a. b. c. d. Live Nonvirulent Strain of S. pneumoniae Mixture of Heat-Killed Virulent and Live Nonvirulent Strains of S. pneumoniae

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1 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. Live Virulent Strain of S. pneumoniae Live onvirulent Strain of S. pneumoniae eat-killed Virulent Strain of S. pneumoniae Mixture of eat-killed Virulent and Live onvirulent Strains of S. pneumoniae olysaccharide coat + Mice die Mice live Mice live a. b. c. d. Mice die heir lungs contain live pathogenic strain of S. pneumoniae 1

2 SIEIFI IKI ypothesis: D is the genetic material in bacteriophage. opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. rediction: he phage life cycle requires reprogramming the cell to make phage proteins. he information for this must be introduced into the cell during infection. est: D can be specifically labeled using radioactive phosphate ( 32 ), and protein can be specifically labeled using radioactive sulfur ( 35 S). hage are grown on either 35 S or 32, then used to infect cells in two experiments. he phage heads remain attached to the outside of the cell and can be removed by brief agitation in a blender. he cell suspension can be collected by centrifugation, leaving the phage heads in the supernatant. 35 S-Labeled Bacteriophages + hage grown in radioactive 35 S, which is incorporated into phage coat Virus infect bacteria Blender separates phage coat from bacteria entrifuge forms bacterial pellet 35 S in supernatant 32 -Labeled Bacteriophages + hage grown in radioactive 32. which is incorporated into phage D Virus infect bacteria Blender separates phage coat from bacteria entrifuge forms bacterial pellet 32 in bacteria pellet Result: When the experiment is done, only 32 makes it into the cell in any significant quantity. onclusion: hus, D must be the molecule that is used to reprogram the cell. Further Experiments: ow does this experiment complement or extend the work of very on the identity of the transforming principle? 2

3 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. hosphate group 2 itrogenous base urines 2 denine itrogenous Base 2 uanine Sugar 1 in R in D yrimidines ytosine (both D and R) 3 hymine (D only) Uracil (R only) 3

4 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. 4 Base 2 hosphodiester bond 2 Base 4

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7 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. hosphate group 4 1 hosphodiester bond carbon sugar 4 1 itrogenous base 2 7

8 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. 2 nm 3.4 nm Minor groove 0.34 nm Major groove Major groove Minor groove 8

9 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. ydrogen bond Sugar Sugar ydrogen bond 3 Sugar Sugar 9

10 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. onservative Semiconservative Dispersive 10

11 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. D E. coli 15 medium E. coli cells grown in 15 medium 14 medium ells shifted to 14 medium and allowed to grow 0 min 0 rounds 20 min 1 round Samples taken at three time points and suspended in cesium chloride solution 40 min 2 rounds Samples are centrifuged 0 rounds 1 round 2 rounds op Bottom Rounds of replication From M. Meselson and F.W. Stahl/S 44(1958):671 11

12 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. emplate Strand ew Strand emplate Strand ew Strand Sugar phosphate backbone D polymerase III yrophosphate 12

13 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. R polymerase makes primer D polymerase extends primer 13

14 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. rigin ermination Replisome rigin ermination ermination ermination ermination rigin rigin Replisome rigin 14

15 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. Supercoiling Replisomes Replisomes o Supercoiling D gyrase 15

16 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. First R primer Lagging strand (discontinuous) pen helix and replicate pen helix and replicate further Second R primer R primer Leading strand (continuous) R primer 16

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18 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. D ligase Lagging strand (discontinuous) R primer D polymerase I kazaki fragment made by D polymerase III Leading strand (continuous) rimase 18

19 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. Replication first round Leading strand (no problem) Lagging strand (problem at the end) Last primer rigin Leading strand Lagging strand rimer removal Replication second round Removed primer cannot be replaced Shortened template 19

20 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. Synthesis by telomerase elomerase elomere extended by telomerase emplate R is part of enzyme elomerase moves and continues to extend telomere ow ready to synthesize next repeat 20

21 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. D with adjacent thymines UV light elix distorted by thymine dimer hymine dimer hotolyase binds to damaged D hotolyase Visible light hymine dimer cleaved 21

22 opyright he Mcraw-ill ompanies, Inc. ermission required for reproduction or display. Damaged or incorrect base Excision repair enzymes recognize damaged D Uvr,B, complex binds damaged D Excision of damaged strand Resynthesis by D polymerase D polymerase 22