DNA as Genetic Material

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Introduction to Genetic Material Definition: Hereditary substance in a cell carrying organism-specific information. Primarily composed of chemical substances with instructions for growth, development, functioning, and reproduction. Mendel's Contributions (1866): Identified "Factors" (Genes/Determiners) as carriers of traits. Chromosomal Composition: Chromosomes contain proteins (60%) and DNA (40%). Genes reside on chromosomes. Characteristics of Genetic Material Stability: Chemically stable to reliably store genetic information over time. Replication: Must replicate faithfully to pass genetic information cell-to-cell and generation-to-generation ($G_0 \rightarrow G_1$). Information Capacity: Carries complex information determining an organism's traits. Expression: Can express itself through RNA and protein synthesis. Mutability/Variation: Allows for mutations, providing genetic diversity and evolution. Location: Found in the nucleus (eukaryotes) or cytoplasm/mitochondria (prokaryotes). Compatibility: Must be compatible with cellular machinery. Is the Genetic Material Protein or DNA? Historical View (Pre-1940s): Proteins were considered genetic material due to their structural complexity and diversity (20 amino acids vs. 4 nucleotides). Proteins were abundant in chromosomes and had diverse functions. DNA: Repetitive polymer of four deoxyribonucleotides (ATP, CTP, GTP, TTP). Less quantity, less diversity led to initial skepticism. Most geneticists focused on "transmission genetics" and passively accepted proteins. Experiments later demonstrated DNA as the genetic material. Experimental Evidences for DNA as Genetic Material 1. Frederick Griffith's Experiment (1928) - Bacterial Transformation Organism: Diplococcus pneumoniae (now Streptococcus pneumoniae ). Strains: SIII (Smooth): Virulent, possessed LPS capsule, caused pneumonia, round colonies. RII (Rough): Avirulent, lacked LPS capsule, non-pathogenic, rough colonies. Steps & Results: Live SIII $\rightarrow$ Mouse dies. Live RII $\rightarrow$ Mouse lives. Heat-killed SIII $\rightarrow$ Mouse lives. Heat-killed SIII + Live RII $\rightarrow$ Mouse dies. Live SIII bacteria recovered from mouse. Conclusion: An "active principle" from heat-killed SIII transformed live RII into virulent SIII. This phenomenon was called bacterial transformation. 2. Avery, MacLeod, and McCarty's Experiment (1944) - Identifying the Transforming Principle Built upon Griffith's findings to identify the molecule responsible for transformation. Hypothesis: Purifying the active molecule from S cell extract would reveal its identity. Developed an in vitro assay to quantify transformation, moving away from mice. Experimental Setup: Grew large quantities of virulent Type III-S pneumococci. Heat-killed cells and created a crude extract. This extract transformed live, non-virulent Type II-R cells into Type III-S cells. Systematic Testing with Enzymes: Treated the active extract with specific enzymes and mixed with live non-virulent (R) strain: Treatment Target Destroyed Transformation Result Conclusion No Treatment - YES Active Proteases (Trypsin) Proteins YES Not Protein Ribonuclease (RNase) RNA YES Not RNA Deoxyribonuclease (DNase) DNA NO It's DNA! DNase Critical Test: When DNase (which specifically cuts DNA) was added, transforming activity completely disappeared. Chemical Analysis: Nitrogen-to-phosphorus ratio matched DNA, not protein. Exhibited UV absorption spectra characteristic of DNA. Reacted positively in tests for deoxyribose sugar. Conclusion: DNA is the fundamental unit of the transforming principle. It carries genetic information and causes heritable change, challenging the "protein dogma". Why Avery-MacLeod-McCarty wasn't immediately accepted: Revolutionary results met with skepticism. Critics argued possible protein contamination or that it only worked in bacteria. Full acceptance came after Hershey-Chase (1952) and Watson & Crick's DNA structure (1953). 3. Hershey and Chase's Experiment (1952) - "Proof that DNA is the Genetic Material" Organism: Bacteriophage T2 (a virus that infects bacteria). Bacteriophage Structure: Simple structure with a protein coat (capsid) and a DNA core. Phages inject genetic material into bacteria to hijack their machinery. Core Idea: Tag and Track Used radioactive isotopes to label protein and DNA separately. Protein Label: Sulfur-35 ($^{35}S$) – found in proteins (methionine, cysteine), NOT in DNA. DNA Label: Phosphorus-32 ($^{32}P$) – key component of DNA phosphate backbone, rare in proteins. Steps & Results: Labeling: Grew phages in media containing either $^{35}S$ (labeling proteins) or $^{32}P$ (labeling DNA). Infection: Allowed labeled phages to infect unlabeled E. coli bacteria. Blender Step: Agitated the mixture in a blender to shear off phage protein coats ("ghosts") from bacterial cells. Centrifugation: Separated heavier bacterial cells (pellet) from lighter phage ghosts and fragments (supernatant). Key Findings: When phages with $^{35}S$-labeled proteins infected bacteria, most $^{35}S$ remained in the supernatant (phage ghosts), and very little was found inside the bacterial pellet. When phages with $^{32}P$-labeled DNA infected bacteria, most $^{32}P$ was found within the bacterial pellet. Bacteriophage progeny carried only radioactive phosphorus ($^{32}P$), not sulfur ($^{35}S$). Conclusion: Only the bacteriophage DNA (labeled with $^{32}P$) entered the bacteria and was used to produce more bacteriophage. This definitively proved that DNA, not protein, is the genetic material. Significance: Provided definitive evidence, confirmed Avery's findings, paved the way for Watson & Crick's DNA structure, and launched modern genetics. Indirect Evidences for DNA as Genetic Material 1. Evidence from Cell Biology: Location Feulgen Stain (1914): Specific staining technique showed DNA is localized exclusively in the nucleus, specifically in chromosomes. Implication: Genetic material must be in the nucleus where chromosomes reside. 2. Evidence from Biochemistry: DNA Content is Consistent Boivin, Vendrely, and Vendrely (1948): Amount of DNA in somatic (body) cells ($2n$) of a species is constant. Amount of DNA in gametes (sex cells) ($n$) is exactly half that of somatic cells. Implication: This reflects Mendelian inheritance. DNA quantity directly correlates with ploidy, while protein content varies greatly. Nuclear division occurs only after DNA duplication during the S phase of Interphase. Diploid DNA amount varies among different species. Other Indirect Evidences DNA has consistent physical/chemical properties across organisms, yet allows for great diversity. Indefinite number of combinations possible with four bases (A, T, G, C). DNA is metabolically the most stable macromolecule. In prokaryotes, DNA is not linked with proteins, yet characters are inherited. Some Organisms Have RNA as Genetic Material Some viruses (e.g., RNA viruses or Ribo Viruses) have ssRNA or dsRNA enveloped by a protein coat. Examples of Human RNA Viruses: SARS, Influenza, Hepatitis C, Measles, Mumps, Rabies. Retroviruses: Rous Sarcoma Virus, HIV. Use Reverse Transcriptase for DNA synthesis in host cell. Examples of Plant RNA Viruses: Tobacco Mosaic Virus (TMV), Papaya Ring Spot Virus (PRV), Potato Leaf Roll Virus (PLRV), Holmesrib-grass Virus (HRV), Alfa-alfa Mosaic Virus (AMV). Evidence in Favor of RNA as Genetic Material (Fraenkel-Conrat & Singer) Experiment: Dissociation and reconstitution of Tobacco Mosaic Virus (TMV). Steps: Dissociated TMV into protein and RNA components using denaturing agents. Separated RNA and protein components. Reconstituted hybrid viruses using TMV RNA and protein coat from a related virus (Brome Mosaic Virus). Result: Hybrid viruses with TMV RNA were still infectious. Conclusion: Genetic information for infectivity and replication is carried by the RNA molecule, while protein serves a structural role. Summary Genes carry information for phenotype expression and are present on chromosomes. Chromosomes contain 60% protein and 40% DNA. Genetic material must be stable, capable of replication, store information for expression, and undergo mutation. Expression is controlled by the Central Dogma: Replication, Transcription, and Translation. Griffith's (1928), Avery et al. (1944), and Hershey & Chase (1952) experiments provided direct evidence for DNA as genetic material. Indirect evidence supports DNA as genetic material. In some organisms (e.g., certain viruses), RNA serves as the genetic material, as shown by experiments on TMV.