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

Cell Entry
- Endocytosis: The cell membrane engulfs the virus.
- Fusion (for enveloped viruses): The viral membrane fuses with the cell membrane.
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.
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.