Cell Biology: Molecular Genetics, Gene Expression, Cell Cycle, and Cancer – Comprehensive Study Notes

Ch 16 – The Structural Basis of Cellular Information: DNA, Chromosomes, and the Nucleus

Chemical Nature of Genetic Material

• Genetic material is the substance responsible for heredity and is composed of DNA, as demonstrated by classic experiments (e.g., Avery-MacLeod-McCarty, Hershey-Chase).

• Key experiments established DNA as the genetic material by showing that only DNA could transform non-virulent bacteria into virulent forms.

DNA Structure

• Watson-Crick model: DNA is a double helix with antiparallel strands held together by complementary base pairing (A-T, G-C).

• Structural variants: DNA can exist in A, B, and Z forms, differing in helical pitch and handedness.

Relaxed and Supercoiled DNA

• Supercoiling: DNA can be overwound (positive supercoil) or underwound (negative supercoil), affecting its compactness and function.

Denaturation and Renaturation

• Denaturation: Separation of DNA strands by heat or chemicals; measured by melting temperature ($T_m$).

• Renaturation: Reannealing of complementary DNA strands.

DNA Packaging

• Bacterial chromosomes: Typically circular, compacted by supercoiling and proteins.

• Bacterial plasmids: Small, circular DNA molecules; types include F (fertility), R (resistance), col (colicinogenic), virulence, metabolic, and cryptic plasmids.

• Eukaryotic DNA packaging: DNA wraps around histones to form nucleosomes, which further fold into chromatin fibers, looped domains, and ultimately heterochromatin.

• Histone code: Post-translational modifications of histones regulate chromatin structure and gene expression.

• Chromatin remodeling: ATP-dependent complexes reposition nucleosomes to regulate DNA accessibility.

• Euchromatin vs. Heterochromatin: Euchromatin is less condensed and transcriptionally active; heterochromatin is highly condensed and inactive.

• Centromere and Kinetochores: Essential for chromosome segregation; contain specific DNA sequences (CEN) and proteins.

• Telomeres: Repetitive DNA at chromosome ends, protecting against degradation.

• G banding: Chromosome staining technique revealing characteristic patterns.

• Repeated DNA: Includes tandem repeats (satellite DNA) and interspersed repeats (SINEs, LINEs).

• Organelle DNA: Mitochondria and chloroplasts contain their own circular DNA.

Nucleus Structure

• Nuclear envelope: Double membrane with outer membrane continuous with ER; supported by nuclear lamina (lamins).

• Nuclear matrix: Internal protein scaffold.

• Nucleolus: Site of rRNA synthesis and ribosome assembly.

• Nuclear pore complexes: Regulate transport of proteins (NLS), RNAs (NES), and involve GEFs and GAPs for transport directionality.

Ch 17 – DNA Replication, Repair, and Recombination

DNA Replication

• Semiconservative and bidirectional replication: Each new DNA molecule contains one old and one new strand; replication proceeds in both directions from the origin.

• Replication initiation: Involves origin recognition (oriC in bacteria), binding of initiator proteins (DnaA, DnaB, DnaC), and recruitment of single-stranded binding proteins (SSB).

• Replication in eukaryotes: Multiple origins, origin recognition complex (ORC), and minichromosome maintenance (MCM) proteins.

• Telomere and telomerase: Telomerase extends telomeres using an RNA template, counteracting shortening during replication.

DNA Damage and Repair

• Mutations: Spontaneous (tautomeric shifts, depurination, deamination) or induced (chemical, physical, biological mutagens).

• Repair mechanisms:

  • Light-dependent repair (photoreactivation)

  • Excision repair (base and nucleotide excision)

  • Mismatch repair

  • Error-prone repair

  • Double-strand break repair (nonhomologous end-joining, homologous recombination, SDSA, Holliday junction resolution)

Mobile Genetic Elements

• Transposons: Class I (retrotransposons) and Class II (DNA-only transposons); can be autonomous or nonautonomous.

• Transposition mechanisms: Replicative or conservative.

• Bacterial and eukaryotic transposons: Composite, noncomposite, and P elements.

Ch 18 – Gene Expression I: The Genetic Code and Transcription

Genetic Code and Information Flow

• Central dogma: DNA → RNA → Protein; with exceptions such as reverse transcription (retrotransposons, retroviruses).

• Genetic code: Triplet, degenerate, nonoverlapping, nearly universal; established by Nirenberg, Matthaei, and Khorana experiments.

Transcription Mechanisms

• Bacterial transcription: Involves RNA polymerase, sigma factor, promoter recognition (-10 and -35 sequences), initiation, elongation (5'→3'), and termination (Rho-dependent or hairpin loop).

• Eukaryotic transcription: Three RNA polymerases (I, II, III), multiple promoter types, general and regulatory transcription factors, preinitiation complex.

RNA Processing and Turnover

• Primary transcript: Precursor to mature RNA; includes rRNA, tRNA, mRNA.

• Processing: rRNA and tRNA processing, mRNA capping (5'), polyadenylation (3'), splicing (spliceosomes, self-splicing, alternative splicing), RNA editing.

• mRNA stability: Influenced by poly(A) tail length, AU-rich elements, and RNA-binding proteins.

Ch 19 – Gene Expression II: Protein Synthesis and Sorting

Translational Machinery

• Ribosomes: Composed of rRNA and proteins; have A, P, and E sites for tRNA binding.

• tRNA: Carries amino acids (aminoacyl-tRNA), matches codon (mRNA) with anticodon (tRNA); wobble hypothesis allows flexibility in base pairing.

• Aminoacyl-tRNA synthetase: Enzyme that attaches amino acids to tRNA.

• mRNA: Can be polycistronic (prokaryotes) or monocistronic (eukaryotes); contains untranslated regions (UTRs), start (AUG) and stop codons (UAG, UAA, UGA).

Mechanism of Translation

• Initiation: Assembly of ribosome on mRNA; involves initiation factors, special start tRNAs (fMet in bacteria, Met in eukaryotes), and recognition sequences (Shine-Dalgarno in bacteria, Kozak in eukaryotes).

• Elongation: Sequential addition of amino acids; peptide bond formation, translocation, and polyribosome formation.

• Termination: Release factors recognize stop codons, release polypeptide.

• Folding and chaperones: Molecular chaperones (Hsp70, Hsp60) assist in proper folding.

Mutations and Translation

• Types: Missense, nonsense, silent, nonstop, indels, larger-scale mutations.

• Suppression: Suppressor tRNAs, nonsense-mediated decay, nonstop decay mechanisms.

Post-Translational Processing and Protein Targeting

• Removal of N-terminal residues, zymogen activation, signal peptide cleavage, chemical modifications, protein splicing.

• Protein sorting: Cotranslational import (ER signal sequence, SRP), posttranslational import (nucleus, mitochondria, chloroplasts), retrieval tags (KDEL), transit sequences, transport complexes (TOM/TIM, TOC/TIC).

Ch 20 – The Regulation of Gene Expression

Bacterial Gene Regulation

• Catabolic pathways: Inducible operons (e.g., lac operon), regulated by inducers (allolactose), repressors, and activators (CAP-cAMP).

• Anabolic pathways: Repressible operons (e.g., trp operon), regulated by end-product repression, leader sequences, and attenuation.

• CRISPR/Cas system: Adaptive immunity in bacteria against foreign DNA.

Eukaryotic Gene Regulation

• Genomic control: Gene amplification/deletion, DNA rearrangement, chromatin remodeling (SWI/SNF), histone modifications, DNA methylation (CpG islands), noncoding RNAs (lncRNA, Xist), epigenetic inheritance (imprinting, PWS, AS).

• Transcriptional control: Core promoters, general and regulatory transcription factors, enhancers, silencers, insulators, DNA response elements (hormone, cAMP, heat-shock), homeotic genes (homeobox, homeodomain).

• Posttranscriptional control: Alternative splicing, mRNA export, mRNA stability (poly(A) tail, AREs), RNA interference (siRNA, miRNA, piRNA), lncRNAs.

• Posttranslational control: Ubiquitin-proteasome pathway, SUMOylation, autophagy (macro-, micro-, chaperone-mediated).

Ch 21 – Molecular Biology Techniques for Cell Biology

DNA Analysis and Manipulation

• Gel electrophoresis: Separation of DNA by size using agarose or polyacrylamide gels; visualized with ethidium bromide.

• Restriction endonucleases: Enzymes that cut DNA at specific palindromic sequences, producing blunt or sticky ends.

• Southern blotting: Detection of specific DNA sequences by hybridization.

• Recombinant DNA technology: Cloning, PCR (Taq polymerase, TA cloning), DNA libraries (genomic, cDNA), DNA sequencing (Sanger method).

Genome Analysis

• Prokaryotic, viral, and eukaryotic genomes (nuclear, mitochondrial, chloroplast).

• Human Genome Project: Map-based and shotgun sequencing, contigs.

• Comparative genomics, C value paradox, phylogenetic tree of life (Bacteria, Archaea, Eukarya).

• Bioinformatics: BLAST, ENCODE, modENCODE.

• Genomics and disease: GWAS, RFLPs, SNPs, haplotypes, VNTRs, DNA fingerprinting.

RNA and Protein Analysis

• Northern blotting, RT-PCR, in situ hybridization (fluorescent probes, digoxigenin), gene expression profiling (microarray, RNAseq).

• Protein analysis: SDS-PAGE, antibodies (polyclonal, monoclonal), Western blotting, chromatography (ion-exchange, gel filtration, affinity), immunoprecipitation, mass spectrometry.

• Protein function: Site-directed mutagenesis, expression vectors, translational fusions, protein-protein interactions (immunostaining, pull-down, co-IP, Y2H, FRET, BiFC).

Gene Function Analysis

• Genetic engineering, transgenic organisms (GMOs), transgenesis methods (Ti plasmid, retrovirus vectors), transcriptional reporters (lacZ, GFP).

• Targeted gene disruption (homologous recombination, genome editing: ZFNs, TALENs, CRISPR/Cas), RNAi, morpholinos.

• Applications: Protein production, crop modification, gene therapy (AAV, RNAi, iPS cells).

Ch 24 – The Cell Cycle and Mitosis

Overview of the Cell Cycle

• Interphase: G1 (growth), S (DNA synthesis), G2 (preparation for mitosis).

• Mitotic phase: Mitosis (nuclear division) and cytokinesis (cytoplasmic division).

• Chromosomes: Chromatin condenses into visible chromosomes; mitotic spindle organizes chromosome movement.

• Cell cycle measurement: Generation time, mitotic index, autoradiography (3H-thymidine), BrdU/EdU labeling, flow cytometry, FACS.

Nuclear and Cell Division

• Mitosis stages: Prophase (spindle formation), prometaphase (spindle attachment), metaphase (alignment), anaphase (chromatid separation), telophase (reformation of nuclei).

• Spindle microtubules: Kinetochore, polar, and astral MTs.

• Cytokinesis: Animal cells (cleavage furrow, contractile ring), plant cells (cell plate, phragmoplast), bacteria (FtsZ ring).

Regulation of the Cell Cycle

• Key transition points: G1-S (restriction point), G2-M, metaphase-anaphase.

• Regulatory molecules: Cyclin-dependent kinases (Cdks), cyclins (G1, S, M), mitosis-promoting factor (MPF).

• Regulation of Cdk-cyclin: Cyclin availability, Cdk phosphorylation, checkpoint mechanisms (Rb, E2F, Mad-Bub, p53).

• Growth factors: RTK-Ras pathway, PI3K-Akt, TGFβ.

• Apoptosis: Programmed cell death (caspases, Bcl-2 family), extrinsic (death receptor) and intrinsic (mitochondrial) pathways.

Ch 25 – Sexual Reproduction, Meiosis, and Genetic Recombination

Sexual Reproduction and Meiosis

• Homologous chromosomes, sex chromosomes, ploidy: Diploid (2n), haploid (n), tetraploid (4n).

• Gametes: Produced by gametogenesis (spermatogenesis, oogenesis), parthenogenesis (asexual reproduction).

• Meiosis: Two divisions (meiosis I – reductional, meiosis II – equational), synapsis, crossing over, synaptonemal complex, nondisjunction (aneuploidy: monosomy, trisomy).

• Genetic variability: Mendel’s laws, chromosome theory, recombination, genetic mapping (map units, centimorgans).

• Bacterial and viral recombination: Transformation, transduction, conjugation, homologous recombination (Holliday junction, SDSA).

Ch 26 – Cancer Cells

How Cancers Arise

• Types: Carcinomas, sarcomas, lymphomas, leukemias.

• Tumor formation: Benign vs. malignant, stem cells, loss of anchorage dependence, telomere maintenance.

• Disruption of cell cycle and apoptosis: Defects in growth factor signaling, restriction point, apoptosis pathways.

• Cancer development: Initiation (mutagen exposure), promotion (proliferation), progression (heterogeneity).

How Cancers Spread

• Angiogenesis: Formation of new blood vessels (VEGF, FGF), balanced by inhibitors (angiostatin, endostatin).

• Invasion and metastasis: Loss of adhesion (E-cadherin), increased motility (Rho GTPases), protease secretion (MMPs), microenvironment, immune surveillance.

Causes of Cancer

• Carcinogens: Chemical (PAHs), physical (UV, ionizing radiation), biological (viruses, bacteria, parasites).

• Genetic mutations: Oncogenes (Ras, Myc, HER2), tumor suppressor genes (Rb, p53, APC), loss of heterozygosity, genetic instability.

• Epigenetic changes: DNA methylation, miRNAs.

Diagnosis, Screening, and Treatment

• Biopsy, tumor grading, screening (Pap smear, mammography, colonoscopy, PSA test).

• Treatments: Surgery, radiation, chemotherapy (antimetabolites, alkylating agents, antibiotics, plant-derived drugs), hormone therapy (tamoxifen), immunotherapy (vaccines, IFNs, ILs), molecular targeting (Herceptin), anti-angiogenic therapy (Avastin), personalized medicine.