Foundations of Biochemistry — Molecular Genetics (Tutorial 6)

The Central Dogma describes the flow of genetic information through transcription and translation.
-DNA stores the original information; it is the "original recipe book".
Transcription: $\text{DNA} \rightarrow \text{mRNA}$
mRNA is a copy of the recipe you’re making.
Translation: $\text{mRNA} \rightarrow \text{protein}$
Protein is the final product, i.e., the food you made.
Overall: DNA (genetic information) -> RNA (transcript) -> Protein (functional product).

In prokaryotes (e.g., bacteria) there are no introns and no mRNA splicing.
A gene is a region of DNA that provides instructions to make an RNA molecule.
A gene has a promoter where transcription factors bind to control transcription.
The promoter itself is not transcribed.
The transcribed region contains untranslated regions (UTRs).
Exons are the translated regions of a gene.
Introns are the untranslated regions that must be spliced out of the mRNA before translation.
Note: The figure in the slides is cited as taken from CELS191.

A gene is expressed or “turned on” when it is transcribed.
Transcription factors are proteins that bind to specific DNA sequences and control the rate of gene expression.
Different transcription factors exist in different cell types, leading to differences in which genes are turned on and under what conditions.
The process of controlling when transcription occurs is called gene regulation.

All cells contain the same DNA.
Gene regulation is essential to create cellular diversity.
Core gene expression: about $\approx 10{,}000$ genes are expressed across cells.
Cell-type-specific expression involves roughly $\approx 1000 \text{ to } 2000$ genes that define a particular cell type.
Different cell types express different transcription factors that control this differential gene expression.

After transcription, a gene needs to be turned into a protein via translation.
There are four unique nucleobases in DNA or RNA.
There are 20 unique amino acids.
The genetic code is read in triplets; amino acids are specified by codons (sets of three nucleotides).
Codons are universal across all life: this is the universal genetic code.
Translation process relies on tRNA matching codons with amino acids during protein synthesis.

A change in DNA sequence can alter the amino acid sequence of a protein.
Some changes are silent (do not change the amino acid) due to codon redundancy (e.g., TGT and TGC both code for cysteine).
Two main types of variation discussed:
- Point variation (single-nucleotide change)
- Frameshift variation (insertions/deletions that shift the reading frame)

Monogenic diseases (Mono + genic): caused by variation in a single gene; often dramatic loss of function or functional disruption; rare (~6% of people).
Polygenic diseases (Poly +genic): influenced by many genes; most traits/diseases are polygenic; variants tend to alter protein function in smaller increments.
Environment can modulate risk in polygenic diseases.

PKU – Phenylketonuria
- Inherited in a recessive manner.
- Requires two copies of the non-functional gene/protein for disease to occur.
Retinitis Pigmentosa
- Inherited in a dominant manner.
- Requires one copy of the non-functional gene/protein for disease to occur.
Note: A diagram of Dominant vs Recessive inheritance is provided in the slides.

Cause: Loss of function of phenylalanine hydroxylase enzyme.
Consequence: Inability to convert phenylalanine (Phe) to tyrosine (Tyr).
Genetic variants: > 950 different possible variants can cause PKU.
Biochemical consequence: Accumulation of Phe and decreased Tyr.
Clinical outcomes: Intellectual disabilities, seizures, behavioral problems, and mental disorders if untreated.
Management: Special diet to keep phenylalanine levels in the bloodstream from becoming excessive and reduce symptoms.

Cause: Rhodopsin receptor gain of function.
Effect: Receptor becomes overly active, causing signaling when it should not occur.
Location: Rod cells in the retina; responsible for detecting light.
Consequence: Night-blindness that can progress to complete blindness.
Mechanism: Receptor contains an “ionic lock” that normally keeps it inactive in the absence of light; variants that interrupt this lock increase receptor activity.

Purpose: Genetic tests determine an individual’s genotype at a particular gene.
A common method is RFLP (Restriction Fragment Length Polymorphism) using PCR, restriction enzymes, and gel electrophoresis to determine genotype.
Genotype: the combination of alleles an individual has for a particular gene.

PCR (Polymerase Chain Reaction) amplifies DNA.
Steps:
1) Heat DNA to $95^{\circ}\text{C}$ to denature strands.
2) Cool to about $60^{\circ}\text{C}$ to allow primers to anneal.
3) Heat to $72^{\circ}\text{C}$ for extension by Taq DNA Polymerase.
4) Repeat steps 1–3 to obtain many copies of DNA.
Taq DNA Polymerase: heat-stable DNA polymerase from Thermus aquaticus.

Restriction enzymes are bacterial proteins that cut DNA at specific sequences.
They serve as a bacterial defense mechanism.
Each enzyme recognizes a specific sequence and cuts at a defined base position.
Recognized sequences are short (4–6 bases) and palindromic (reads the same 5'->3' forward and reverse).

A specific variant (MODY2) creates a new restriction site that is recognized by HindIII.
DNA containing the MODY2 variant will be cut by HindIII; DNA without the variant will not be cut.
Gel electrophoresis visualizes different fragment patterns, illustrating genotype.
Practical experience: an example will be performed in Lab 4.

Cancer is a collection of related diseases where cells fail to respond to signals that regulate growth and death, leading to uncontrolled proliferation.
It results from an accumulation of mutations, many arising during DNA replication.
Mutations in cancer are somatic mutations (not population-level natural variation) and accumulate with age due to more cell divisions.

Genes that encode proteins promoting cell growth and division.
Proto-oncogenes are normal versions with the potential to become oncogenes when mutated.
Oncogenes typically arise from dominant gain-of-function mutations that promote growth when not appropriate.

Genes encoding proteins that prevent uncontrolled cell growth by inhibiting cell division.
Mutations are recessive and involve loss of function.
To lose function, both copies (alleles) of the tumor suppressor gene must be inactivated (two-hit hypothesis).
Absence of tumor suppressor activity can relieve restraint on cell growth.