Genetics Final Exam Condensed PDF

CHAPTER 15: DNA DAMAGE, REPAIR AND MUTATION Point Mutations:change in a single base pair, often due to base substitution. Types of Point Mutations: ○ Transition: pyrimidine changes to another pyrimidine, or a purine changes to another purin...

CHAPTER 15: DNA DAMAGE, REPAIR AND MUTATION Point Mutations:change in a single base pair, often due to base substitution. Types of Point Mutations: ○ Transition: pyrimidine changes to another pyrimidine, or a purine changes to another purine. usually don't have a large effect b/c they don't change the shape of the DNA helix. more common ○ Transversion: A pyrimidine changes to a purine or vice versa creates energetically unfavorable mismatch and harder to escape DNA repair systems. Consequences of Point Mutations in Coding Sequences Mutation type Change to AA Sequence Effect on protein Effect on phenotype silent(synonymous) No change due to redundancy in the No change in protein structure. No change genetic code missense(conservative) change to an AA with similar little effect on protein function Usually no or minimal change properties ( + → + or - → -) if the AA properties are similar. missense(non-conservative) change to AA with diff properties disrupting protein's structure/ May lead to a disease or altered (ex.., + → - or polar → non-polar) function function. nonsense creates stop codon, leading to early Results in a truncated protein, Often loss-of-function phenotype, termination of protein synthesis. usually nonfunctional. possibly causing disease. Frameshift Alters the protein length due to protein that is longer or Likely to cause nonfunctional indels shorter protein, resulting in disease or a severe phenotype. Regulatory Mutations in promoter or enhancer prevent gene expression, cause a complete loss of gene regions affect gene expression leading to no mRNA or protein function, leading to diseases or being produced developmental issues Ras Protein and Point Mutations Ras is involved in cell signaling and proliferation. Normal conditions: ○ Ras is inactive when bound to GDP ○ active when bound to GTP ○ regulated by GTP-exchange factor (GEF) and GAP (GTPase activating protein)--- inactivates Ras. In cancer: ○ mutation in Ras (e.g., replacing glycine with valine) ○ prevents GAP from converting GTP to GDP ○ keeping Ras active and promoting constant cell division. Consequences of Point Mutations in Non-Coding Regions Mutation type Description Effect on gene expression Effect on phenotype Regulatory regions Mutations in promoter or enhancer Can increase, decrease, or Altered gene expression may lead to (enhancers/promoters) regions affect transcription. completely block gene expression. developmental issues or diseases. Splicing Mutations at exon/intron boundaries defective mRNA and protein, as result in nonfunctional proteins, or within introns can disrupt splicing. improper splicing results in potentially causing diseases incorrect exon combinations microRNA binding sites Mutations in the 3' UTR can prevent continuous translation of mRNA, lead to overexpression of protein, microRNA from regulating gene preventing mRNA degradation or contributing to diseases or expression. translational inhibition dysregulation of cellular processes. Causes of Spontaneous Mutations Tautomeric Shifts: Bases can switch between common and rare forms (tautomers) ○ causing mispairing during DNA replication. ○ This results in transition mutations. Depurination: loss of a purine (A or G) base from DNA backbone ○ leading to improper base pairing. Deamination: Loss of an amino group from a base (e.g., cytosine deaminating to uracil) ○ leading to a mismatch. Oxidation: Oxygen radicals produced during metabolism can modify bases ○ causing mutations such as thymine glycol or oxoguanine, which mispair with incorrect bases. Trinucleotide Repeat Expansions Huntington’s Disease: Caused by trinucleotide repeat expansions (CAG) in the HTT gene. ○ Slippage during DNA replication ↑ repeats, leading to longer polyglutamine stretches in protein cause aggregation and disease. Other Diseases: ○ Kennedy Disease: CAG repeats in androgen receptor gene cause progressive muscle weakness. ○ Fragile X Syndrome: CGG repeat expansion in FMR1 gene leads to mental impairment & autism. Larger repeats (>200) cause hypermethylation, silence transcription and loss of function. Base Modification by Alkyl Groups Ethyl Methanesulfonate (EMS): Common mutagen used especially with C. elegans and Drosophila. ○ Causes alkylation (addition of an alkyl group). Methylnitrosonitrosoguanidine (MNNG):Modifies bases to alter hydrogen bonding. ○ Ex. G typically pairs with C, but after alkylation, it pairs with T, leading to transition mutations. Base Damage by Bulky Adducts Aflatoxin B1: potent mutagen produced by Aspergillus fungus found in corn and peanuts. ○ Forms bulky adducts that block DNA replication and transcription. ○ Breaks glycosidic bond between ribose sugar and guanine, replacing it with bulky adducts. ○ Affects DNA polymerase and RNA polymerase II, preventing them from functioning properly.  Benzo(a)pyrene (Diol epoxide): Produced by internal combustion engines. ○ Similarly causes bulky adduct formation, leading to transcription and replication blockage. Incorporation of Base Analogs:Chemicals that resemble normal nitrogen bases and get incorporated into DNA. 5-bromouracil (5-BU): Analog of Thymine—causes pairing with A ○ Bromine replaces methyl group, altering base interactions and electrons 2-aminopurine (2-AP): Analog of Adenine. ○ Mis-pairs with thymine, causing AT to GC transition mutations. ○ G can also be affected when protonated, causing mispairing and a GC to AT transition. Intercalating Agents: slips between bases Proflavin, Acridine Orange: Chemicals slip b/ween base pairs of DNA, causing indels DNA polymerase misreads sequence, leading to mutations. Used in mutagenesis studies to identify mutagenic compounds. Ames Test:detect mutagenic compound using Salmonella typhimurium strain–can’t synthesize histidine(mutation) Developed by Bruce Ames. bacteria can revert to histidine-synthesis ability through a second mutation (reversion). 1. Test setup: Grow culture on plates lacking histidine. Control: Colonies form only if bacteria revert from his- to his+ (spontaneous reversion). Experimental: Soak a filter disc in a suspected mutagen and place it on the plate. If mutagen accelerates reversion (mutation), you will see an increase in bacterial growth. 2. Some compounds become mutagenic only after being processed in the liver. Liver enzymes are added to simulate metabolism (S9 extract from mouse liver). If a compound is mutagenic, after metabolism, the reversion rate will increase. Types of Mutations Tested: ○ Some Salmonella strains revert through base-pair substitution. ○ Other strains require frameshift mutations. Physical Mutagens Ionizing Radiation: Includes X-rays and gamma rays. ○ High energy, short wavelength. ○ Causes free radicals, leading to: Base deletions, Single-strand breaks in DNA, Cross-linking, Chromosomal breaks, oxidative damage and mutations in DNA repair. ○ Example: Marie Curie's work on radon radiation. Non-Ionizing Radiation (UV light): Lower energy, longer wavelength. ○ Cause thymine dimers,make cross-linked thymine pairs that block DNA replication/transcription. ○ UV light will create bonding between two T to make cyclobutane ring ○ 6-4 photoproducts: Another type of damage from UV light. Importance of DNA Repair  Errors in DNA structure must be repaired to avoid mutations, which can lead to cancer or other diseases. Key players: DNA polymerases (e.g., Pol I and Pol III) and exonuclease activities. 1. Detection: DNA repair systems identify irregularities in DNA structure(abnormal bps or wrong DNA size). 2. Removal: The damaged or incorrect DNA section is removed. 3. Synthesis: Correct DNA is synthesized to replace the damaged portion. Nobel Prize winners (2015) for DNA repair mechanisms: ○ Tomas Lindahl – Base-excision repair. ○ Paur Modrich – Mismatch repair. ○ Aziz Sancar – Nucleotide excision repair. ○ contribution to linking repair system breakdown that lead to DNA replication blockage and cancer Base-Excision Repair (BER): Fixes minor base damage (e.g., alkylation, oxidation, deamination). ○ DNA glycosylases, AP endonuclease, and DNA polymerase I. ○ Removes damaged bases and replaces them with normal bases. ○ Includes direct repair mechanisms for specific damage types Photolyase: Light-dependent enzyme that splits thymine dimers. O6-methylguanine(MGMT): Repairs alkylated bases by transferring methyl or ethyl group to itself, inactivating enzyme (suicide enzyme). ○ Bacteria: O-oxoguanine: Caused by oxidative damage. DNA Glycosylases remove damaged bases, creating an AP (apurinic/apyrimidinic) site. AP Endonuclease and DNA phosphodiesterase open the site, and DNA polymerase I fills the gap. Uracil removal: Uracil is removed from DNA when cytosine is deaminated. ○ Eukaryotes: Same as in bacteria, but with additional steps: DNA polymerase can insert 1 nucleotide before excising the AP site. DNA ligase fills in the gap. In longer gap, DNA pol inserts 2-10 nucleotides, creating a flap removed by flap endonuclease followed by ligase to complete the repair. 5-methylcytosine: Deamination converts to T leading to a C-T transition mutation. Nucleotide Excision Repair (NER): DNA replication is stalled due to DNA damage or transcription blockage. Bulky adducts like thymine dimers are removed. Key Proteins: XPF-ERCC1 endonuclease: Cleaves phosphodiester bond 5' to the damage. XPG: Cleaves bond 3' to the damage. These cleave a 27-nucleotide region containing the damage. Repair Process: 1. Excision: damaged DNA is excised. 2. Synthesis: Gap is filled by RFC (Replication Factor C), PCNA (Proliferating Cell Nuclear Antigen), and DNA polymerase. 3. Ligation: Phosphodiester backbone is sealed. Thymine Dimer Repair: thymine dimer forms, replication is stalled, and XPE or XPC bind to damaged site. Bacterial Nucleotide Excision Repair 1. UVR-B helicase separates strands and releases UvrA. 2. UvrB recruits UvrC (endonuclease) to cleave 8 nucleotides 5' and 4-5 nucleotides 3' of damage.  3. UvrD helicase and DNA polymerase I excise the damaged region. 4. DNA polymerase I fills the gap, and DNA ligase seals the backbone. Mismatch Repair (MMR): Fixes mismatched base pairs that are missed by proofreading during DNA replication. Corrects replication errors missed by DNA pol III proofreading DNA polymerase III typically makes 1 error per 10 million base pairs. Loss of mismatch repair increases mutation rates and is associated with hereditary forms of colon cancer. 1. Recognition: MutS protein recognizes distortion in the DNA caused by the mismatch. MutS recruits MutL and MutH proteins to the site. 2. Nicking the Daughter Strand: MutH nicks newly synthesized strand at GATC methylation site. daughter strand not yet methylated (only after replication), so it is identified as incorrect strand. 3. DNA Unwinding: UVRD (a helicase) binds to nick and unwinds DNA around the mismatch ○ carry an exonuclease to excise the incorrect region. parental strand is protected during this process. 4. Excision and Repair: section of the daughter strand containing the mismatch is excised. DNA polymerase III synthesizes the correct DNA to fill the gap. DNA ligase seals the phosphodiester backbone. Defects in DNA Repair Systems and Diseases Xeroderma Pigmentosum (XP): Leads to skin cancer, extreme sensitivity to UV light, and premature aging. ○ disorder caused by defective nucleotide excision repair (NER). Cockayne Syndrome: Affects development, causing dwarfism, mental retardation, and premature aging. ○ Results from defects in NER. ○ CSA or CSB proteins mutated, preventing RNA pol II from transcribing through thymine dimers. ○ TFIIH (transcription factor) binds and helicase unwind the region around the dimer. ○ XPF and RPA are recruited to cleave the damaged region. ○ DNA polymerase synthesizes new DNA, and the backbone is ligated. Translesion Synthesis(SOS Mechanism): Allows DNA replication to continue past lesions or damage in the template strand, preventing replication fork stalling and cell death. 1. replicative polymerase (such as DNA Pol III) stalls when it encounters DNA damage or lesions 2. Ubiquitination of the Beta Clamp: ○ beta clamp (or PCNA, proliferating cell nuclear antigen) is ubiquitinated, changing conformation. ○ marks the clamp for the recruitment of TLS polymerase (e.g., DNA Pol V). 3. TLS polymerase (e.g., DNA Pol V) replaces the replicative polymerase. ○ larger active site and can accommodate damaged bases but does not prioritize accuracy. ○ synthesizes past lesion, incorporating incorrect nucleotides to continue replication lacks proofreading activity (no 3’-5’ exonuclease). 4. Resumption of Normal Replication: ○ Once TLS polymerase has bypassed the lesion, ubiquitination of the beta clamp is reversed. ○ The regular replicative polymerase resumes synthesis, continuing normal DNA replication. Double-Stranded Break Repair Mechanisms  caused by X-rays or free radicals–dangerous bc disrupt DNA’s ability to use complementarity for repair. breaks can be repaired through two main mechanisms: 1. Non-Homologous End Joining (NHEJ): Quick repair, but often mutagenic due to errors at the break site Ku70/80 proteins bind to DNA ends. Artemis' processes ends to blunt them. Ends are ligated together, potentially causing errors. 2. Homologous Recombination (HR): Precise repair, with potential genetic exchange between chromatids. Accurate repair using a homologous template (e.g., sister chromatid). D-loop forms with homologous DNA. Holliday junction is resolved by resolvase enzymes. SDSA (Synthesis-Dependent Strand Annealing) uses helicases and polymerase for accurate repair. Differences Between Cancer Cells and Normal Cells 1. Rapid Division and Metabolism: Cancer cells divide quicker and have a higher metabolic rate 2. Invasiveness: Cancer cells can invade surrounding tissues. 3. Secretion of Proteins or Hormones: angiogenic factors promote growth of new blood vessels (angiogenesis) to supply oxygen and nutrients to the tumor. 4. Uncontrolled Growth: keep dividing in response to signals from neighboring cells(no contact inhibition). Genetic Changes in Cancer Cells 1. Oncogenes (Gain of Function, Dominant Mutations) ○ Oncogenes are mutated proto-oncogenes (normal genes that regulate cell growth). ○ Mutations in 1 allele of oncogene lead to constant activation, driving uncontrolled cell division. ○ Ex. Mutations in growth factor receptors or signaling pathways. 2. Tumor Suppressor Genes (Loss of Function, Recessive Mutations) ○ normally prevent uncontrolled cell growth and promote apoptosis. ○ For cancer to develop, both alleles of a tumor suppressor gene must be mutated. ○ Ex. Mutations in BRCA1 and BRCA2 are linked to breast cancer. Functions of Oncogenes vs. Tumor Suppressors Oncogenes (Gain of Function): ○ Promote cell-cycle progression. ○ Inhibit apoptosis. ○ Drive cancer by maintaining constant activity (e.g., growth factors, transcription regulators). ○ growth factor receptors, signal transduction proteins, transcription and apoptosis regulators Tumor Suppressors (Loss of Function): ○ Inhibit cell-cycle progression. ○ Promote apoptosis. ○ Promote DNA repair. ○ loss leads to cancer (e.g., mutations in tumor suppressors like p53). Large-scale Chromosomal Variation Polyploidy: Extra complete sets of chromosomes (e.g., 3n = triploidy)  ○ Found in spontaneously aborted human fetuses Aneuploidy: Abnormal number of chromosomes (too many or too few) ○ Trisomy (2n + 1): Extra copy of a chromosome ○ Monosomy (2n - 1): Loss of one copy of a chromosome ○ Origin: meiotic nondisjunction (chromosomes fail to separate properly after metaphase) Common Human Aneuploidies Condition Chromosome symptoms/features Turner syndrome X(monosomy) - short stature, spaced nipples, aorta constriction, poor breast development, no period Klinefleter syndrome XXY - tall stature, no frontal baldness, poor beard growth, small testes, breast development Triple X syndrome XXX - normal female development(third X chromosome likely inactivated) Down syndrome Trisomy 21 - mild to moderate intellectual disability, short stature, heart defects Patau syndrome Trisomy 13 - severe developmental issues, often fatal by infancy Edwards syndrome Trisomy 18 - severe developmental delays, most die shortly after birth Robertsonian Translocation: A fusion of chromosomes 21 and 14. ○ lead to trisomy 21 if a parent carries translocation (partial chr. 21 attached to chr. 14). ○ Unaffected children may be normal or translocation carriers. ○ Lethal trisomy 14 (insufficient chromosomes) may occur from this type of translocation. Origins of Chromosomal Rearrangements Type Description examples/mechanisms Non-allelic recombination - Crossing over b/ween non-homologous chromosomes Rapid rearrangements due to repetitive - often due to repetitive DNA DNA(deletions. duplications) Deletions Loss of chromosomal segment Cri du chat(chr 5 deletion) William syndrome(chr 7) Duplications extra copies of a chromosomal segment Can occur during unequal crossing over Inversions Reversal of a chromosomal segment - paracentric(one arm) - pericentric(spans centromere) Translocations Exchange of chromosomal segments b/ween may lead to fusion genes or gene misexpression non-homologous chromosomes Deletions: Causes of Deletions: 1. Heterozygous Reciprocal Translocation: parent carrying a reciprocal translocation (a swap of chromosome segments) may pass on a chromosome with deletions due to improper segregation.  2. Recombination in a Heterozygote: During meiosis, recombination in a heterozygote may lead to deletions. 3. Pericentric Inversion:chromosome with pericentric inversion can cause deletions when homologous chromosomes undergo recombination. William Syndrome Deletion: 1.5 Mb on chromosome 7 ○ Removes 17 genes, bounded by 2 copies of PMS gene (DNA repair protein) ○ Leads to aberrant development of the nervous system—Hypersocial behavior, singing ○ Autosomal dominant ○ Origin: Arises spontaneously in a normal parent’s gonad (not seen in parents) ○ Mechanism: Unequal crossing over b/ween sister chromatids, leading to deletion and duplication Inversions Paracentric Inversion: Inversion on one arm of the chromosome (one side of centromere). ○ May not affect gene function or may disrupt it. ○ Breakpoints between genes may be insignificant or cause gene loss. ○ More likely to cause lethal deletions ○ Result: Crossing over at inversion loop forms a dicentric bridge Chromosomes attempt to align, causing a circular molecule. Segregation at anaphase I cause acentric fragments loss & forming terminal deletions. second meiotic event splits the centromere, producing lethal deletion products. Pericentric Inversion: Inversion spans across centromere, affecting both chromosome arms. ○ May cause gene disruption. ○ Result: Crossover in the inversion loop leads to deletions and duplications During meiosis I, incorrect chromosome combinations occur. By the end of meiosis II, products may include: Normal product Duplication of A arm, deletion of D arm Duplication of D arm, deletion of A arm Inversion product Breakpoints: ○ Between genes: May lead to loss of function of the affected gene. ○ Within genes: result in chimeric protein, that may be beneficial or bad through evolution. Example: Cri du Chat syndrome may arise from deletions due to pericentric inversions. Mechanisms of Chromosomal Rearrangements 1. Unequal Crossing Over:Leads to duplications and deletions between chromosomes. ○ William Syndrome: Deletion caused by unequal crossing over. 2. Inversions: ○ Paracentric Inversion: Only one arm is affected; can lead to deletions during meiosis. ○ Pericentric Inversion: Spans the centromere; may result in duplications and deletions. ○ Meiosis Impact: chromosomal instability, generating deleted or duplicated regions 3. Reciprocal Translocation: Parts of chromosomes exchange places; may be balanced (no loss of genetic material) or unbalanced. Reciprocal Translocation Behavior during Meiosis  Independent Assortment: Chromosomes assort independently during meiosis, but translocated chromosomes may not follow normal patterns. Segregation Types: ○ Adjacent-1 Segregation:Leads to inviable gametes with duplications and deletions. Ex. Duplication of one chromosome arm and deletion of the other after translocation. ○ Alternate Segregation: Produces viable gametes with balanced translocations. Ex.One chromosome arm translocated and the other retained. Cancer by Somatic Translocation Burkitt Lymphoma: Relocation of an oncogene (MYC) next to a new regulatory element. ○ Chromosome 8 contains the MYC gene. ○ Chromosome 14 contains the IG (Immunoglobulin) gene. ○ Translocation occurs between chromosomes 8 and 14. ○ MYC gene comes under control of the IG regulatory region (Reg^IG), causing constant activation of MYC, which drives tumor formation. Chronic Myelogenous Leukemia (CML): hybrid oncogene from transloc. b/ween chr. 9 and 22. ○ Chromosome 9 (ABL gene) and chromosome 22 (BCR1 gene) are split and translocated. ○ fusion of the ABL and BCR1 genes create hybrid protein that acts as an unregulated kinase, driving uncontrolled cell growth. CHAPTER 12: REGULATION OF TRANSCRIPTION IN EUKARYOTES Transplantation Experiments Dorsal Lip of Blastopore (Spemann's Organizer) ○ Experiment: Transplant dorsal lip of blastopore to ventral region of recipient embryo. Observed two separate dorsal lips and induction of a secondary embryonic axis. Resulted in the formation of a Siamese twin in Xenopus laevis embryos. ○ Why Xenopus laevis? pigmented eggs, allowing clear identification of the dorsal surface (black). Limb Bud Development and Zone of Polarizing Activity (ZPA) Setup: Transplant ZPA from posterior to anterior limb bud position in chick embryos. Result: Extra digits form with reverse polarity (mirror image of original digits). Sonic Hedgehog (Shh): responsible for reverse polarity and formation of extra digits. Polydactyly: Misexpression of Shh in atypical limb regions induces extra digits. Mutations in cis-acting regulatory elements controlling Shh expression (not coding region). Properties of Cis-acting Mutations: ○ Phenotypes are often dominant due to regulatory effects in cis. ○ Specific regulatory elements affected; other gene functions may remain normal. Homeotic Mutants in Drosophila Homeotic Genes: Control the identity of body parts. ○ Wildtype: One pair of wings on the second thoracic segment, haltere on the third. ○ Bithorax Mutant: Wings develop on the third thoracic segment (misexpression of wing genes). ○ Antennapedia Mutant: Legs develop where antennae normally are (misexpression of leg genes).  Hox Genes: group of related genes that control the body plan of an embryo along the anterior-posterior ○ Two complexes in Drosophila: Bithorax (controls thoracic and abdominal segments) and Antennapedia (controls head and thoracic segments). ○ Gene Order: The order on the chromosome corresponds to the body region from head to tail. Methods for Gene Expression Visualization In Situ Hybridization: Detect mRNA localization. Antibody-based protein expression and cDNA clones for gene identification. Hox Gene Expression ○ Spatially restricted domains: Hox genes express early in embryogenesis, controlling segment. ○ Mutagenesis screens (Wieschaus, Lewis, Nüsslein-Volhard): Discovered maternal and zygotic genes controlling Hox gene expression. Drosophila Development: Gene Gradients 1. Maternal Genes: ○ Bicoid: Expressed at the anterior; establishes gradient to activate anterior genes. ○ Nanos: Expressed at the posterior; creates gradient for posterior genes. 2. Gap Genes: ○ Kruppel, Knirps, Giant, Hunchback: Control large segment areas. ○ Bicoid mutants: Missing anterior; Hunchback gene activated. 3. Pair-rule Genes: ○ Even-skipped: Affects odd-numbered segments. ○ Fushi tarazu: Affects even-numbered segments. ○ Stripe formation: Controlled by maternal and gap gene combinations. 4. Segment Polarity Genes: Dictate anterior vs. posterior borders of segments. ○ Engrailed and Wingless are key in segment patterning. Hedgehog Signaling Pathway Engrailed: Expresses Hedgehog (Hh), a paracrine signaling factor. Patched: Receptor for Hh on neighboring cells, stimulates Wingless (Wg) production. Feedback Loop: Wg binds to Frizzled receptors on engrailed cells, stabilizing expression boundaries. MicroRNA and Translational Control MicroRNA (miRNA): Regulates mRNA stability and translation, derived from genes or introns. ○ Processed by Dicer to form double-stranded RNA, then unwound by RISC. ○ Lin-4 and let-7 (e.g., in C. elegans): Control transitions b/ween developmental stages by repressing specific protein expressions. Let-7 miRNA: ○ In mammals, it plays a crucial role in cell differentiation, division cycle, limb/brain development. ○ Tumor suppressor function: Loss or overexpression linked to cancers (e.g., lung, breast). ○ Target genes: Ras, Myc, Cyclin D, CDK6. mRNA-binding Proteins: GLD-1 represses translation in posterior cells by binding glp-1 mRNA. Translational Control in Cell Lineage Determination  Example: GLP-1 mRNA in C. elegans Anterior Cells: GLP-1 protein expressed. Posterior Cells: Translation blocked by GLD-1 protein binding to mRNA. Experimental Techniques for Gene Expression In Situ Hybridization (mRNA Localization): 1. Synthesize single-stranded RNA probe complementary to target mRNA. 2. Incubate with fixed embryos/tissues. 3. Add enzyme-conjugated antibodies and substrates for visualization. Immunolocalization (Protein Expression): 1. Inject protein into the host to produce antibodies. 2. Incubate tissues with primary and fluorochrome-conjugated secondary antibodies. 3. Visualize under fluorescence microscope.