DNA is the Genetic Material
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1 Lecture#1 DNA is the Genetic Material Readings: Griffiths et al (2004) 8th Edition: Chap. 1, 2-4; Chap. 7 pp Problems: Chap. 7: 1-25, 26, 27 Genetics has been approached from two directions. Mendel, Morgan and Sturtevant were interested in genes in terms of how they encode traits and specifically, how they are inherited. However, the other question was what are gene made of, from a chemical perspective. Whatever the substance was, it must be capable of containing the information necessary to convert one phenotype to another, and have that change be permanently heritable. What were the key experiments that lead us to the conclusion that DNA is the Genetic Material? 1. Griffiths Heat killed cells can transform bacteria. Genes are made of DNA? 2. Avery, MacLeod, & McCarty DNA transforms bacteria 3. Hershey & Chase DNA (not protein) is the genetic material 4. The structure of DNA is a double helix, which is compatible with its role as the genetic material. Frederick Griffith in "Basic Research" with bacterium Streptococcus pneumonia (diplococcus), which causes pneumonia in humans 2 strains: smooth (S) and rough (R). Figure 7-2 (8th) 8-1(7th) a) S - smooth colonies - virulent (causes pneumonia) ->inject -> kills mice b) R - rough colonies - non-virulent (don't cause pneumonia) ->inject -> live mouse c) heat killed S-strain cells ->injected into-> mouse -> lives \ polysaccharide coat not cause of death d) -> heat killed S-strain cells + R-strain live cells -> dead mouse. (Whereas 30 control mice R-strain lived) Further Griffith"s found that: - live cells could be recovered from these dead mice -> had - S-smooth colonies - virulent upon subsequent injections Conclude: something in the S-cell debris could convert (transform) R -> S -Process called transformation - "Transforming Principle" 1
2 Figure 7-2. The presence of heat-killed S cells transforms live R cells into live S cells. Notes: First, they did not need mice as the R and S phenotype was enough. Second, you will do the same in the lab (transformation), but using a plasmid containing an antibiotic resistance marker. Avery, MacLeod & McCarty 1944 Fig 7-3 (8th) 8-2(7th) Purified cell molecules and test each for ability to transform - purified the transforming substance - chemical composition agreed with that of DNA - had High MW (500,000) - protease treatment -> activity intact - treatment with ribonuclease -> activity intact - treatment with deoxyribonuclease -> activity lost Transforming substance is DNA -> DNA is the gene, add to R-cells -> S-cells DNA in vitro - transform But at the time there was reluctance to accept DNA as the genetic material Transforming substance - contaminated with other chemical that is really the activity - proteins were thought to be more likely since the complexity of DNA is only 4 simple bases. Proteins are more complex. Class Question: Q.) DNA may only induce a change - just a mutagenic agent? Is it possible that DNA is not actually the genetic material itself? 2
3 Fig 7-3 (8th) 8-3(7th) DNA is the agent transforming the R strain into virulence. Experiment of Hershey & Chase Bacteriophage (bacterial virus) T2 - very simple -> Protein coat outside - DNA inside Therefore the genetic material is either protein or DNA Hershey & Chase used different radioisotopes to follow each of these components during the process of infection of the bacteria and replication of the Phage. Radioisotopes: 32P to follow DNA; P not found in protein 35S labels protein; S not found in DNA Incorporated radioisotopes into phage in separate cultures - infected E.coli with many virus per cell - after sufficient time ->separated phage ghosts & cells Result 35S protein -> ghosts - protein never entered the cell 32P DNA -> did enter the cells Furthermore 32P could be recovered from the phage progeny ---> For bacteriophage T2 the genetic information is in DNA not protein Conclusion: 1- DNA is the hereditary material 2- But this was still not immediately accepted as proof that genes were DNA. 3
4 Figure 7-4 The Hershey-Chase experiment. If DNA is the hereditary material then: 1) Cells must be able to replicate DNA. How? Mold? Template? 2) Carries hereditary information. New processes needed to explain these. 3) Transfer information so as to control a cell's activity. How can a molecule carry info? 4) Must be able to change (mutate). Carry one information type then another? How? What were the basic facts that were known to Watson and Crick at the time of their discovery. DNA has a double-helix structure J. Watson & F. Crick (Nobel Prize in 1962; Watson, Crick, & Wilkins) 1953 Cambridge Univ. No experiments - work was theoretical - few facts -> synthesized the correct answer Few facts where: Biochemists -> DNA composed of 4 bases + sugar deoxyribose (form nucleotides) + phosphoric acid X-ray diffraction data (from Rosalind Franklin and Maurice Wilkins) suggested DNA was: - 2 long strands twisted around one another - thickness of ~ 20 Ångstrom Chargaff (1950) -> 4
5 - amounts of A = T, G=C - T and C small molecules - A and G are larger molecules Problem solved Put it all together -> Double Helix Model Fig 7-9, (8th) - 2 chains of nucleotides held together by phosphodiester bonds (phosphate) - 2 chains held together by hydrogen bonds in an antiparallel manner Accounts for: 1) X-ray diffraction data 2) Chargaff"s ratios A+G = (C+T) A=T G=C Implications of the structure Obvious explanation of 3 of 4 previous requirements of DNA being the genetic material are inherent in the structrure: 1) Replicate DNA with A:T G:C pairs - complementary pair -> exact duplication 2) Information carried as the sequence of bases How?-> details determined later 3) If sequence is the information then changes in sequence -> Changes in information -> mutations I expect you to know that A,G = purines and CT = pyrimidines, why they pair well (size, electrostatic interactions) and that G:C pair more tightly that A:T. 5
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