Lecture 23: DNA is the genetic

Sugar-phosphate Nitrogenous Here are a few figures from backbone bases material

5′ end O– CH 5′ 3 O P CH H O James Watson your book that correspond to O 2 O 1′ O– 4′ H N N and Francis H H H H 3′ 2′ O H Crick with their Thymine (T) Dr. Tierney’s lecture material DNA model O H H O P O CH2 N O N for Oct. 28. – O H H N H H H N N H H Adenine (A) O H H O P O CH H N 2 O H – O H H N N H H H O Cytosine (C)

O 5′ O P O CH2 H N O 1′ O – 4 O ′ H H N Phosphate (a) Rosalind Franklin (b)Franklin’s X-ray diffraction H H N 3′ 2′ N H DNA nucleotide Photograph of DNA OH H Sugar (deoxyribose) N H 3′ end H Guanine (G)

Figure 16.2 Can the genetic trait of pathogenicity be transferred between bacteria? Figure 16.3 Viruses infecting a bacterial cell

E X P E R I M E N T Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below: Phage head Living S Living R Heat-killed Mixture of heat-killed S cells (control) cells (control) cells (control) S cells and living R cells

Tail

Tail fiber

RESULTS Mouse dies Mouse healthy Mouse healthy Mouse dies DNA m n

Living S cells 0

are found in 0 blood sample. Bacterial 1 cell C O N C L U S I O N Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells.

1 Hershey and Chase experiment Is DNA or protein the genetic material of phage T2?

E X P E R I M E N T In their famous 1952 experiment, Alfred Hershey and Martha Chase used radioactive sulfur Figure 16.7 The double helix and phosphorus to trace the fates of the protein and DNA, respectively, of T2 phages that infected bacterial cells.

1 Mixed radioactively 2 Agitated in a blender to 3 Centrifuged the mixture 4 Measured the 5 end labeled phages with separate phages outside so that bacteria formed radioactivity in G C ′ the bacteria from the a pellet at the bottom of O bacteria. The phages the pellet and OH A T P Hydrogen bond infected the bacterial cells. bacterial cells. the test tube. the liquid –O 3′ end O T A OH H2C O Radioactive Empty Radioactivity T A Phage 1 nm protein protein shell (phage protein) O O O CH in liquid G C 2 35 P O S labels proteiBnascterial cell 3.4 nm –O O– C G O P O H2C O O G C Batch 1: Phages were DNA A T O CH grown with radioactive Phage C G O O 2 P O 35 DNA –O O– sulfur ( S), which was O P incorporated into phage H C O O O Centrifuge 2 C G protein (pink). T A Radioactive O O CH2 Pellet (bacterial O DNA T A – P O O O– cells and contents) O P A T O 32 H2C O P labels DNA A T O A T O CH Batch 2: Phages were OH 2 O grown with radioactive O– 3′ end P 32 phosphorus ( P), which G C O O was incorporated into 0.34 nm A T 5′ end phage DNA (blue). Centrifuge Radioactivity (a) Key features of DNA structure (b) Partial chemical structure (c) Space-filling model Pellet (phage DNA) in pellet Only labeled phosphate showed up in cells, so DNA must be the genetic material

Figure 16.8 BHase pairing in DNA

N N H O CH3 Purine + Purine: too wide

N N H N Sugar N N

O Sugar Adenine (A) Thymine (T)

Pyrimidine + pyrimidine: too narrow H

N O H N

N N H N Purine + pyrimidine: width Sugar Consistent with X-ray data N N

N H O Sugar H Guanine (G) Cytosine (C)

2 Figure 16.10 Three alternative models of DNA replication Figure 16.9 A model for DNA replication: the First Second Parent cell replication replication

(a) Conservative basic concept model. The two parental strands reassociate after acting as templates for new strands, thus restoring T A T A T A A T A T A T the parental double helix. C G C G C G C G C G C G T A T A T A T A T A T A (b) Semiconserva- tive model. A T T T A T A A A T A T The two strands G C G C G C C G C G C of the parental molecule G separate, and each functions as a template (a) The parent molecule has two (b) The first step in replication is (c) Each parental strand now (d) The nucleotides are connected for synthesis of complementary strands of DNA. separation of the two DNA serves as a template that to form the sugar-phosphate a new, comple- Each base is paired by hydrogen strands. determines the order of backbones of the new strands. mentary strand. bonding with its specific partner, nucleotides along a new, Each “daughter” DNA A with T and G with C. complementary strand. molecule consists of one parental (c) Dispersive strand and one new strand. model. Each strand of both daughter mol- ecules contains a mixture of old and newly synthesized DNA.

Figure 16.11 Does DNA replication follow the conservative, semiconservative, or dispersive C O N C L U S I O N Meselson and Stahl concluded that DNA replication follows the semiconservative model by comparing their result to the results predicted by each of the three models in Figure 16.10. model? The first replication in the 14N medium produced a band of hybrid (15N–14N) DNA. This result eliminated E X P E R I M E N T Matthew Meselson and Franklin Stahl cultured E. coli bacteria for several generations the conservative model. A second replication produced both light and hybrid DNA, a result that eliminated on a medium containing nucleotide precursors labeled with a heavy isotope of nitrogen, 15N. The bacteria the dispersive model and supported the semiconservative model. incorporated the heavy nitrogen into their DNA. The scientists then transferred the bacteria to a medium with only 14N, the lighter, more common isotope of nitrogen. Any new DNA that the bacteria synthesized would be First replication Second replication lighter than the parental DNA made in the 15N medium. Meselson and Stahl could distinguish DNA of different densities by centrifuging DNA extracted from the bacteria. Conservative 1 Bacteria 2 Bacteria model cultured in transferred to medium medium containing containing 15N 14N Semiconservative RESULTS model

Less 3 DNA sample 4 DNA sample dense centrifuged centrifuged after 20 min after 40 min Dispersive (after first (after second More model replication) replication) dense The bands in these two centrifuge tubes represent the results of centrifuging two DNA samples from the flask in step 2, one sample taken after 20 minutes and one after 40 minutes.

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