DNA: The Genetic Material

• Search for genetic material---is it composed of protein/DNA or RNA?

• Griffith’s Transformation Experiment• Avery’s Transformation Experiment• Hershey-Chase Bacteriophage Experiment• Tobacco Mosaic Virus (TMV) Experiment• Lederberg and Tautum conjugation expt.

• Chemistry of DNA: composition and structure

• Double-helix model of DNA - Watson & Crick

Search for the genetic material:

1. Biologically useful and stable source of information2. Ability to replicate accurately and transmitted across generations3. Capable of change4. Express itself to make other biomolecules

Timeline of events:• 1900 Chromosomes shown to contain hereditary information,

later shown to be composed of protein & nucleic acids.

• 1928 Griffith’s Transformation Experiment

• 1944 Avery’s Transformation Experiment• 1946 Lederberg and Tautum’s conjugation experiment• 1952 Hershey-Chase Bacteriophage Experiment

• 1953 Watson & Crick propose double-helix model of DNA

• 1956 First demonstration that RNA is viral genetic material.

How do we know that all of our genetic information comes from

DNA?

• What type of experiment would you design to determine that DNA is the source of all genetic information?

Griffith’s Experiment with S. pneumoniae and the accidental discovery of transformation (1928)

Frederick GriffithFrederick Griffith

Rough smoothRough smooth

Streptococcus pneumoniaeGram postive bacteriaCommonly Exist as diplococci

Smooth coloniesRough colonies

Griffith’s Experiment with S. pneumoniae and the accidental discovery of transformation

Griffith’s Experiment did not prove that DNA was responsible for transformation

How would you design an experiment to prove that DNA was

responsible for transformation?

Avery, McCarty, and MacLeod Repeated Griffith’s Experiment

(1944)

Oswald AveryOswald Avery MaclynMaclyn McCartyMcCarty Colin MacLeodColin MacLeod

CarbohydratesCarbohydrates LipidsLipids ProteinsProteins RNARNA DNADNA

Heat kill Smooth Type cellsHeat kill Smooth Type cells——breakbreak------isolate RNA, DNA, etcisolate RNA, DNA, etc

Add each to Rough type bacteriaAdd each to Rough type bacteria----------only DNA could transform them to S only DNA could transform them to S typetype

CarbohydratesCarbohydrates LipidsLipids ProteinsProteins RNARNA DNADNA

To the HeatTo the Heat--Killed Smooth Type, added enzymes that Killed Smooth Type, added enzymes that destroyeddestroyed……

Add to RAdd to R--cellscells……only only DNaseDNase inactivated inactivated transformationtransformation

Conclusion:Conclusion:

DNA was the DNA was the transforming factor!transforming factor!

Bacteriophage = Virus that attacks bacteria and replicates by invading a living cell and using the cell’s molecular machinery.

Fig. 2.4Structure of T2 phage

Bacteriophagesare composed ofDNA & protein

Hershey-Chase Bacteriophage Experiment - 1953

Fig. 2.5: Life cycle of virulent T2 phage:

1. T2 bacteriophage is composed of DNA and proteins:

2. Set-up two replicates:

• Label DNA with 32P• Label Protein with 35S

3. Infected E. coli bacteria with two types of labeled T2

4. 32P is discovered within the bacteria and progeny phages, whereas 35S is not found within the bacteria but released with phage ghosts.

Fig. 2.6: Hershey-Chase Bacteriophage Blender Experiment - 1953

Alfred Hershey

The Hershey-Chase results reinforced the Avery, McCarty,

and MacLeod conclusion:

DNA carries the genetic code!

Gierer & Schramm Tobacco Mosaic Virus (TMV) Experiment - 1956Fraenkel-Conrat & Singer - 1957

Demonstrated that RNA is the genetic material of TMV.

Conclusions about these early experiments:

Griffith 1928 & Avery 1944:

DNA (not RNA) is transforming agent.

Hershey-Chase 1953:

DNA (not protein) is the genetic material.

Gierer & Schramm 1956/Fraenkel-Conrat & Singer 1957:

RNA (not protein) is genetic material of some viruses, but no known prokaryotes or eukaryotes use RNA as their genetic material.

Alfred HersheyNobel Prize in Physiology or Medicine1969

Chemistry of DNA1869: Fred Miescher isolated nuclein(acidic with high phosphorous content)1880: Emil Fischer identified purines and pyrimidines1910: Kossel identified nucleotides (base+sugar+phosphate)Got the Nobel Prize1950: Alexander Todd discovered that 2 nucleotides are linkedby 3’ to 5’ phosphodiester bond1953: Watson and Crick propose double-helical model of DNA

The Race to Discover DNA’s Structure

The Race to Discover DNA’s Structure

LinusLinus PaulingPauling 1940s1940s

Discovered the alphaDiscovered the alpha-- helical structure of helical structure of proteins.proteins.

1. Base composition studies indicated double-stranded DNA consists of ~50% purines (A,G) and ~50% pyrimidines (T, C)

2. amount of A = amount of T and amount of G = amount of C

3. %GC content varies from organism to organism

Erwin ChargaffErwin ChargaffExamples: %A %T %G %C %GC

Homo sapiens 31.0 31.5 19.1 18.4 37.5Zea mays 25.6 25.3 24.5 24.6 49.1Drosophila 27.3 27.6 22.5 22.5 45.0Aythya americana 25.8 25.8 24.2 24.2 48.4

“Chargaff’s rule”

A = T & C = G

The Race to Discover DNA’s Structure

Maurice WilkinsMaurice Wilkins Rosalind FranklinRosalind FranklinXX--Ray diffraction image of DNA Ray diffraction image of DNA

taken by Franklin in 1951taken by Franklin in 1951

Conclusion-DNA is a helical structure with distinctive regularities, 0.34 nm & 3.4 nm.

X-ray diffraction studies by Rosalind Franklin & Maurice Wilkins

The Race to Discover DNA’s Structure

19501950

ChargaffChargaff’’s Rule: s Rule: Equal amounts of Equal amounts of AAdenine and denine and TThymine, hymine, and equal amounts of and equal amounts of GGuanine and uanine and CCytosineytosine

Why do you think Why do you think the bases match up the bases match up

this way?this way?

PurinePurine + + PurinePurine = Too wide= Too wide

PyrimidinePyrimidine + + PyrimidinePyrimidine = Too Narrow= Too Narrow

PurinePurine + + PyrimidinePyrimidine = Perfect Fit from X= Perfect Fit from X--ray dataray data

James Watson and Francis Crick propose double-helical model of DNA

Double Helix Model of DNA: Six main features

1. Two polynucleotide chains wound in a right-handed (clockwise) double-helix.

2. Nucleotide chains are anti-parallel: 5’

3’3’

5’

3. Sugar-phosphate backbones are on the outside of the double helix, and the bases are oriented towards the central axis.

4. Complementary base pairs from opposite strands are bound together by weak hydrogen bonds.

A pairs with T (2 H-bonds), and G pairs with C (3 H-bonds).

5’-TATTCCGA-3’3’-ATAAGGCT-5’

5. Base pairs are 0.34 nm apart. One complete turn of the helix requires 3.4 nm (10 bases/turn).

6. Sugar-phosphate backbones are not equally-spaced, resulting in major and minor grooves.

Nucleotide = monomers that make up DNA and RNA (Figs. 2.8)

Three components

1. Pentose (5-carbon) sugarDNA = deoxyriboseRNA = ribose(compare 2’ carbons)

2. Nitrogenous base

PurinesAdenineGuanine

PyrimidinesCytosineThymine (DNA)Uracil (RNA)

3. Phosphate group attached to 5’ carbon

Nucleotides are linked by phosphodiester bonds to form polynucleotides.

Phosphodiester bond

Covalent bond between the phosphate group (attached to 5’ carbon) of one nucleotide and the 3’ carbon of the sugar of another nucleotide.

This bond is very strong, and for this reason DNA is remarkably stable. DNA can be boiled and even autoclaved without degrading!

5’ and 3’

The ends of the DNA or RNA chain are not the same. One end of the chain has a 5’ carbon and the other end has a 3’ carbon.

5’ end

3’ end

Type B-DNA

Other DNA forms include:

A-DNA:Right-handed double helix with 11 bases per turn; shorter and wider at 2.2 nm diameter. Exists in some DNA-protein complexes.

Z-DNA:Left-handed double helix with 12 bases per turn; longer and thinner at 1.8 nm diameter.

Type A, B, and Z conformations of DNAFig. 2.14

1962: Nobel Prize in Physiology and Medicine

James D.Watson

Francis H.Crick

Maurice H. F.Wilkins

What about?Rosalind Franklin