud off, forming new viral particles without destroying the cell.
   • Cell Lysis (for non-enveloped viruses): The virus makes many copies of itself in the cell, then destroys the cell to release the viral particles.

Viral Replication Summary

• Viruses enter cells, make copies of themselves, and then exit to spread to other cells.

Why New Vaccines Every Year?

• Some vaccines, like MMR or chickenpox, only need to be administered once.
• Flu and COVID vaccines require annual updates.

Antigenic Drift

• Antigenic drift: Gradual accumulation of mutations in viral genes.
• Influenza has HA and NA glycoproteins on its surface.
• COVID has the spike protein on its surface.
• We develop antibodies against these proteins.
• Influenza and COVID are RNA viruses.
• RNA-dependent RNA polymerase has a higher error rate than DNA polymerase.
• Mutations in viral RNA lead to changes in the surface proteins.
• Antibodies become less effective over time due to these mutations.

Antigenic Shift

• Antigenic shift: Abrupt, major change in a virus, often due to viral recombination.
• Zoonotic infection: A virus that can jump from animals (e.g., birds) to humans.
• Viral recombination: When a host cell is infected with both human and non-human viruses, the viruses can recombine their genetic material.
• This can create a new virus with the worst qualities of both viruses (e.g., bird flu genes with human surface antigens).
• COVID is an example of antigenic shift.

Bird Flu Concerns

• Current concerns: Bird flu potentially jumping to humans.
• Bird flu has a high fatality rate (e.g., 52% in previous outbreaks).
• Urging people to get flu shots to prevent recombination events.

Homework

• Homework will be posted on Southern blotting and VDJ recombination.