1. Review
Search for genetic material---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
Today’s Lesson
Organization of DNA/RNA in chromosomes

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
1944 Avery’s Transformation Experiment
1953 Hershey-Chase Bacteriophage Experiment
1953 Watson & Crick propose double-helix model of DNA
1956 Gierer & Schramm/Fraenkel-Conrat & Singer
Demonstrate RNA is viral genetic material.

3. Frederick Griffith’s Transformation Experiment - 1928
“transforming principle” demonstrated with Streptococcus pneumoniae
Griffith hypothesized that the transforming agent was a “IIIS” protein.

4. Oswald T. Avery’s Transformation Experiment - 1944
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.
Structure of T2 phage
DNA & protein

6. 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.
Hershey-Chase Bacteriophage Experiment
1969: Alfred Hershey

8. Gierer & Schramm Tobacco Mosaic Virus (TMV) Experiment - 1956
Fraenkel-Conrat & Singer
Used 2 viral strains to demonstrate RNA is the genetic material of TMV

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.

10. Nucleotide = monomers that make up DNA and RNA
Three components
1. Pentose (5-carbon) sugar
DNA = deoxyribose
RNA = ribose
(compare 2’ carbons)
2. Nitrogenous base
Purines
Adenine
Guanine
Pyrimidines
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!
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

13. James D. Watson & Francis H. Crick - 1953 Double Helix Model of DNA
Two sources of information:
Base composition studies of Erwin Chargaff
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
(Chargraff’s rules)
%GC content varies from organism to organism
Examples: %A %T %G %C %GC
Homo sapiens
Zea mays
Drosophila
Aythya americana

14. James D. Watson & Francis H. Crick - 1953 Double Helix Model of DNA
Two sources of information:
X-ray diffraction studies - Rosalind Franklin & Maurice Wilkins
Conclusion-DNA is a helical structure with
distinctive regularities, 0.34 nm & 3.4 nm.

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).
e.g., 5’-TATTCCGA-3’
3’-ATAAGGCT-3’
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.

18. 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

19. 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

20. 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.
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)
2. Looped domains

21. More about genome size:
C value = total amount of DNA in the haploid (1N) genome
Varies widely from species to species and shows no 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 ,000,000,000
Drosophila melanogaster ,000,000
Mus musculus 3,454,200,000
Canis familiaris 3,355,500,000
Equus caballus ,311,000,000
Homo sapiens 3,400,000,000

22. 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 (which is negatively charged)
5 main types: H1, H2A, H2B, H3, H4
~equal in mass to DNA
evolutionarily conserved
Histones leave the DNA only transiently during DNA replication.
They stay with the DNA during transcription. By changing shape
and position, nucleosomes allow RNA-synthesizing polymerases
to move along the DNA.
Non-histones all the other proteins associated with DNA differ markedly in
type and structure
amounts vary widely

23. 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.

25. More about different types 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.

26. 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 (e.g., TTA|TTA|TTA)