Lecture Notes on DNA, Genetics, and Infectious Diseases

The genome comprises a large circular chromosome and plasmids.
The information within the genome dictates the organism's potential.
Genomes are fluid and can undergo changes, which is crucial for infectious diseases.
Sharing of genetic information among bacteria can lead to new or enhanced pathogens, particularly concerning antibiotic resistance.

Proteins play a crucial role in packaging DNA within cells.
HU proteins are important for DNA packaging in bacteria and act like bacterial histones.
DNA strands are wrapped around histones (or HU in bacteria) and folded to create chromosome structures.

Base pairing is fundamental to DNA function.
The sequence of bases dictates the function through RNA and protein.
To access the information, DNA strands must be separated.
The analogy of packing a backpack for a trip illustrates how DNA is tightly packed but still accessible for processing.
Specific signals allow strands to be pulled out, processed, and returned.

Genes are segments of DNA that serve as sentences with specific functions.
Epigenetics demonstrates how factors other than nucleotide order control information content.
Example 1: Mouse pups that are licked by their mothers have altered methylation of the glucocorticoid receptor gene, leading to reduced stress. Licking removes methylation, activating the gene.
Example 2: The Agouti gene in mice can cause obesity and diabetes. Feeding the mouse vitamin B12 and folic acid changes the methylation state, turning off the gene and reversing the adverse effects.
Epigenetics can be inherited and is influenced by environmental factors (e.g., famine, abuse, smoking).
Smoking causes targeted modifications on genes that increase the likelihood of cancer.
Epigenetic modifications, not changes in DNA sequence, control gene expression (on or off).
This regulation is crucial for virulence; pathogens activate certain genes based on environmental conditions (e.g., temperature).
Yersinia pestis regulates genes in the flea throat to block swallowing at 28 degrees Celsius, enhancing transmission. In the mammalian host at 37 degrees Celsius, different genes are activated to cause disease.

DNA replication relies on base pairing (A with T, G with C).
The process involves opening the DNA, adding complementary bases, and reassembling the strands.
Accuracy in replication is crucial to maintain the correct function; errors can lead to altered protein functions.
Enzymes involved in replication can recognize bumps caused by incorrect base pairings (e.g., double ring pairing with a double ring) and correct them.
This proofreading ensures accurate reproduction.

New bases are added to the three prime (3') hydroxyl end.
When synthesizing a new strand, the direction is from the five prime (5') end to the 3' end; reading occurs from 3' to 5'.
Chemistry facilitates base pairing, with phosphate separation providing energy for bond formation.
Polymerases facilitate the connection of bases during DNA replication.
Proteins keep strands separated.
Helicases unwind the DNA strands.
The rate of replication is fast: 50 bases per second in human cells and 500 bases per second in bacteria.
The error rate is low due to proofreading mechanisms.

A balance between perfect replication and errors is essential for adaptation.
Without the potential for change, organisms cannot adapt to new environments (e.g., antibiotic resistance).
Mutations are random and not induced by the antibiotic itself.
Bacterial evolution is clonal and rapid, leading to quick displacement of less adapted organisms. A slight advantage allows certain bacteria to wipe out the rest of the population in the area.
Too many errors can break the cellular machinery, leading to non-viable organisms.
Housekeeping genes, essential for survival, must function correctly.
Selection also plays a vital role because defective organisms are displaced by functional ones.

Sanger sequencing is a baseline method used for various applications but has limitations (800 bases at a time).
It relies on DNA replication principles.
The process involves single-stranded DNA, primers, DNTPs, and dideoxynucleotides (ddNTPs).
ddNTPs are chain terminators, labeled with different colors for each base (A, T, G, C).
AZT (azidothymidine) is an example of a chain terminator used to treat HIV; it lacks a 3'-OH group, preventing further DNA synthesis.

HIV's life cycle involves attachment to CD4 receptors, fusion with the cell membrane, and reverse transcription of RNA into DNA.
Reverse transcriptase preferentially incorporates AZT, terminating DNA synthesis.
Protease inhibitors block the cleavage of polyproteins into functional proteins.
Multiple drug therapies targeting different stages of the viral life cycle are used to manage HIV.
COVID-19 (SARS-CoV-2) also involves a protease that cuts polyproteins; drugs like Paxlovid inhibit this protease.

DNA is copied using a primer and DNTPs, with a few ddNTPs.
When a ddNTP is incorporated, synthesis stops, and the fragment is labeled with a color.
Fragments of different lengths are generated, each ending with a colored ddNTP.
These fragments are separated by size using gel electrophoresis.
A camera detects the colors as the fragments pass through the gel, determining the DNA sequence.
Gel electrophoresis separates DNA fragments by size, with smaller fragments moving faster.
DNA has a negative charge due to the phosphate backbone, facilitating movement through the gel.

The case involves a 42-year-old female and a 36-year-old male from Rhode Island with fever, rash, diarrhea, and bleeding gums/urine.
Both had organ transplants three weeks prior and were on immunosuppressive drugs.
An 8-year-old girl from Massachusetts with a lung transplant also showed similar symptoms, including neurological issues and retinal lesions.
A 25-year-old male from Massachusetts with a kidney transplant exhibited mild diarrhea and petechiae.
The first three patients died, and autopsies revealed hepatocellular necrosis.
Blood and tissue samples were sent to the CDC for analysis, which involved special staining and reverse transcriptase PCR.
The investigation identified Lymphocytic Choriomeningitis Virus (LCMV), a rodent-borne viral infection.
The virus was linked to a recent hamster acquisition by a family member of the donor.

The family member who was the caretaker of the hamster tested positive for antibodies to LCMV.
The hamster itself tested positive for LCMV via viral isolation, RT-PCR, and immunohistochemistry.
Genetic sequencing confirmed that it was the same virus.
The hamster was traced back to a pet store in Rhode Island, which led to a wholesaler in Ohio.
The outbreak was linked to wild rodents accessing the wholesale facility and transmitting the virus to pet animals.
Shipping records were used to contact affected pet stores and purchasers of the animals.
If immunocompromised or severely stressed, they will show symptoms. The ones who are only carriers are called hypoallergenic and can be asymptomatic.
A case of rabies virus transmitted through organ donation was mentioned as a similar example, noting also that some viruses have this tropism for nervous tissue.