mutations and genetic engineering
Environment, Gene Regulation & Evolutionary Pressure
gene-expression regulation: Short-term / minor environmental shifts
genetic change: Long-term / permanent shifts →
Mutation (universal option)
Horizontal gene transfer (restricted to unicellular organisms)
Natural-selection paradigm
Mutant alleles pre-exist in populations → Environmental pressure selects which variant succeeds Illustrative examples
Antibiotic plate: susceptible \rightarrow die, resistant minority \rightarrow dominant; remove drug + time ⇒ susceptible regain dominance (energetic cost of resistance)
Island bird beak tale: thick-beak (nut eater) vs thin-beak mutant; volcanic eruption kills fruit trees → thin-beak lineage out-competes; underscores selection, not directed mutation
Genetic Change in Bacteria
Mutations
Spontaneous (random polymerase “typos”) – trillions of mitoses/day in humans
point mutation: single base pair change
most common
mutation during replication so offspring has one with change (mutant) one without (wildtype) → called vertical gene transfer
outcomes: (increasing severity)
silent mutation (same amino acid)
missense mutation (diff amino acid, least predictable outcome of all)
nonsense mutation (prd. stop codon = truncated / shortened protein)
deletions / additions: of nucleotides
impact depends: # deleted & location
outcome:
frameshift mutation → often nonsense mutations (don’t work)
Induced by mutagens (chemical, physical)
defn: anything that mutates base pairs
HGT
Mutagens
chemical mutagens
Base Modifiers (most potent bc can change at any point)
Covalently alter existing bases and will modify existing base pair to induce mutation without replication event
→ chemical alkylating agents causes the base pair to slightly change form → cell repair mechanisms will see → swap to new base pairs (opposite, so originally GC, now AT)
clinical use: ethylene oxide
package equipment ➔ flood chamber ✓ EO gas ✓ 2–3 h ➔ mutations overwhelm microbes
not used anymore! not safe! : strong carcinogen
cause substitutions
Base Analogs
Structural mimics incorporated during replication only and can cause binding to the wrong base
not much practical implication
cause substitutions
Intercalating Agents
chemicals that insert into the double helix and cause strain → breaking backbone → deletion / addition mutations (the only chemical mutagen that can do this!)
extremely toxic thus not so potent! however the ntercelating agent itself will halt the transcription and thus is lethal
ex: methylene blue!!!!
radiation mutagens
Non-ionizing Radiation (UV)
problem: penetration
microwave, visible light, etc → low energy
covalently bonds adjacent thymines from the same strand (like their holding hands) → polymerase cannot pass this point so no transcriptions
distorts molecule… (does not actually damage)
bacteria can repair with photolyase by using visible light as catalyst (thus close curtains)
UV and human health (as E increased, penetration lowers)
UV-A: lowest energy so can penetrate deeper into the body and cause damage
UV-B: dense irregular CT → “ sun-aging”
UV-C: cannot penetrate skin (worst is sunburn)
only one used in clinical work bc safe!
Ionizing Radiation
problem: containment
so much energy so extremely dangerous → break that backbone of DNA (single and double strands) and can happen in multiple places uncontrolled → lethal
X-rays
cannot penetrate to sterilize the metal of a needle
Gamma Rays
can penetrate to sterilize metal needle
need extreme protective measures for safety so only good in industry use
clinically: diagnostic imagining, sterilization in industrial context!
Consequences of Mutations – General Framework
A single mutation can be:
Neutral (no phenotypic effect).
Deleterious (truncated or inactive product).
Adventitious/beneficial (helpful).
Real-world biology is rarely “all good” or “all bad”; many mutations show mixed effects depending on environment or genetic context.
ex: sickle cell anemia is both deleterious and adventitious bc resistant to malaria
Cancer: Core Concepts
Not “one disease”; rather thousands of genetically unique disorders and no patients have the same outcome
No single magic bullet therapy; heterogeneity prevents a universal cure.
Multi-hit model
At least two mutations required to initiate a tumor; usually hundreds accumulate.
Hallmarks of cancer
first 2 present in all cancers
Cell-Proliferation- cell cannot stop dividing
Apoptosis- cell apop. gene turned off so ignored immune response
factors that play a role in the outcome (p53 gene → 50% cancers linked)
replicative immortality- old cells dividing→ even more mutations bc they’re old and have mutations/ not functioning
proliferative- prolif & suck all nutrients, put pressure on vital organs
angiogenesis- tumor develops blood flow and siphens resources
metastasis- parts break off → enter blood → start in new area
stage 4 cancer: can no longer target… lethal.
Genes that Drive Cancer
Proto-oncogenes: born w/… for cell proliferation → becomes oncogenes when the mutation occurs
Myc- most lymphoma
Ras- most pancreatic cancers
Tumor Supression genes: apoptosis genes (p53)
BRCA (breast cancer - can be found in blood test)
BRCA 1 & BRCA 2
Mutant Isolation via Bacterial Plating
Positive selection:
to find antibiotic mutant
stamp master plate onto nutrient plate ± antibiotic; colonies that grow on antibiotic plate = resistant mutants that study
Negative selection:
stamp master plate and 2 other plates (w and w/o histidine) → see which grow where
histidine plate produces auxotroph
a nutritional mutant
compared to the prototroph (non mutated version) for testing
Ames Test (Carcinogen Screen)
Start with his^- auxotroph of Salmonella.
Incubate with suspected mutagen + rat‐liver extract ➔ plate w/o histidine -. see if the bacteria can ‘revert’ back to their native state which indicates that they can be carcinogenic
Horizontal Gene Transfer (HGT)
putting extrachromosomal dna into a bacterial dna
Plasmids- easy to introduce, cells normally make and transfer these
chromosomal info- difficult to introduce, requires a homologous recombination meaning it is similar enough to almost overlap the preexisting
3 mxns DNA transfer between bacteria (no reproduction involved)
Transformation: uptake of naked DNA from enviro and incoorp.
• Conjugation: plasmids transfer bet. bacteria via type-4 pilus (bridge)
• Transduction: virus pcks host DNA making viral
Transformation
clinically relevant: very favorable!
naked DNA into cell → finds homologous region → if not degradge
plasmid uptake into cell → stable w/i cell automatically
favorable bc effective
organisms vary what they like! E. Coli takes it all, S. phneumoniae only likes it’s species
Conjugation
clinically: cause extremely fast transmission → antibitoic resistance within 2-4 hours
Gram - allows for conjugation at further distance
Gram+ needs mating bridge, basically uses pilus to pull other bacteria close and then they clev membranes and will insert replicated plasmid
Plasmids: possible clinical use to use another plasmid that is tighter bound and will target the same promoter causing production of diff protein instead
antibiotic resistance
antibiotic synthesis
increase virulence
increase toxins
Transduction
transfer genetic material w/ bacteriophage
Types of transduction:
Generalized (“lytic bacteria"): bacteriophage injects DNA into host cell → destroys host DNA & replicates using host machinery and resources → produces transducing particle: the cut bacterial DNA mistakenly enters new created phage→ upon infection of a new host, this DNA can integrate into the host genome, resulting in genetic variation and potentially new traits in the recipient bacteria.
ex: common cold
requires homologous recombination → usually fails.
Specialized (temperate/lysogenic phage): integrate into host genetics by using integrase enzyme to create the homologous region to enter DNA → cell will cut it out, including couple bases on each end of the DNA to ensure gone → now incorporated into transducing particle and can infect other cells with new DNA
ex: herpes
Genetic Engineering
PCR amplify gene of interest (GOI) with sticky ends; cycles = denature → anneal → extend using heat-stable polymerase (e.g., Taq).
Cut plasmid & GOI with same restriction enzyme (e.g., EcoRI: \text{GAATTC} palindromic site).
Ligate to form recombinant plasmid.
Transform bacteria.
Screen colonies:
• Antibiotic plate selects cells that received any plasmid.
• Blue/white screening: lacZ in multiple-cloning site—interrupted \Rightarrow white (recombinant); intact \Rightarrow blue (native plasmid).
Agrobacterium-Mediated Plant Engineering
Agrobacterium\ tumefaciens TI plasmid drives inserted gene into plant chromosome.
Uses: stable transgenic crops (drought, pest, heat resistance) or transient expression bioreactors (vacuum-infiltrate wounded leaves; short-term overproduction of therapeutics, then harvest & discard plant).
Key Applications of Genetic Engineering
Nucleic acids: gene therapy (e.g., sickle-cell cure), diagnostic probes.
Recombinant microbes: industrial enzymes, vaccines, insulin, bioremediation.
Transgenic plants: stress-tolerant foods, higher yield cotton, plant-made vaccines/ drugs.
Transgenic animals:
• AquAdvantage salmon (fast-growing).
• Humanized mice (Regeneron) for drug testing.
• Xenografting: editing pigs to provide human-compatible organs.
Essential Terms & Equations
Auxotroph \neq prototroph.
Homologous (reciprocal) recombination vs. non-reciprocal (lab-forced).
PCR amplification is exponential: copies = 2^{n} after n cycles.