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How Do Genes Work? Are Genes Composed of DNA or Protein? Chromosomes, the known carriers of genes, are made of DNA and proteins Proteins are more variable in structure than DNA, which was not thought to be sufficiently complex to code for all the operations of a cell. DNA = a polymer made of 4 different types of nucleotides (adenine, cytosine, guanine, thymine) Protein = a polymer made of 20 different types of amino acids Experiments showing DNA is the genetic material ROUGH COLONY (R) SMOOTH COLONY (S) There are two strains of Streptococcus pneumoniae. R strain is benign (Lacks a protective capsule, recognized and destroyed by host’s immune system) S strain is virulent (Polysaccharide capsule prevents detection by host’s immune system) Experiments by Griffiths, 1928 Conclusion: Experiments by Avery, MacLeod and McCarty (1944) Heat-killed S cells Add proteinases Add proteinases Sample should Sample should Contain contain NO PROTEIN NO PROTEIN 1. Remove lipids and carbohydrates from a solution of heat-killed S cells. Proteins, RNA, and DNA remain. Lipids Carbohydrates Add RNAse Add ribonuclease Sample should Sample should Contain contain NO RNA NO RNA Add Add DNAse deoxy- 2. Treat solutions with Sample Sampleshould should enzymes to destroy Contain proteins, RNA, or DNA. contain NODNA DNA NO ribonuclease 3. Add a portion of each sample to a culture containing R S cells S cells No S cells cells. Observe if appear appear appear transformation Transformation occurs No transformation occurs occurs. Add R cells Add R cells Add R cells Conclusion: Transformation cannot occur unless DNA is present. Therefore, DNA must be the hereditary material. Are Genes Composed of DNA or Protein? Transformation experiments provide the first evidence that genes are DNA. • Griffith: material from dead virulent Streptococcus can transform benign Streptococcus into a virulent strain. • Avery et al.: extracted material from dead virulent Streptococcus and treated it with enzymes to destroy either DNA or RNA or proteins. Only DNA-destroying enzymes prevented transformation, hence DNA is the genetic material. Are Genes Composed of DNA or Protein? Not everyone was convinced Hershey and Chase (1952): T2 virus experiments convinces skeptics that DNA is the genetic material since the material injected by the virus into host cells is DNA, not protein. Bacteriophages are viruses that infect bacterial cells How do viruses work? Virus particle Virus Virus particle particle Host cell genome Host Host cell cell genome genome New viruses released 4. Particles assembled inside host. 1. Viral genome 1. 1. Viral Viral genome genome enters host cell. enters enters host host cell. cell. Protein 3. Viral particles produced DNA DNA mRNA mRNA 2. 2.Viral genome Viral genome is isreplicated and replicated and transcribed. transcribed. Experiments by Hershey and Chase (1952) Protein contains sulfur, but not phosphorous DNA contains phosphorous, but not sulfur Growing viruses with radioactive sulfur will label proteins but not DNA Growing viruses with radioactive phosphorous will label DNA but not proteins Experiments by Hershey and Chase (1952) The structure and replication of genetic material DNA structure was determined by James Watson and Francis Crick using data from Rosalind Franklin In order to pass on genetic material from parent (cell) to offspring (cell), the genetic material must be duplicated: DNA replication Sister chromatids Centromere One chromosome (Unreplicated state) DNA replication One chromosome (Replicated state) Figure 16.0 Watson and Crick Figure 16.4 Rosalind Franklin and her X-ray diffraction photo of DNA P Free DNA nucleotides are called dNTPs Structure of dNTPs P P 5' CH2 Base O 4' 1' 2' 3' OH Review of DNA structure: DNA and RNA are polymers made of nucleotide subunits Nucleotide Pyrimidines NH2 O – O P O 5 O N Nitrogenous base 1 4 O Phosphate 2 3 group 5-carbon sugar Ribose OH HO CH2 O 4 C H H 1C 3 2 H C C H OH OH Deoxyribose N N O H Cytosine (C) NH2 N N H3C N N NH N O H Uracil (U) 5 HO CH2 O OH 4 C H H 1C 3 2 H C C H OH H O NH N – 5 O O H Thymine (T) Purines O N N NH N H H Adenine (A) Guanine (G) NH2 5' A single strand of nucleotides is made when a phosphodiester bond is formed between the 3’ C of one nucleotide and the 5’ C of another 3' DNA is double stranded with the 2 strands held together by hydrogen bonds between complementary bases 3´ 5´ 5´ DNA is a double helix. 3´ T T A G C C T G A A T A A C G G C A T T A C G C T A T G C T A G C T A 3´Cartoon of 5´ base pairing 5´ 3´ A G C CG T T C G G A T A T G A A T G C A T 5´ Cartoon of double helix 3´ 5´ 3´ Space-filling model of double helix Figure 16.6 Base pairing in DNA Unnumbered Figure (page 292) Purine and pyridimine Distinguishing between Models of DNA Replication Three different models of how DNA might replicate were proposed based on DNA structure. • Semi-conservative replication • Conservative replication • Dispersive replication Figure 16.8 Three alternative models of DNA replication Distinguishing Between Models of DNA Replication The Meselsohn and Stahl experiment determines which model is correct. • was fed to growing E. coli cells to mark DNA (“heavy” DNA), then cells were switched to 14N. 15N • Newly synthesized DNA will incorporate 14N (“light” DNA. 15N 15N 15N 14N 14N 15N Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 1) Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 2) Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 3) Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 4)