1. Chapter 2 - DNA: The Genetic Material
Search for genetic material---is it composed of nucleic acid or protein/DNA or RNA?
Griffith’s Transformation Experiment
Avery’s Transformation Experiment
Hershey-Chase Bacteriophage Experiment
Tobacco Mosaic Virus (TMV) Experiment
Nucleotides - composition and structure
Double-helix model of DNA - Watson & Crick
Organization of DNA/RNA in chromosomes
Prokaryotes
Eukaryotes

2. Search for the genetic material:
Stable source of information
Ability to replicate accurately
Capable of change
Timeline of events:
1890 Weismann - substance in the cell nuclei controls development.
1900 Chromosomes shown to contain hereditary information, later shown to be composed of protein & nucleic acids.
1928 Griffith’s Transformation Experiment (incorrectly guessed protein!)
1944 Avery’s Transformation Experiment (DNA not RNA)
1953 Hershey-Chase Bacteriophage Experiment (DNA not protein)
1953 Watson & Crick propose double-helix model of DNA
1956 First demonstration that RNA is viral genetic material.

3. Fig. 2.2: Frederick Griffith’s Transformation Experiment - 1928
“transforming principle” demonstrated with Streptococcus pneumoniae
Griffith hypothesized that the transforming agent was a “IIIS” protein. But this was only a guess, and Griffith turned out to be wrong.

4. Oswald T. Avery’s Transformation Experiment - 1944
Fig. 2.3:
Oswald T. Avery’s Transformation Experiment
Determined that “IIIS” DNA was the genetic material responsible for Griffith’s results (not RNA).
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

5. Hershey-Chase Bacteriophage Experiment - 1953
Bacteriophage = Virus that attacks bacteria and replicates by invading a living cell and using the cell’s molecular machinery.
Fig. 2.4
Structure of T2 phage
Bacteriophages
are composed of
DNA & protein

6. Fig. 2.5: Life cycle of virulent T2 phage:

7. T2 bacteriophage is composed of DNA and proteins:
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 Experiment
Alfred Hershey

8. Gierer & Schramm Tobacco Mosaic Virus (TMV) Experiment - 1956
Fig. 2.7 (2nd edition)
Gierer & Schramm Tobacco Mosaic Virus (TMV) Experiment
Fraenkel-Conrat & Singer
Demonstrated that RNA is the genetic material of TMV.
Numerous viruses uses RNA as the genetic material. But no known prokaryotes or eukaryotes have RNA as their genetic material.

9. 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 Hershey
Nobel Prize in Physiology or Medicine
1969

10. Nucleotide = monomers that make up DNA and RNA (Figs. 2.8)
Three components
1. Pentose (5-carbon) sugar
DNA = deoxyribose
RNA = ribose
(compare 2’ carbons)
2. Nitrogenous base
Purines (2 rings)
Adenine
Guanine
Pyrimidines (1 ring)
Cytosine
Thymine (DNA)
Uracil (RNA)
3. Phosphate group attached to 5’ carbon

11. Nucleotides are linked by phosphodiester bonds to form polynucleotides.
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!
No kidding, you can autoclave a mouse and get good PCR!
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.

12. 5’ end
3’ end
Fig. 2.9

13. Structure of DNA
James D. Watson/Francis H. Crick 1953 proposed the Double Helix Model based on two sources of information:
Base composition studies of Erwin Chargaff (Chargaff’s Rules)
indicated double-stranded DNA consists of ~50% purines (A,G) and ~50% pyrimidines (T, C)
amount of A = amount of T and amount of G = amount of C
%GC content varies from organism to organism
Examples: %A %T %G %C %GC
Homo sapiens
Zea mays
Drosophila
Aythya americana

14. Structure of DNA
James D. Watson/Francis H. Crick 1953 proposed the Double Helix Model based on two sources of information:
X-ray diffraction studies by Rosalind Franklin & Maurice Wilkins
Conclusion-DNA is a helical structure with
distinctive regularities, 0.34 nm & 3.4 nm.
Fig. 2.11

15. Double Helix Model of DNA: Six main features
Two polynucleotide chains wound in a right-handed (clockwise) double-helix.
Nucleotide chains are anti-parallel: 5’  3’
3’  5’
Sugar-phosphate backbones are on the outside of the double helix, and the bases are oriented towards the central axis.
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’
Base pairs are 0.34 nm apart. One complete turn of the helix requires 3.4 nm (10 bases/turn).
Sugar-phosphate backbones are not equally-spaced, resulting in major and minor grooves.

16. Fig. 2.13

17. Fig. 2.12 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.

18. Type A, B, and Z conformations of DNA
Fig. 2.14

19. Stop and Play the Video

20. James D. Watson Francis H. Crick Maurice H. F. Wilkins What about?
1962: Nobel Prize in Physiology and Medicine
James D.
Watson
Francis H.
Crick
Maurice H. F.
Wilkins
What about?
Rosalind Franklin

21. RNA possesses uracil (U) not thymine (T)
A pairs with U and C pairs with G
Examples:
mRNA messenger RNA
tRNA transfer RNA
rRNA ribosomal RNA
snRNA small nuclear RNA
miRNA micro RNA
siRNA small interfering RNA
RNA secondary structure:
single-stranded
Function in transcription
(RNA processing) and translation
Yeast Alanine tRNA

22. Organization of DNA/RNA in chromosomes
Genome = chromosome or set of chromosomes that contains all the
DNA an organism (or organelle) possesses
Viral chromosomes 1. single or double-stranded DNA or RNA
2. circular or linear
3. surrounded by proteins
TMV T2 bacteriophage  bacteriophage
Prokaryotic chromosomes
1. most contain one double-stranded circular
DNA chromosome
2. others consist of one or more chromosomes
and are either circular or linear
3. typically arranged in arranged in a dense
clump in a region called the nucleoid

23. Problem:
Measured linearly, the Escherichia coli genome (4.6 Mb) would be 1,000
times longer than the E. coli cell.
The human genome (3.4 Gb) would be 2.3 m long if stretched linearly.
Fig. 2.15
Chromosome released from lysed E. coli cell.

24. Stop and Play the Video Prokaryotic chromosome structure---solutions:
Supercoiling DNA double helix is twisted in space about its
own axis, a process is controlled by
topoisomerases (enzymes).
(occurs in circular and linear DNA molecules)
Stop and Play the Video
Fig. 2.17

25. Looped domains
Fig. 2.18

26. Eukaryotic chromosome structure
Chromatin complex of DNA and chomosomal proteins
~ twice as much protein as DNA
Two major types of proteins:
Histones abundant, basic proteins with a positive charge
that bind to DNA
5 main types: H1, H2A, H2B, H3, H4
~equal in mass to DNA
evolutionarily conserved
Non-histones all the other proteins associated with DNA
differ markedly in type and structure
amounts vary widely
>> 100% DNA mass
<< 50% DNA mass

27. Packing of DNA into chromosomes:
Level 1 Winding of DNA around histones to create a nucleosome structure.
Level 2 Nucleosomes connected by
strands of linker DNA like
beads on a string.
Level 3 Packaging of nucleosomes into
30-nm chromatin fiber.
Level 4 Formation of looped domains.
See Fig. 2.20

29. Fig. 2.21

30. Fig. 2.23

31. Fig Metaphase chromosome depleted of histones maintains its shape with a nonhistone protein scaffold.

32. Two other regions of DNA you should know about:
Centromeric DNA (CEN) Center of chromosome, specialized sequences function with the microtubles and spindle apparatus during mitosis/meiosis.
Telomeric DNA At extreme ends of the chromosome, maintain stability, and consist of tandem repeats. Play a role in DNA replication and stability of DNA.
Fig. 2.25

33. Repeated DNA:
Unique-sequence DNA Often referred to as single-copy and usually code for genes.
Repetitive-sequence DNA May be interspersed or clustered and vary in size.
SINEs short interspersed repeated sequences ( bp)
LINEs long interspersed repeated sequences (>5,000 bp)
Microsatellites short tandem repeats
<---TTATTATTATTATTATTATTA--->

34. More about genome size:
C value = total amount of DNA in the haploid (1N) genome
Varies widely from species to species and shows no simple relationship to structural or organizational complexity.
Examples C value (bp)
 ,502
T ,900
HIV ,750
E. Coli ,639,221
Lilium formosanum 36,000,000,000
Zea mays 5,000,000,000
Amoeba proteus 290,000,000,000
Drosophila melanogaster ,000,000
Mus musculus 3,454,200,000
Canis familiaris 3,355,500,000
Equus caballus 3,311,000,000
Homo sapiens 3,400,000,000