Discovering the material for heredity: DNA Ch. 13 Biology In Focus AP Biology 2014

Time Line Frederick Griffith (1928) Griffith discovers that bacteria can change from one form to another. Oswald Avery & colleagues (1944) follow up Griffith’s earlier discovery and conclude that the transforming factor is DNA. Rosalind Franklin & Maurice Wilkins (1950) provide evidence that DNA is in the form of a double helix. Edwin Chargaff (1951) publishes that the nitrogenous bases of DNA occur in a ratio, with equal amounts of adenine and thymine, and cytosine and guanine. Alfred Hershey & Martha Chase (1952) Hershey and Chase conduct experiments proving that DNA was the hereditary material. James Watson & Francis Crick (1953) publish the three dimensional structure and composition of DNA.

“ Ancient ” History By the 1920's chromosomes were suspected as carriers of genetic information based on observations of mitosis. Biochemical studies of chromosome composition demonstrated that they were composed of % nucleic acid and % protein. It was generally believed that PROTEINS would prove to be the carriers of genetic information. WHY?

Genetic Transformation Discovered Fred Griffith unwittingly discovered transformation. He showed that some “active genetic substance” could be transferred from a dead bacteria capable of causing disease to a live harmless bacteria – making this bacteria dangerous. How did he accomplish this?

Griffith’s Experiment Griffith was attempting to develop a vaccine for Streptococcus pneumoniae. Griffith was attempting to develop a vaccine for Streptococcus pneumoniae. There were two strains of Streptococcus, one of which was harmless to people. The other strain caused pneumonia. The term for the ability of an organism to cause disease is virulence. There were two strains of Streptococcus, one of which was harmless to people. The other strain caused pneumonia. The term for the ability of an organism to cause disease is virulence.

More on Streptococcus… Strain #1: S strain – W– W– W– Was called S strain because it formed smooth colonies on a petri dish culture. –H–H–H–Had a polysaccharide coat that protected it from attack by the immune system. –W–W–W–Was virulent.

Still More on Streptococcus… Strain #2: R strain –W–W–W–Was called R strain because it formed rough colonies on a petri dish culture. –D–D–D–Did not have the polysaccharide coat that protected it from attack by the immune system. –W–W–W–Was avirulent (harmless).

Griffith and later Oswald Avery & his colleagues perform the following experiment… Live S strain injected into a mouse yields a dead mouse. Live S strain injected into a mouse yields a dead mouse. Live R strain injected into a mouse yields a live mouse. Live R strain injected into a mouse yields a live mouse. BUT… what if you heat kill the S strain and add it to the live R strain? BUT… what if you heat kill the S strain and add it to the live R strain?

“ Something ” (we now know this to be DNA) from the virulent S strain of bacteria had been able to transform the harmless R strain into a mouse killer!

Avery & colleagues… Avery and his colleagues repeated Griffith ’ s experiment but added an additional step. Avery and his colleagues repeated Griffith ’ s experiment but added an additional step. First they added a protein-destroying enzyme to the heat-killed S strain. Mice still died. First they added a protein-destroying enzyme to the heat-killed S strain. Mice still died. They repeated the experiment but the second time added a DNA-destroying enzyme to the heat-killed S strain. The mice didn ’ t die! They repeated the experiment but the second time added a DNA-destroying enzyme to the heat-killed S strain. The mice didn ’ t die! The “ transforming factor ” had to be DNA!! The “ transforming factor ” had to be DNA!!

Alfred Hershey & Martha Chase Even after Avery ’ s experiments, scientists were still skeptical about the possibility that DNA was the “ stuff of heredity. ” Even after Avery ’ s experiments, scientists were still skeptical about the possibility that DNA was the “ stuff of heredity. ” In 1952, Alfred Hershey & Martha Chase performed an elegant series of experiments which proved that DNA was the genetic material – using a household blender! In 1952, Alfred Hershey & Martha Chase performed an elegant series of experiments which proved that DNA was the genetic material – using a household blender!

Hershey and Chase used?  Hershey and Chase used the bacteriophage, a virus which infects and kills bacterial cells.  Viruses infect living cells and then multiply inside these cells, producing millions of copies of the virus which then explode the cell, releasing these copies to go out and infect more cells.

Experimental Predictions If the virus carried the instructions for making copies of itself (its genetic material) in the form of protein, then the virus would have to inject its protein into the bacteria. If the virus carried the instructions for making copies of itself (its genetic material) in the form of protein, then the virus would have to inject its protein into the bacteria. If the virus carried the instructions for making copies of itself (its genetic material) in the form of DNA, then the virus would have to inject its DNA into the bacteria. If the virus carried the instructions for making copies of itself (its genetic material) in the form of DNA, then the virus would have to inject its DNA into the bacteria.

The Experiment Use a batch of virus with radioactive protein shells ( 35 S). Use a batch of virus with radioactive protein shells ( 35 S). Use a second batch of virus with radioactive DNA ( 32 P). Use a second batch of virus with radioactive DNA ( 32 P). Allow each to infect bacteria (E. coli), then remove the virus on the outside using a blender. Allow each to infect bacteria (E. coli), then remove the virus on the outside using a blender. Collect the E. coli bacteria and see whether they contain 35 S (radioactive protein) or 32 P (radioactive DNA) on the inside. Collect the E. coli bacteria and see whether they contain 35 S (radioactive protein) or 32 P (radioactive DNA) on the inside.

Results E. coli bacteria in the pellet contained virtually no 35S. The offspring of the virus (its progeny) contained lots of 32P-labelled DNA. Conclusion: DNA carried the genetic information!!

What did we know about DNA? We knew that it was composed of chains of four nucleotides – containing four different nitrogenous bases. –A–A–A–Adenine –T–T–T–Thymine –C–C–C–Cytosine –G–G–G–Guanine

What is a nucleotide?

Nitrogenous Bases

One strand = nucleotides covalently linked, forming a sugar-phosphate backbone

Purines & Pyrimidines The bases with one ring are pyrimidines. The bases with one ring are pyrimidines. –Cytosine –Thymine The bases with two rings are purines. The bases with two rings are purines. –Adenine –Guanine

Chargaff ’ s Rule 1949 – Edwin Chargaff noticed that in every analysis of DNA that he performed, the amount of adenine present always equaled the amount of thymine and that the amount of cytosine always equaled the amount of guanine – Edwin Chargaff noticed that in every analysis of DNA that he performed, the amount of adenine present always equaled the amount of thymine and that the amount of cytosine always equaled the amount of guanine. There could be different overall amount of C & G and A & T – but these pairs of bases were always present in equal ratios! There could be different overall amount of C & G and A & T – but these pairs of bases were always present in equal ratios! This is called Chargaff ’ s Rule. This is called Chargaff ’ s Rule.

Rosalind Franklin & Maurice Wilkins Rosalind Franklin, a talented X-ray crystallographer working with Maurice Wilkins, developed X- ray diffraction images of DNA. Rosalind Franklin, a talented X-ray crystallographer working with Maurice Wilkins, developed X- ray diffraction images of DNA. Her data indicated that DNA existed in the form of a double helix. Her data indicated that DNA existed in the form of a double helix.

James Watson & Francis Crick

What they knew… Chargaff’s Rule: Adenine & thymine occur in equal amounts, as does cytosine and guanine. Rosalind Franklin’s data indicated that the molecule was a double helix.

What they finally figured out… DNA is a two-stranded double helix - like a twisted ladder! DNA is a two-stranded double helix - like a twisted ladder! The legs of the ladder are composed of the phosphates and deoxyribose sugars of the nucleotides (phosphate-sugar backbone). The legs of the ladder are composed of the phosphates and deoxyribose sugars of the nucleotides (phosphate-sugar backbone). The rungs are composed of the nitrogenous bases, which stick together by hydrogen bonding. The rungs are composed of the nitrogenous bases, which stick together by hydrogen bonding. A hydrogen bonds with T and G hydrogen bonds with C A hydrogen bonds with T and G hydrogen bonds with C

Base-pairing between the two strands of DNA

Yeah DNA!!

DNA structure cliff notes Composed of two chains, or strands, of nucleotides (A, T, C and G) Composed of two chains, or strands, of nucleotides (A, T, C and G) The nucleotides in one strand are connected by covalent bonds The nucleotides in one strand are connected by covalent bonds The two strands are held together by weak hydrogen bonds – A bonds with T and C bonds with G The two strands are held together by weak hydrogen bonds – A bonds with T and C bonds with G In cells, the DNA curls around forming a double-helix – but we often visualize DNA as a flat ladder because it is a little easier to see the nucleotides… In cells, the DNA curls around forming a double-helix – but we often visualize DNA as a flat ladder because it is a little easier to see the nucleotides…

Strands run in opposite directions = antiparallel