BIOSCI 202 Genetics: Lectures 11-12 Causes and Effects of Mutations; Lab, Regulation, Transposons (Fill-in-the-Blank Flashcards)

Mechanisms of Mutation

• Small-scale mutations arise from three main sources:
  • Errors during DNA replication and repair
  • Spontaneous chemical changes of bases that alter pairing
  • Induced chemical changes from external mutagens
• Diagrammatic idea: mutations become stably incorporated into DNA through replication and repair processes, creating heritable changes.

Replication mutations

• DNA polymerase fidelity is extremely high, due to proofreading activity; failures during replication can lead to mutations.
• If proofreading or other fidelity checks fail, replication errors become permanent mutations in the genome.

Repair mutations

• The cell has multiple DNA damage repair pathways; many repairs occur during replication when DNA is damaged.
• Repair DNA polymerases often have higher error rates than normal polymerases, so repair processes can be error-prone, paradoxically increasing mutation rates during repair.

Spontaneous base chemical changes

• Cytosine deamination (spontaneous) is an example:
  • Cytosine can deaminate to uracil, and because uracil pairs with adenine, this causes C⇒T transitions after replication.
• Methylcytosine deamination (spontaneous):
  • In CpG regions, methylated cytosine deaminates to thymine, leading to C→T transitions over time.
  • Specific enzymes detect and repair G:T mispairs to reduce this mutation type.

Induced Mutations

• Incorporation of base analogs:
  • Base analogs substitute for natural bases and can cause mispairing; e.g., 2-amino-purine (2-AP) can pair with T or C and act as a mutagen.
• Establishment of mutations via mispairing after mutagen exposure:
  • 5-bromouracil can pair with A or G; full mutation may require at least two rounds of replication to fix.
• Alkylating mutagens (EMS, etc.):
  • EMS alkylates bases, causing mispairing (e.g., G pairs with T due to modification).
• Damage that prevents pairing (DNA adducts):
  • Benzo[a]pyrene from cigarette smoke binds irreversibly to G; replication often results in T rather than G after repair.

Radiation-induced mutation

• Radiation causes DNA breaks; large-scale mutations (duplications/deletions, inversions, translocations) arise from breaks.
• Large-scale mutations tend to have larger phenotypic effects, so radiation is a powerful mutagen.

Establishment and fixation of mutations

• Mutations can be established during replication and replication-associated repair, and then fixed in daughter genomes.

Effects of mutations: overview

• Mutation effects are context-dependent: mutations in regions critical for function are more likely to affect phenotype; in other regions, effects may be negligible.
• The more nucleotides a mutation affects, the greater the chance of an effect, but context matters.

Effects of Mutation – 1. Single nucleotide variants (SNVs)

• SNVs in coding regions can be:
  • Synonymous (silent) if they do not change the encoded amino acid due to redundant genetic code.
  • Nonsynonymous (missense) if they change the amino acid; may be conservative (similar amino acid) or non-conservative (different).
  • Nonsense if a stop codon is created.
• SNVs arise from redundancy in the genetic code; multiple codons can code for the same amino acid.
• The chemical properties of amino acids and the 3D structure of proteins are critical for function.

Effects of Mutation – 1. SNVs (continued)

• The amino acid substitution can be conservative or non-conservative, affecting protein folding, stability, or activity.
• Nonsense mutations truncate proteins, often severely affecting function.

Effects of Mutation – 2. Indels (insertions/deletions)

• Indels can cause frameshift mutations if they occur in coding regions, altering the reading frame and downstream amino acids.
• Indels must occur in coding regions to affect protein sequence; some indels may occur in non-coding regions with no direct protein effect.
• Question: Is it possible to get indels that do not cause frameshifts? (Yes, if the indel is in multiples of 3 nt, preserving the reading frame.)

Effects of Mutation – 3. Regulatory mutations

• Mutations in regulatory regions (e.g., promoters) can alter transcription levels.
• Example: promoter mutations affecting the β-globin gene promoter (CACCC/CACACCC/ATAAA motifs) can reduce transcription.
• Regulatory mutations influence gene expression levels rather than protein sequence.

Effects of Mutation – summary: context matters

• Effects are generally context-dependent: a mutation in a functionally important region is more likely to affect phenotype.
• More nucleotides affected by a mutation increases the chance of an effect, but context remains decisive.

Continuum of mutation effects

• Mutations span a continuum: neutral, deleterious, and beneficial.
• Neutral mutations do not affect organismal function; deleterious reduce fitness; beneficial improve function, though beneficial mutations are relatively rare.
• Bank et al., 2014 note this continuum in Trends in Genetics (2014) 30:540–6.

Conservation as a clue to function

• DNA conservation across species can indicate functional regions.
• Highly conserved regions are likely functionally important; mutations there are often deleterious and not observed in populations.
• Kuhn et al., 2012 discuss conservation in Briefings in Bioinformatics 14:144–61.

Beneficial mutations in populations

• Beneficial mutations can sweep through populations to fixation; demonstrates that new advantageous variants can arise and spread.
• A video example illustrates these dynamics; real-world data show resistance mutations can preexist and spread under selection.

Origin of variation

• A key question: Are beneficial mutations typically induced by environmental stress or do they occur by chance (pre-existing variation)?
• The fluctuation test (Luria & Delbrück, 1943) tested whether resistance mutations arise in response to phage exposure or pre-exist.

Fluctuation test to decide the origin of mutations

• Classic experiment used E. coli with resistance to bacteriophage T1.
• Predictions:
  • If mutations are induced by phage exposure, resistant colonies should appear with similar numbers across all tubes (same number of bacteria, same phage pressure, same mutation rate).
  • If mutations are pre-existing, the number of resistant colonies should vary considerably between tubes; earlier mutations yield more resistant descendants.
• Results: The data do not fit a Poisson distribution expected if mutations were induced; instead, they show substantial variation consistent with pre-existing (spontaneous) mutations.
• Conclusion: Most mutations are spontaneous and resistance mutations are pre-existing before exposure.

Modern genome sequencing and standing variation

• Modern sequencing reveals substantial standing variation; humans average ~20 million base differences from the reference genome, with ~100–200 de novo mutations per individual.
• Most genetic diseases are caused by pre-existing variants rather than new mutations.

Summary of learning objectives (mutations)

• Describe mechanisms that can cause mutations.
• Diagram how mutations can be stably incorporated into DNA.
• Define synonymous, non-synonymous, and frameshift mutations.
• Predict effects of different mutations in various genomic contexts with justification.
• Explain the continuum of mutation effects from deleterious to neutral to beneficial.
• Explain how mutation location informs functional importance of genome regions.
• Explain the fluctuation test and how it demonstrates pre-existing mutations in populations.

Laboratory: One-step growth experiment (phage biology)

• Aim: Monitor the infection cycle of bacterial viruses (phages).
• Phage structure: Head contains viral DNA; tail for attachment; injects DNA and hijacks host machinery to replicate and lyse the cell.
• Life cycle steps: Phage attaches to bacterium, injects DNA, host machinery makes phage components, phage particles assemble, host cells lyse releasing phage.
• Time-dependent data: Latent period, burst size, and plaque-forming units (Pfus) are measured at various time points.
• Synchronizing infection: Use cyanide (KCN) to temporarily stop bacterial growth, infect non-growing cells, then relieve inhibition to synchronize infection.
• Workflow: Involves growth tubes, dilutions, and plaque assays to quantify phage at different times.
• Plaque assay: Mix infected bacteria with indicator bacteria on soft agar and count plaques at different time points.
• Measurements: Relative phage titre over time, latent period, and burst size derived from data.

Regulation of Gene Expression in Bacteria

• Gene regulation is critical and can be achieved via multiple mechanisms, including transcriptional control, operons, and global regulation.
• Transcriptional control includes the Lac operon, the Arabinose operon, negative vs positive control, cis vs trans effects, and the logic of gene regulation.
• Global regulation includes sigma factors and consensus sequences; control of large gene networks via shared regulatory proteins.

Promoters and transcription initiation

• Promoters in E. coli typically have two key elements bound by RNA polymerase to initiate transcription:
  -35 region: TTGACA; spacer; -10 region: TATAAT; spacer; start site N17; N7 positions before transcription start site.
• Strong promoters bind RNA polymerase more effectively; weak promoters bind less well and transcribe more slowly.
• Proper spacing between -35 and -10 is crucial; optimal spacing allows RNA polymerase to contact both regions.
• Example promoter strength: recA promoter (strong) vs araBAD promoter (weak).

Genetic switches: regulators of transcription

• Promoter determines where transcription begins; Operator is the repressor binding site; Activator binding site is for activators.
• Activators/repressors respond to environmental cues; two key sites in proteins: DNA-binding site and allosteric site.

The Lac operon (negative regulation with catabolite repression)

• Structure: lacI (repressor) controlling lacZ (β-galactosidase), lacY (permease), lacA (transacetylase).
• In absence of lactose: repressor binds operator, transcription off.
• In presence of lactose (allolactose as the actual inducer): repressor binding to operator is alleviated, enabling transcription.
• Discovery: Jacob & Monod showed that adding lactose induces a large increase in β-galactosidase expression.
• Mutations:
  • Oc mutations: constitutive expression; repressor cannot bind operator (cis-acting).
  • lacI- mutations: defective repressor cannot bind operator; operon is always on (trans-acting for the operator in the chromosome).
  • I+ and I- interactions show cis vs trans effects; lacI+ can repress in trans if present with a wild-type background.

Catabolite repression of lac operon

• Expression requires two signals: lactose present (derepresses repressor) and glucose absent (positive regulation via CAP-cAMP).
• cAMP level inversely related to glucose; CAP-cAMP complex enhances RNA polymerase binding to the promoter, increasing transcription when glucose is scarce.
• Mechanism: CAP-cAMP bends DNA and assists RNA polymerase binding to -10/-35 sites, elevating transcription.

Regulation of transcription: global control and sigma factors

• Sigma factors direct RNA polymerase to specific promoter sequences; multiple sigma factors exist to regulate sets of genes under different conditions.
• E. coli primary sigma factor is sigma 70; alternative sigma factors regulate stress responses, sporulation, etc.
• Concept: a single sigma factor can govern many genes; switching sigma factor expression reprograms entire gene networks.

Promoter consensus sequences

• Consensus sequences reflect the most common nucleotides at each position across promoters; used to predict promoter strength.
• Example: -35 box consensus: TNATATT (some positions variable); -10 box consensus: TATAAT (typical core).
• A site that closely matches the consensus tends to have stronger transcription initiation.

The Arabinose operon: dual positive and negative control

• AraC acts as an activator (positive control) and, in the absence of arabinose, functions as a repressor via DNA looping that prevents RNA polymerase access.
• In the presence of arabinose, AraC changes conformation and promotes transcription, in part by recruiting Mediator and looping DNA to bring transcription machinery to the promoter.
• Additional positive control via CAP-cAMP; glucose absence increases cAMP and CAP activity.

Three-dimensional genome organization and chromatin (contrast with bacteria)

• Eukaryotes rely on chromatin structure to regulate transcription.
• DNA is packaged into chromatin; access to DNA is regulated by nucleosome positioning and histone modifications.
• Transcriptional default state in bacteria is on; in eukaryotes, it is off due to nucleosome occupancy blocking access.
• Enhancers and promoters function in 3D space; Mediator complexes and looping bring distant regulatory elements into proximity with promoters.

Chromatin and epigenetic regulation

• Euchromatin (active) vs heterochromatin (silenced): chromatin remodeling and histone modifications govern accessibility.
• Histone tails can be post-translationally modified (e.g., methylation, acetylation) to activate or repress transcription.
• Activating marks: histone acetylation (e.g., H3K9ac, H3K27ac) and certain methylations (e.g., H3K4me3, H3K36me3, H3K79me).
• Repressive marks: H3K9me, H3K27me, H4K20me, HP1-associated heterochromatin.
• Chromatin states include active euchromatin, poised euchromatin, facultative heterochromatin, constitutive heterochromatin.
• Boundary/insulator elements can block spread of heterochromatin.

Position effect variegation (PEV)

• Introduced genes may be variably expressed depending on chromatin context (heterochromatin vs euchromatin) where the gene resides.
• Classic Drosophila example: white gene inversion leads to variegated expression due to heterochromatin spreading.
• Indicates importance of chromatin state in regulating gene expression.

Three-dimensional genome organization in nucleus

• Genome organized into 3D territories and loops; topologically associating domains (TADs) group genes with similar expression patterns.
• 3D organization is a key layer of gene regulation beyond linear DNA sequence.

GAL system in yeast: a case study in eukaryotic transcriptional control

• GAL genes (GAL1, GAL2, GAL7, GAL10) are not in an operon in Saccharomyces cerevisiae.
• GAL4 is a regulatory transcription factor; GAL4 binds UASG (upstream activating sequences) to promote transcription when galactose is present and glucose is absent.
• GAL80 acts as a repressor that binds Gal4 in the absence of galactose; when galactose is present, GAL3 binds galactose, sequestering GAL80 away from Gal4 and enabling transcription.
• Gal4 can recruit Mediator and preinitiation complex to initiate transcription via looping.
• Yeast is diploid; mutations can be analyzed in heterozygous and homozygous backgrounds.

Gal4 as a tool for synthetic gene control

• Gal4 can be used as a DNA-binding domain to drive transcription of genes linked to UAS sequences.
• Swapping Gal4’s DNA-binding domain with other domains allows transcriptional control of different target genes.

Transcriptional regulation and chromatin interaction: GAL activation and chromatin

• Gal4 activation involves chromatin modifiers that unwind DNA from nucleosomes, enabling RNA polymerase II binding and transcription initiation.
• This demonstrates that transcriptional activation can occur via changes in chromatin structure, not just promoter sequence.

Regulation of Gene Expression in Eukaryotes

• Similarities with prokaryotes include promoter-based control and transcription factors; but eukaryotes differ in operon absence, intron processing, and nuclear separation of transcription/translation.
• Transcriptional regulation in eukaryotes heavily involves promoters, enhancers, silencers, and three-dimensional genome organization.
• The GAL system is a canonical example illustrating enhancer-mediated control and chromatin-wide regulation.

Epigenetic and post-transcriptional regulation

• DNA methylation: methylation of CG dinucleotides in promoters typically represses transcription and can be inherited through DNA replication.
• RNA-based regulation: small RNAs (siRNA, miRNA) guide RISC to target mRNAs, leading to degradation or translational repression.
• Alternative splicing: many genes have introns; splicing can produce multiple isoforms, contributing to protein diversity and regulation across tissues.

Small RNA regulation of gene expression

• siRNA/miRNA pathway:
  • Long dsRNA is processed by Dicer into short dsRNA fragments (20–24 nt).
  • RISC binds one strand and pairs with complementary mRNA, resulting in degradation (siRNA) or translational repression (miRNA).
• Let-7 and miR-34 examples show roles in differentiation and repression of pluripotency factors.

Post-transcriptional control and translation

• mRNA stability affects protein output; untranslated regions and regulatory elements influence half-life.
• Post-translational control includes protein stability and modifications that affect activity, localization, or interactions.

Transposons (Transposable Elements, TEs)

• Barbara McClintock discovered transposons in maize; later work showed transposons are widespread across life.
• Transposons are DNA sequences that catalyze their own movement within genomes via transposases.
• They can be classified by mechanism and copy number:
  • Class I: retrotransposons — transpose via an RNA intermediate (transcribed, reverse transcribed, and inserted back into genome).
  • Class II: DNA transposons — move via a DNA intermediate; can be conservative (cut-and-paste) or replicative (copy-and-paste).
• Autonomous transposons encode transposase and can transpose themselves; non-autonomous rely on transposases from autonomous elements.
• Inverted repeats at termini are important for transposase recognition; direct repeats are created as target-site duplications upon insertion.
• Transposons can cause mutations by inserting into genes or regulatory regions; they can also rearrange the genome through recombination between transposon copies.

Transposon types and features

• In bacteria: insertion sequences (IS elements) are simple transposons; composite transposons contain genes flanked by IS elements (e.g., Tn1, Tn5, Tn10, Tn3).
• Target site duplications: insertions generate short direct repeats flanking the inserted element due to staggered cuts and resynthesis.
• Inverted repeats (IRs) are recognition sites for transposases; transposases mediate excision and insertion.
• Class I vs Class II distinction reflects transposition mechanism and requirement for RNA intermediates (Class I) vs DNA transposition (Class II).

Transposons in the human genome

• Retrotransposons dominate the human genome:
  • LINEs (Long Interspersed Nuclear Elements) — autonomous; encode reverse transcriptase; >21% of the genome.
  • SINEs (Short Interspersed Nuclear Elements) — non-autonomous; rely on LINE machinery; >13% of the genome; most derive from tRNA genes; Alu is the most abundant SINE.
  • LTRs (Long Terminal Repeat retrotransposons) — related to retroviruses; make up >8% of the genome; some are endogenous retroviruses (ERVs).
• Alu elements are a major SINE family derived from 7SL RNA gene; over a million copies.
• ERVs and other retrotransposons contribute to genome evolution and regulation of host genes, including placental genes and early development programs.

Transposon activity and host suppression

• Transposons are often silenced by the host genome via heterochromatin formation, DNA methylation, and small RNA silencing pathways to limit movement.
• Transposition rates are typically low in humans (e.g., about one LINE transposition per 95–270 live births).
• In mammals, stem cells and early development can see transient upregulation of transposon transcription, the reasons for which are still debated.
• Genome defense mechanisms include DNA methylation, histone modifications, heterochromatin formation, and small RNA pathways that target transposons for silencing.

Transposons as drivers of evolution and genome structure

• Transposons can move genes between plasmids and genomes, reshaping gene content and regulatory landscapes.
• They can facilitate antibiotic resistance gene spread in bacteria via mobilization and horizontal transfer.
• Transposons contribute to regulatory innovation by providing promoters, enhancers, and other regulatory elements.
• The genome contains many transposon remnants; current activity is limited by host repression, but regulatory co-option can benefit host organisms.

Key terms and concepts (quick glossary)

• Synonymous (silent) mutation: a nucleotide change that does not alter the amino acid encoded.
• Nonsynonymous (missense) mutation: a nucleotide change that alters the amino acid; may be conservative or non-conservative.
• Frameshift mutation: insertion/deletion not in multiples of 3 that shifts the reading frame.
• Promoter: DNA region where RNA polymerase binds to initiate transcription.
• Operator: DNA sequence bound by a repressor to regulate transcription.
• Activator: protein that increases transcription by aiding RNA polymerase binding.
• Cis-acting element: DNA sequences that influence regulation on the same molecule (e.g., Oc).
• Trans-acting factor: diffusible proteins (e.g., LacI) that regulate targets other than their own gene.
• CAP-cAMP: regulatory complex that activates transcription under glucose-poor conditions.
• Enhancer: distal regulatory DNA sequence that increases transcription via chromatin looping.
• Silencer: DNA element that represses transcription.
• TADs: Topologically associating domains; higher-order genome organization influences gene expression.
• Epigenetics: heritable changes in gene expression not due to changes in DNA sequence (e.g., DNA methylation, histone modifications).
• siRNA/miRNA: small RNAs guiding RISC to degrade or repress target mRNA.
• Spliceosome: complex that removes introns from pre-mRNA in eukaryotes, enabling alternative splicing.
• Bursts, latent period, and Pfus (plaque-forming units): key phage infection metrics in one-step growth experiments.
• Recombination frequency: ext{RF} = rac{N{ ext{recombinant}}}{N{ ext{total}}}; used to map distances in phage genetics.

Important formulas and quantitative notes

• Recombination frequency in phage genetics: ext{RF} = rac{N{ ext{recombinant}}}{N{ ext{total (progeny)}}}
• Burst size (phage biology): approximately the number of phage produced per infected bacterial cell; can be estimated as
  ext{Burst size} \, ext{≈} \, rac{ ext{Pfus produced after lysis}}{ ext{Number of infected cells}}
• Direct repeats and target site duplications upon transposon insertion:
  • Insertion creates short direct repeats flanking the transposon (typically 3–30 bp): ext{Direct repeat length} \sim 3 ext{–}30\,\text{bp}
• Transposon insertion mechanics:
  • Target site duplication results from staggered cuts at the insertion site followed by repair synthesis.
  • Distinguish direct repeats from inverted repeats: direct repeats flank the inserted element; inverted repeats are at the termini of the transposon and are recognition sites for transposases.
• Consensus sequences (for promoter elements):
  • -35 box consensus example: ext{TNATATT} (positions with variability indicated by N)
  • -10 box consensus example: often around ext{TATAAT} or related sequences; spacing is crucial for RNA polymerase binding.

Connections to foundational principles and real-world relevance

• Mutation mechanisms illuminate why genomes contain a spectrum of variation, from neutral to deleterious to beneficial, shaping evolution and adaptation.
• The fluctuation test demonstrates that many mutations are pre-existing, informing how populations adapt to pathogens and environmental changes.
• Transposons illustrate genome dynamism: they can drive evolution by reshaping gene regulation and creating genetic diversity, while host defense mechanisms keep their activity in check.
• Epigenetic and post-transcriptional regulation add layers of control beyond DNA sequence, enabling cell-type–specific expression patterns and development.
• Understanding operons, transcription factors, and chromatin dynamics underpins modern biotechnology, synthetic biology, and medical genetics (e.g., gene therapy strategies, CRISPR-based regulation, and epigenetic therapies).

Practical implications and ethical considerations

• Epigenetic inheritance and DNA methylation raise questions about heritability of expression states beyond the DNA sequence.
• Transposon activity and genome editing carry implications for genome stability, cancer biology, and long-term evolutionary trajectories.
• Use of small RNAs as gene-silencing tools offers powerful research and therapeutic potential, accompanied by safety and off-target concerns.
• The knowledge of bacterial regulation (lac operon) informs synthetic biology designs, metabolic engineering, and antibiotic resistance monitoring.

Quick reference guide (study-ready bullets)

• Three sources of mutation: replication/repair errors, spontaneous base changes, induced mutagens.
• Spontaneous base changes: cytosine deamination (C→U → potential C→T), methylcytosine deamination (mC→T, especially in CpG sites).
• Induced mutagens: base analogs (e.g., 2-AP), alkylating agents (EMS), carcinogens (benzo[a]pyrene) requiring repair (often G→T substitutions).
• Radiation induces large-scale changes; point mutations vs large-scale events differ in phenotypic impact.
• SNVs: synonymous vs nonsynonymous (conservative vs non-conservative) vs nonsense; frameshifts from indels.
• Regulatory mutations alter transcription levels; examples include promoter variants in the β-globin gene.
• Continuum of mutation effects: neutral, deleterious, beneficial; most beneficial mutations are rare.
• Conservation as a functional proxy; highly conserved regions likely functionally important.
• Fluctuation test: pre-existing mutations vs induced mutations; supports spontaneous variation.
• Lac operon: negative regulation by LacI; catabolite repression by CAP-cAMP; glucose presence lowers cAMP, reducing CAP activity.
• Arabinose operon: dual positive and negative control via AraC, AraI/AraO, and 3D genome interactions; galactose condition changes regulate transcription.
• Eukaryotic regulation: promoters, enhancers, and the 3D genome architecture (TADs); chromatin states govern accessibility; histone modifications and DNA methylation modulate transcription.
• GAL system as a case study of enhancer-based regulation and chromatin remodeling in yeast.
• Transposons: IS elements, composite transposons, autonomous vs non-autonomous; class I vs class II; effects include insertions, rearrangements, and gene mobility.
• Human genome TE landscape: LINEs, SINEs (Alu), and LTRs/ERVs; transposons shape genome structure and regulation while being tightly repressed by host defenses.
• Recombination in phage genetics uses a frequency-based approach to map distances; complementation tests help determine gene relationships.