BIOL 3010: Gene Mutation and DNA Repair (Chapter 14)

Gene Mutation and DNA Repair

Xeroderma Pigmentosum (XP)

Description: A genetic disorder characterized by extreme sensitivity to sunlight, leading to severe skin lesions, mottled redness (erythema), irregular pigment changes, and an increased risk of nodular cancers.
Example: A $4$ -year-old boy presented with marked skin lesions on his face, including two nodular cancers on his nose, highlighting the impact of cellular injury from sunlight.
Underlying Cause: Patients with XP lose the ability to perform Nucleotide Excision Repair (NER), a DNA repair mechanism. Complementation studies using heterokaryons suggest that as many as $7$ different genes can be involved in the disorder.

Nobel Prize in Chemistry $2015$ – DNA Repair

Awarded jointly to Tomas Lindahl, Paul Modrich, and Aziz Sancar "for mechanistic studies of DNA repair."
- Tomas Lindahl: Discovered Base Excision Repair (BER).
- Paul Modrich: Discovered Mismatch Repair (MMR).
- Aziz Sancar: Discovered Nucleotide Excision Repair (NER).
These topics are central to Chapter $14$ of Klug and Cummings in BIOL $3010$ . (http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2015/press.html)

Genetic Variation

Definition: The diversity among different genomes that gives rise to different individuals within a species.
Mechanism: Genomes are dynamic and change over time. These changes are heritable, meaning they can be passed through the germline.
Sources:
- Mutation: The "ultimate" source of genetic variation, involving changes in DNA at the nucleotide level or higher chromosomal levels.
- Recombination: Also contributes significantly to genetic variation.
Types of Mutations:
- Spontaneous Mutations: Occur naturally in nature, primarily due to errors in DNA replication.
- Induced Mutations: Result from exposure to external physical or chemical factors that damage DNA (e.g., Hermann Muller's $1927$ work with X-rays).
Example: Yoandri Hernandez Garrido, known as "Twenty-Four," who is proud of his extra digits, serves as a real-world example of genetic variation (though the specific genetic cause isn't detailed, it illustrates phenotypic diversity).

Classification of Gene Mutations

Gene mutations can be categorized in various ways (refer to Table $14.1$ for examples):

Somatic Mutations: Occur in somatic cells and are not inherited by offspring. They are most likely to be visible if dominant.
Gametic Mutations: Occur in germline cells and are heritable. They can be recessive or X-linked.
Nutritional/Biochemical Mutations: Impact an organism's ability to synthesize essential nutrients (e.g., prototrophs vs. auxotrophs, discussed in Chapter $13$ ).
Behavioral Mutations: Alter an organism's behavior, often challenging to discern their genetic basis.
Regulatory Mutations: Affect the over- or under-expression of a gene.
Lethal Mutations: Cause the organism to be unable to survive to reproduce.
Conditional Mutations: Their phenotypic expression depends on specific environmental conditions (e.g., temperature-sensitive mutations).
Neutral Mutations: Have a negligible effect on the organism's fitness level, often with no discernible impact on the gene product or its expression.
Note on "Deepfake Mutations": A cautionary note against misinformation or artificially generated content related to mutations.

Molecular Basis of Mutation

Genetic information is read in triplets, known as codons.

Transition: A point mutation where a pyrimidine (C, T) is replaced by another pyrimidine, or a purine (A, G) is replaced by another purine.
Transversion: A point mutation where a pyrimidine is replaced by a purine, or a purine is replaced by a pyrimidine.
Missense Mutation: A base-pair substitution that results in a codon coding for a different amino acid, leading to an altered protein sequence.
Nonsense Mutation: A base-pair substitution that changes an amino acid codon into a stop codon, leading to premature termination of protein synthesis and a truncated protein.
Frameshift Mutation: An insertion or deletion of one or more nucleotide pairs (not multiples of three) that alters the reading frame of the gene, leading to a completely different protein sequence downstream of the mutation and often a premature stop codon.

Spontaneous Mutations: Origins

Spontaneous mutations are changes in nucleotide sequences that occur naturally, often during DNA replication, and are not attributed to external factors.

Tautomeric Shifts (Figures $14-2$ and $14-3$ ):
- Description: Purines and pyrimidines exist in alternative, less frequent forms called tautomers, which differ by a single proton shift.
- Mechanism:
  - The standard keto form of thymine and guanine, and amino form of adenine and cytosine, are common.
  - Less frequent enol forms (thymine, guanine) and imino forms (adenine, cytosine) can transiently exist.
  - If these tautomeric forms exist during DNA replication, they can base-pair anomalously with non-complementary bases (e.g., thymine (enol) pairs with guanine (keto); cytosine (imino) pairs with adenine (amino)).
  - This anomalous pairing during replication can lead to transition mutations (e.g., a T-A base pair mutating to C-G, as illustrated in Figure $14-3$ ).
Oxidative Damage:
- Description: Damage to DNA bases caused by reactive oxygen species (ROS) such as superoxides ( $O<em>2^{\cdot-}$ ) and hydrogen peroxide ( $H</em>2O_2$ ).
- Mechanism: These species modify bases, leading to mispairing during DNA replication and subsequent mutations.
Apurinic Sites (AP Sites) and Deamination (Figure $14-4$ ):
- Apurinic (AP) Sites: Spontaneous loss of a purine base (adenine or guanine) from the DNA backbone, creating a site where no base is present. This leads to an inadequate template during replication, and any nucleotide can be inserted opposite the AP site, often resulting in a mutation.
- Deamination: The removal of an amino group from a base, converting it into a keto group.
  - Cytosine to Uracil (C $\rightarrow$ U): Deamination of cytosine forms uracil. Uracil is typically found in RNA and pairs with adenine, whereas cytosine pairs with guanine. If not repaired, a C-G base pair can become a T-A base pair after replication.
  - Adenine to Hypoxanthine (A $\rightarrow$ Hypoxanthine): Deamination of adenine forms hypoxanthine. Hypoxanthine resembles guanine and pairs with cytosine instead of thymine. This can lead to an A-T base pair becoming a G-C base pair.
- Consequence: Both AP sites and deamination alter normal base-pairing efficiencies, leading to potential mutations.

Induced Mutations: Chemical and Radiation Damage

Induced mutations result from the influence of extraneous factors, either natural or artificial agents, such as radiation and UV light.

Chemical Mutagens

Base Analogs (Figure $14-5$ ):
- Description: Chemicals structurally similar enough to standard nitrogenous bases to be incorporated into DNA during replication.
- Example: $5$ -bromouracil ( $5$ -BU) is an analog of thymine. In its common keto form, $5$ -BU pairs normally with adenine. However, in its rare enol form, $5$ -BU pairs anomalously with guanine, leading to A-T to G-C transitions.
Alkylating Agents:
- Description: Chemicals that donate an alkyl group (e.g., methyl, ethyl) to amino or keto groups within nucleotides. These were among the first chemical mutagens identified (e.g., during WWI).
- Example: Ethyl methanesulfonate (EMS) is a common alkylating agent.
- Mechanism: Alkylation alters base-pairing properties, frequently causing transition mutations (e.g., G-C to A-T or A-T to G-C).
Acridine Dyes and Frameshift Mutations (Figure $14-6$ ):
- Description: Planar molecules that can wedge (intercalate) themselves between adjacent base pairs in the DNA double helix.
- Example: Acridine orange.
- Mechanism: Intercalation causes distortion of the DNA helix, leading to slippage or improper pairing during replication. This can result in the addition or deletion of a single base pair, thereby causing frameshift mutations downstream of the insertion/deletion.

Radiation-Induced Mutations

UV Radiation (Figures $14-7$ , $14-8$ ):
- Description: Shorter wavelength, high-energy ultraviolet radiation is mutagenic. Sunburn, for instance, is caused by UVA and UVB wavelengths damaging tissue and DNA.
- Major Effect: Induction of pyrimidine dimers, primarily thymine dimers ( $T=T$ ), between adjacent pyrimidine bases along the same DNA strand.
- Mechanism: The covalent crosslinks between atoms of the pyrimidine rings distort the DNA helix, impeding normal DNA replication and transcription.
Ionizing Radiation (X-rays, gamma rays, cosmic rays) (Old Figure $14-9$ ):
- Description: High-energy radiation that penetrates tissue and causes atoms to lose electrons, creating highly reactive "free radicals" (e.g., hydroxyl radicals).
- Mechanism:
  - Free radicals interact with DNA, leading to various types of damage, including point mutations.
  - Can also directly break the sugar-phosphate backbone, causing chromosome aberrations such as deletions, inversions, and translocations (breakage of DNA backbone).
- Target Theory ( $1924$ ): Proposed that X-rays cause damage at single sites (mutation), and their effect is cumulative, meaning repeated exposure or higher doses increase the likelihood of mutation.
- Electromagnetic Spectrum: Figure $14-7$ illustrates the relationship between wavelength and energy, showing that shorter wavelengths (like gamma and X-rays) correspond to higher energy.

Single Gene Mutations and Human Diseases

Single base pair changes (Single Nucleotide Polymorphisms, SNPs) can lead to serious genetic disorders. The OMIM database currently catalogs over $5000$ human genetic diseases.

Impact of SNPs:
- Approximately $30\%$ of SNPs result in stop codons (nonsense mutations).
- About $15\%$ of SNPs cause abnormal RNA splicing.
Case Studies of Mutations in Human Genes:
- Beta-thalassemia (Reduced or Missing Hemoglobin) (Table $14.3$ ):
  - Description: A diverse group of genetic blood disorders caused by mutations in the HBB gene, leading to reduced or absent beta-globin chains of hemoglobin.
  - Mutation Types:
    - $5\text{'}$ upstream region: Single base-pair mutations (e.g., T $\to$ A transition in TATA box at $-30$ ) decrease gene transcription, causing severe disease (estimated more than $100$ types of mutations in total).
    - mRNA CAP site: Single base-pair mutation (A $\to$ C transversion at $+1$ ) reduces mRNA levels.
    - $5\text{'}$ untranslated region: Single base-pair mutations decrease transcription and translation, causing mild disease.
    - ATG translation initiation codon: Single base-pair mutations alter the mRNA AUG sequence, preventing translation and leading to severe disease.
    - Exons $1$ , $2$ , and $3$ coding regions: Missense and nonsense mutations, and mutations creating abnormal mRNA splice sites. Severity varies.
    - Introns $1$ and $2$ : Single base-pair transitions and transversions reduce or abolish mRNA splicing or create abnormal splice sites, affecting mRNA stability. Most cause severe disease.
    - Polyadenylation site: Single base-pair changes in the AATAAA sequence reduce mRNA cleavage and polyadenylation efficiency, yielding long or unstable mRNAs; disease is mild.
    - Insertions, Deletions, Duplications: Short insertions, deletions, and duplications throughout and surrounding the HBB gene alter coding sequences, create frameshift stop codons, and affect mRNA splicing.
- Trinucleotide Repeats:
  - Description: Expansions of specific three-nucleotide sequences within or near genes.
  - Mechanism: The number of repeats often increases in successive generations, a phenomenon called genetic anticipation. Loss of CAG repeats (encoding glutamine) can lead to long tracts of glutamine, causing proteins to aggregate.
  - Examples:
    - Huntington disease: CAG repeat. Normal: $6$ – $35$ repeats. Affected: $36$ – $129$ repeats.
    - Myotonic dystrophy: CTG repeat. Normal: $5$ – $35$ repeats. Affected: >200 repeats.
    - Fragile X Syndrome: CGG repeat. Normal: $6$ – $50$ repeats. Affected: >200 repeats.
    - Spinolumbar Muscular Dystrophy: CAG repeat. Normal: $10$ – $30$ repeats. Affected: $35$ – $60$ repeats.
  - Location: Mutations can occur in coding regions or in upstream/downstream regulatory regions; the mechanism for expansion is complex and not fully understood.
- ABO Blood Type Alleles:
  - $I^A$ and $I^B$ Alleles: Differ by $4$ common nucleotide substitutions that lead to changes in the amino acid sequence of the glycosyltransferase enzyme.
  - $i^O$ Allele: Caused by a deletion of one nucleotide, leading to a frameshift. This results in an abnormal and truncated (shorter than normal) glycosyltransferase protein, which is non-functional, leading to the O blood type (see Chapter $4$ notes).
- Muscular Dystrophy (MD):
  - Types: Common and severe Duchenne Muscular Dystrophy (DMD) and milder Becker Muscular Dystrophy (BMD).
  - Gene: The dystrophin gene is very large, spanning $2$ million base pairs, and produces a $14$ kb mRNA message encoding a $3685$ amino acid protein.
  - Mutations: Most mutations cause premature termination of the protein.
    - Approximately $65\%$ of DMD cases involve substantial deletions in the gene, which typically change the reading frame.
    - In BMD, deletions are also common, but they often do not change the reading frame, leading to a partially functional, albeit shorter, protein and a milder phenotype.

DNA Repair Systems

Organisms have evolved sophisticated DNA repair systems, which are essential for survival, to counteract mutations and maintain genomic integrity.

Repair During DNA Replication

DNA Polymerase Proofreading:
- Mechanism: DNA polymerase enzymes possess a $3'\to 5'$ exonuclease activity that allows them to detect and remove incorrectly paired nucleotides immediately after their insertion during replication.
- Efficiency: This proofreading function catches approximately $99\%$ of errors, resulting in an error rate of about $1$ in $10^{-7}$ base pairs.
Mismatch Repair (MMR) (Discovered by Paul Modrich):
- Mechanism: Repairs nucleotide mismatches that escape DNA polymerase proofreading. In bacteria, the old DNA strand (template) is methylated, distinguishing it from the newly synthesized, unmethylated strand.
- Process: Repair enzymes recognize the mismatch, bind to the unmethylated new strand, an endonuclease cuts the backbone, an exonuclease removes a segment of nucleotides, and DNA polymerase fills the gap again, followed by DNA ligase to seal the nick. The new strand is then methylated.

Post-Replication Repair

RecA-mediated Homologous Recombination Repair (Figure $14-10$ ):
- Mechanism: If DNA polymerase encounters a lesion that blocks its progression (e.g., a pyrimidine dimer) and leaves a gap, RecA protein (found in E. coli) promotes recombination. It allows the non-mutated sister strand to be used as a template to fill the gap on the newly synthesized strand opposite the lesion.
- Nature: This is a form of homologous recombination repair, utilizing genetic information from an undamaged homologous DNA molecule.
SOS Repair (In E. coli) (Figure $14-9$ ):
- Description: A last-resort, error-prone repair system induced in response to extensive DNA damage that overwhelms other repair mechanisms.
- Mechanism: Conditions are created that allow the insertion of nucleotides into the damaged region even if they do not correctly base pair, promoting DNA synthesis past the lesion. This is error-prone but allows cell survival.
- Complexity: Involves as many as $20$ different enzymes.

Direct Repair

Photoactivation Repair (in E. coli and many Eukaryotes, but not Humans) (Figure $14-11$ , $14-10$ from old text):
- Mechanism: A photoreactivation enzyme (PRE) directly cleaves pyrimidine dimers induced by UV radiation.
- Requirement: The enzyme is only active in the presence of blue light.
- Significance: It is not an essential activity in E. coli, as a null PRE mutation is not lethal, indicating other repair pathways can compensate.

Excision Repair

These systems are conserved across prokaryotes and eukaryotes.

General Mechanism:
1. Excision: A nuclease removes the damaged base or nucleotide segment.
2. Filling the Gap: DNA Polymerase I (in prokaryotes) fills the resulting gap using the complementary strand as a template.
3. Ligation: DNA ligase seals the nick in the DNA backbone.
- Evidence: polA1 cells, which lack functional DNA polymerase I, are sensitive to UV radiation and unable to fill gaps after excision of dimers, highlighting POL I's role.
Base Excision Repair (BER) (Discovered by Tomas Lindahl) (Figure $14-10$ ):
- Mechanism: Primarily repairs small, non-helix-distorting base lesions (e.g., deaminated bases, oxidized bases, alkylated bases).
- Process:
  1. DNA Glycosylase: An enzyme that recognizes and excises an incorrect or damaged base by cleaving the glycosidic bond between the base and the sugar, leaving an AP site.
  2. AP Endonuclease: Cleaves the phosphodiester backbone just $5'$ to the AP site and removes the deoxyribose sugar.
  3. DNA Polymerase and DNA Ligase: DNA polymerase (e.g., Pol I in E. coli, Pol $\beta$ in eukaryotes) fills the single-nucleotide gap, and DNA ligase seals the remaining nick.
Nucleotide Excision Repair (NER) (Discovered by Aziz Sancar) (Figures $14-13$ , Figure $14-12$ from old text):
- Mechanism: Repairs "bulky" lesions that distort the DNA helix, such as pyrimidine dimers (from UV) and bulky chemical adducts.
- Process:
  1. Damage Recognition: Repair proteins (e.g., UvrA, UvrB in E. coli) recognize the distortion.
  2. Dual Incision: UvrC nuclease makes two cuts in the phosphodiester backbone, one on each side of the lesion (e.g., $12$ nucleotides are removed in E. coli).
  3. Excision: The segment containing the lesion is removed (e.g., UvrD helicase).
  4. Filling the Gap and Ligation: DNA polymerase (e.g., Pol I) fills the gap using the undamaged strand as a template, and DNA ligase seals the nick.
- Clinical Significance: Defects in NER are the underlying cause of Xeroderma Pigmentosum (XP), where patients lose the ability to perform this critical repair, leading to severe sun sensitivity and skin cancers.

Double-Stranded Break (DSB) Repair in Mammals

These pathways are crucial for repairing breaks in both strands of the DNA helix, which are particularly dangerous as they can lead to chromosomal rearrangements and cell death.

Homologous Recombination (HR) (Figure $14-13$ ):
- Mechanism: Uses an undamaged homologous DNA molecule (typically a sister chromatid during the S or G2 phase of the cell cycle) as a template to accurately repair the break.
- Process: The broken ends are resected, and recombination proteins (like Rad51) facilitate strand invasion into the homologous template, followed by DNA synthesis and ligation to restore the original sequence.
- Timing: Occurs in late S or early G2 phase of the cell cycle when sister chromatids are available (see Chapter $9$ ).
- Clinical Significance: Defects in the HR pathway are linked to genetic predisposition to breast and ovarian cancers (e.g., BRCA1 and BRCA2 mutations).
Nonhomologous End Joining (NHEJ) (Figure $14-13$ ):
- Mechanism: A "last resort" or "error-prone" pathway that directly ligates the broken DNA ends without requiring a homologous template.
- Timing: Occurs primarily in the G1 phase of the cell cycle, before DNA replication when a sister chromatid is not available.
- Process:
  1. Proteins (e.g., Ku proteins, DNA-PKcs) bind to the broken DNA ends.
  2. The ends are processed (sometimes involving trimming of nucleotides), and then joined together by DNA ligase (e.g., ligase IV).
- Consequence: This pathway often results in the loss of some DNA sequence at the repair site, making it error-prone.
- Risk: If multiple chromosomes have double-stranded breaks, NHEJ can incorrectly join ends from different chromosomes, leading to translocations and genomic instability.

The Ames Test

The Ames test is a widely used biological assay developed by Bruce Ames to assess the mutagenicity of various chemical compounds.

Principle: It utilizes specific strains of Salmonella typhimurium that are auxotrophic for histidine (i.e., they cannot synthesize histidine and require an external supply for growth). These strains carry point mutations in genes required for histidine synthesis.
Methodology:
1. Potential mutagen is mixed with the auxotrophic bacteria.
2. The mixture is plated on a medium lacking histidine.
3. The number of revertant mutations (bacteria that have reverted to prototrophy and can grow on the histidine-deficient medium) is counted.
Results Interpretation: An increase in the number of revertant colonies in the presence of the test compound, compared to control plates without the compound, indicates that the compound is mutagenic.
Role of Liver Enzymes: Many test compounds are not mutagenic in their original form but are metabolized into mutagenic derivatives by liver enzymes. Therefore, liver extracts (e.g., S9 fraction) are often added to the test system to mimic the metabolic processes that occur in the mammalian body, providing a more relevant assessment of potential mutagenicity (Figure $14-14$ ).

Transposable Elements (Transposons)

Transposable elements are DNA sequences that can move from one location to another within a genome, often causing mutations in the process. Their discovery earned Barbara McClintock the Nobel Prize in $1983$ , more than $30$ years after her initial findings in $1940$ .

Discovery by Barbara McClintock ( $1902$ - $1992$ )

Organism: Cytogeneticist who discovered "transposable" or "mobile" genetic elements in corn (Zea mays).
Methodology: By conducting specific crosses of corn, McClintock observed variable phenotypic effects (e.g., variegated kernel color) that changed across generations, which she attributed to the movement of these elements.
Significance: The mutable phenotypic effects provided an excellent genetic system to directly observe changes in genotype and phenotype due to transposition.

Types of Transposable Elements

Insertion Sequence (IS) Elements (in Bacteria) (Figure $14-15$ ):
- Description: Generally short, less than $2500$ nucleotides long.
- Content: Contain only genes necessary for their own transposition (e.g., transposase).
- Characteristics: Cause unstable mutations (i.e., revert with high frequency) and have short inverted terminal repeats.
Transposons (Tn Elements) (in Bacteria):
- Description: Larger than IS elements.
- Content: Contain genes unrelated to transposition (e.g., antibiotic resistance genes) in addition to those required for transposition.
- Discovery: Certain lac-mutations in E. coli (unable to utilize lactose) showed unusually high reversion rates. Sequence comparisons revealed the loss of extra DNA (insertion elements) from the gene, explaining the reversion.
Activator (Ac) and Dissociator (Ds) Elements (in Corn) (Figures $14-16a$ , $14-17b$ , $14-17c$ , Old Figure $14.20$ ):
- Ac (Activator) Element: An autonomous element (approximately $4563$ bp) that encodes a functional transposase enzyme. It contains $11$ bp inverted terminal repeats.
- Ds (Dissociator) Element: A non-autonomous element. It is a derivative of Ac that has internal sequences missing, meaning it lacks a functional transposase gene. Ds elements can only transpose if Ac is also present in the genome to provide the transposase function in trans.
- Phenotypic Effects:
  - Ds-a, Ds-b, Ds-c: Various derivatives of Ac with different internal sequences removed.
  - Double Ds: Occurs when one Ds element is inserted into another Ds element.
  - Chromosome Breakage: When Ac is present, Ds may transpose. If Ds is located near genes (e.g., W gene for pigment synthesis), its excision can lead to chromosome breakage and loss of gene expression, producing a mutant effect.
  - Gene Inhibition/Restoration: Ds can transpose into a gene (e.g., W gene), inhibiting its function and producing a mutant phenotype. If Ds later "jumps" out of the gene, wild-type expression of the gene can be restored, leading to mosaic phenotypes (e.g., variegated corn kernels).

Transposons in Humans

Alu Family (SINES):
- Description: Short Interspersed Elements (SINEs), approximately $200$ to $300$ bp long, with over $500,000$ copies in the human genome (originally named because they are cleaved by the restriction enzyme Alu1).
- Characteristics: Found in all primates and rodents. Have a highly conserved $40$ bp segment and are flanked by $7$ - $20$ bp direct repeats. Alu sequences can also be represented in some transcripts.
- Activity: Their clustering varies between and within individuals, providing evidence for active transposition.
LINEs (Long Interspersed Elements): Longer retrotransposons.
Impact on Human Mutations: Transposons can cause mutations in humans, although rarely in modern-day humans.
- Example 1: One known instance involved a new mutation in the Factor VIII gene (on the X-chromosome) that caused hemophilia. This LINE element was not present on the X chromosome of either parent but was found on their chromosome $22$ , suggesting a germline transposition event.
- Example 2: A SINE insertion in the BRCA2 gene was also found to cause cancer.

Retrotransposons

Mechanism: Move within the genome via a "copy-and-paste" mechanism, meaning an RNA intermediate is involved.
Nature: Resemble retroviruses (e.g., they have genes for reverse transcriptase) but typically do not form infectious viral particles.
Types: Two main types are classified based on the presence or absence of Long Terminal Repeats (LTRs).
- LTR Retrotransposons: Have LTRs at their ends (e.g., Copia in Drosophila).
- Non-LTR Retrotransposons: Lack LTRs (e.g., LINEs and SINEs in humans).
Process (Figure $14-17$ ):
1. The genes within the retrotransposon (e.g., for reverse transcriptase) are transcribed into RNA.
2. The RNA is then reverse-transcribed into DNA.
3. This newly synthesized DNA copy is then inserted into a new genomic location.

Copia Retrotransposon in Drosophila (Figure $14-18$ )

Example: Insertion of a copia element into exon $2$ of the white gene (which has $6$ exons) in Drosophila.
Consequence: This insertion leads to a prematurely terminated mRNA and a non-functional gene product, altering eye pigmentation.
The white ( $w^{\text{+}}$ ) Gene: Discovered in $1910$ by Thomas Hunt Morgan, it encodes a subunit of an ATP-binding cassette (ABC) transporter. This transporter is crucial for loading pigment granules and depositing their content into pigment cells of the compound eyes, ocelli, Malpighian tubules, and testis. Additionally, the White protein transports bioamines, neurotransmitters, metabolic intermediates, and second messengers, suggesting broader "housekeeping" functions in the central nervous system beyond its classical role in eye pigmentation.

Transposons, Mutations, and Evolution

Transposons have a wide range of effects on genes and play a significant role in evolution.

Effects on Genes:
1. Alter Coding Region: Insertion directly into a coding region can alter the amino acid sequence, sometimes leading to beneficial changes.
2. Affect Expression of Nearby Genes: Insertion near a gene can modify its expression levels, potentially in a positive way.
3. Premature Termination: Insertion into an intron may interfere with splicing, causing premature termination of the gene transcript.
4. Aberrant Polyadenylation: Insertion can lead to altered or aberrant polyadenylation of a transcript.
5. Altered Upstream Expression: Insertion upstream of a gene can significantly alter its expression patterns.
6. Genetic Rearrangements: Two transposons located near each other can facilitate the movement or recombination of the genetic material between them.
Frequency of New Transpositions: New germline transpositions occur in approximately $1/100$ human births.
Evolutionary Impact: All these effects have contributed to the evolution of many genes and their associated traits.
- Immune System: Recombination-activating genes (RAG) genes, critical for immune system diversity, are thought to have evolved from ancient transposases.
- Antibiotic Resistance: In bacteria, transposons frequently carry antibiotic resistance genes, facilitating their rapid spread among bacterial populations and contributing to the evolution of "superbugs"—a significant problem in public health.
- Evolution of Eukaryotic Genomes: DNA transposons and retrotransposons are major drivers of the evolution and shaping of eukaryotic genomes.

Transposons in Drosophila - Hybrid Dysgenesis (Figure $14-18$ )

Description: A phenomenon where certain crosses between specific strains of Drosophila melanogaster produce hybrids with a "deterioration in quality" (dysgenesis), exhibiting frequent mutations, chromosome breakage, and sterility.
Discovery: In $1977$ , Margaret and James Kidwell (Rhode Island) and John Sved (Australia) identified this phenomenon.
Cytotypes:
- P Cytotype: Strains that contain active P elements (transposons).
- M Cytotype: Strains that lack active P elements and also lack cytoplasmic factors that repress P element transposition.
Crosses and Outcomes:
- P female x P male: Normal offspring.
- M female x M male: Normal offspring.
- P female x M male: Normal offspring (the P element repressors from the P female's cytoplasm prevent dysgenesis).
- M female x P male: Dysgenic offspring (the M female lacks repressors, allowing P elements from the P male's germline to transpose uncontrollably, leading to widespread genomic instability and dysgenesis).
P Elements: The element responsible for hybrid dysgenesis was isolated from the white gene by Simmons and Lim in $1980$ . P elements are not found in M cytotypes and are mobile DNA transposons.
Transposase Expression: The transposase enzyme encoded by P elements is expressed specifically in the germline, explaining why the effects of dysgenesis are heritable and impact reproductive cells.

Chapter Questions and Assignments

Refer to assigned questions in the "In Class schedule Part $2$ " file within the Part $2$ course folder.
Complete Mastering Genetics Assignments for this chapter, due three days after the in-class problem-solving session.
Old multiple-choice questions for this chapter will be posted online for practice.