1. Search for the Genetic Material
Seminal characteristics of genetic material
First, it must be stable enough to store information for long periods
Second, it must be able to replicate accurately
Finally, it must be capable of change to allow evolution to proceed
Scientists believed for a long time that chromosomes were the carriers of heredity.
A chromosome is composed of chromatin, which is 20% DNA and 80% Protein
So which is the heritable material, DNA Or Protein? Experiments will provide the answer…

2. Authored by Peter J. Russell
CHAPTER 2 DNA: The Genetic Material

3. Of note: Microbiologists commonly refer to bacterium as ‘bugs’

4. Griffith’s Transformation
They used different strains of the bug Streptococcus pneumoniae to infect mice
R type (no capsule): non virulent
S type (capsule): virulent
Notice the appearance of the smooth strain colonies; this tells us why the smooth strain is so virulent. The surface character of the bug allows it to evade the immune system (clever little bastards!)

5. Conclusion: Something was passed from the dead smooth bugs
Figure 2.2 Griffith’s transformation experiment. Mice injected with type IIIS pneumococcus died, whereas mice injected with either type IIR or heat-killed type IIIS bacteria survived. When injected with a mixture of living type IIR and heat-killed type IIIS bacteria, however, the mice died.
Conclusion: Something was passed from the dead smooth bugs
to the living rough ones, allowing them to transform their surface

6. MacLeod, McCarty and Avery’s Transformation
They broke open dead cells of S. pneumoniae, purified the components and determined which was capable of transforming live cells
Conclusion: nucleic acids act as the transforming agent.

7. Hershey and Chase’s Bateriophage Assay
In this very clever and elegant Nobel Prize winning assay, Escherichia coli bacterium were infected by selectively radiolabeled bacteriophage T2
Bacteriophages are viruses that only infect bacteria (we just call them ‘phage’)
Phages replicate by a lytic life cycle
Viruses have the genetic material (nucleic acid) enclosed within a protein coat.
Nucleic Acid: radiolabeled with 32P; Protein: with 35S
Lets roll to the experimental footage…

8. Figure 2.4 Electron micrograph and diagram of bacteriophage T2 (1 nm = 10-9m).

9. Animation: Phage T2 Reproductive Cycle
Figure 2.5 Lytic life cycle of a virulent phage, such as T2.
Animation: Phage T2 Reproductive Cycle

10. EXPERIMENT Animation: Hershery-Chase Experiment Empty protein shell
Radioactivity (phage protein) in liquid
Radioactive protein
Phage
Bacterial cell
Batch 1: radioactive sulfur (35S)
DNA
Phage DNA
Centrifuge
Radioactive DNA
Pellet (bacterial cells and contents)
Batch 2: radioactive phosphorus (32P)
Centrifuge
Radioactivity (phage DNA) in pellet
Pellet
Animation: Hershery-Chase Experiment

11. Animation: DNA and RNA Structure
Nucleic Acid Composition and Structure
Nucleic Acids include deoxyribonucleic and ribonucleic forms (note the difference – why?).
It was known that deoxyribonucleic acid (DNA) is a nucleic acid polymer of deoxynucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group
Animation: DNA and RNA Structure

12. Nucleoside: sugar + base Nucleotide: nucleoside + phosphate
Summary
Nucleoside: sugar + base Nucleotide: nucleoside + phosphate
Polynucleotides: formed by phosphodiester (covalent) bond between the phosphate of one nucleotide and the sugar of the second nucleotide.
Figure 2.10 Chemical structures of DNA and RNA. (a) Basic structures of DNA and RNA nucleosides (sugar plus base) and nucleotides (sugar, plus base, plus phosphate group), the fundamental building blocks of DNA and RNA molecules. Here, the phosphate groups are orange, the sugars are red, and the bases are brown. (b) A segment of a polynucleotide chain, in this case a single strand of DNA. The deoxyribose sugars are linked by phosphodiester bonds (shaded) between the3’ carbon of one sugar and the 5’ carbon of the next sugar.

13. 2 Classes of Nitrogenous Bases
Purines: double-ringed, includes A and G
Pyrimidines: single-ring, includes C, U and T
Figure 2.9 Structures of the nitrogenous bases in DNA and RNA. The parent compounds are purine (top left) and pyrimidine (bottom left). Differences between the bases are highlighted.

14. The DNA Double Helix
Watson and Crick deduced the structure of DNA (milestone in Biology) without carrying out a single experiment.
Their structure had to be designed in a way so that it could explain the 3 properties of genetic material:
Able to self-replicate
Serves as the heritable unit
Has the ability to change
Their determination of the structure of DNA was based on the following experimental data...

15. In 1950, Erwin Chargaff reported that DNA composition varies from one species to the next. This evidence of diversity made DNA a more credible candidate as the genetic material (all species are different)
Chargaff’s rules state that in any species there is an equal number of A and T bases, and an equal number of G and C bases (why? – think about it)

16. Maurice Wilkins and Rosalind Franklin used a technique called X-ray crystallography to study and visualize the molecular structure of DNA
Figure 16.6 Rosalind Franklin and her X-ray diffraction photo of DNA

17. Franklin’s X-ray crystallographic images of DNA enabled James Watson to deduce the helical structure of DNA
Franklin had concluded that there were two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior
The X-ray images also enabled Watson to deduce the spacing (or length) between the nitrogenous bases, the width of the helix and the specific base-pairing therein (affirming Chargaff’s findings)
The width suggested that the DNA molecule was made up of two strands, forming a double helix

18. Animation: DNA Double Helix
Figure 16.1 How was the structure of DNA determined?
Animation: DNA Double Helix

19. 2 Polynucleotide chains are wound around in a right-handed double helix
Chains are held together by hydrogen bonding between the bases
Sugar-phosphate linkage forms the backbone; bases point inward

20. Nucleotides are spaced 0. 34 nm apart. Each turn is 3
Nucleotides are spaced 0.34 nm apart. Each turn is 3.4 nm and therefore has 10 bases/turn. The diameter of the helix is 2 nm
Sugar phosphate backbones are in opposite directions = antiparallel

21. Two hydrogen bonds between A and T
Base pairing
Two hydrogen bonds between A and T
Three hydrogen bonds between C and G
Figure 2.14 Structures of the complementary base pairs found in DNA. In both cases, a purine pairs with a pyrimidine: (a) The adenine–thymine bases, which pair through two hydrogen bonds. (b) The guanine–cytosine bases, which pair through three hydrogen bonds.

22. Reason for base pairing between A-T and G-C
Purine + purine: too wide
Pyrimidine + pyrimidine: too narrow
Purine + pyrimidine: width consistent with X-ray data of Wilkins and Franklin

23. Environment and DNA Structure
A DNA
dehydrated form
not found in a cell
right handed helix
10.9 bases/turn
Diameter 2.2 nm
B DNA
hydrated form
physiological
right handed helix
10 bases /turn
Diameter 2.0 nm
Z DNA
unknown function
zig-zag shape
left handed helix
12 bases/turn
Diameter 1.8 nm

24. Structure of RNA RNA structure is very similar to that of DNA.
It is a polymer of ribonucleotides (the sugar is ribose rather than deoxyribose
Three of its bases are the same (A, G, and C) while it contains U rather than T
RNA is single-stranded, but internal base pairing can produce secondary structure in the molecule
Some viruses use RNA for their genomes. In some it is dsRNA, while in others it is ssRNA

25. Chromosomal Organization of DNA
Cellular DNA is organized into a haploid set of linear chromosomes
A genome is the chromosome or the set of chromosomes containing all the organisms DNA
Mitochondrial and Chloroplast DNA is also present (endosymbiosis and evolution)
Prokaryotic genomes are circular

26. Viral Chromosomes The viral genome is highly variable DNA or RNA
Double or single stranded
Linear or circular
Single or segmented molecule
Figure 2.17 Electron micrograph and diagram of bacteriophage .

27. Prokaryotic chromosomes
One circular double stranded DNA chromosome is present in the nucleoid region of the cytoplasm
Other minor chromosomes are smaller mobile elements called plasmids (vectoral transfer of nuclear material)
Supercoiling (next slide…) and looping (later slide…) occurs

28. Animation: Supercoiling
A note on supercoiling and topoisomerase…
Animation: Supercoiling
B-form DNA (physiological) is relaxed
If turns of the helix are removed and circularization occurs, the molecule twists to compensate for the added tension back to an energetically favorable conformation
This process is supercoiling, and is negative or positive in character; relative to the number of base pairs/turn (read in book – very well explained!)

29. Animation: DNA Packing
Eukaryotic chromosomes
Eukaryotes have a diploid number of linear chromosomes
The cell cycle influences chromosome form
In G1, each chromosome is a singular structure
In S, individual chromosomes duplicate
During M phase, duplicated chromosomes segregate to daughter nuclei
Chromatin is a complex of DNA and protein, and is found in the nucleus of eukaryotic cells. Histones are the proteins responsible for the first level of DNA packing in chromatin, which is further organized into fibers and then a metaphase chromosome with non-histone proteins
Animation: DNA Packing

30. DNA associating proteins
DNA is associated with two protein types:
Histones:
-4 types initiate condensation
-H1 type further compacts
through nucleosome linkage
-have a positive charge
-tail modification effects
Nonhistones:
-broad and big group
-have a negative charge
-bind histones and DNA
-variable binding profile
Chromatin undergoes folding and looping throughout the cell cycle…
Figure 2.24 Basic eukaryotic chromosome structure. (a) Histone core for the nucleosome. (b) Diagram of nucleosomes in “beads-on-a-string” chromatin. A nucleosome is (c) Chromosome condensation brought about by the binding of histone H1.

31. Changes in Chromatin Structure
Nucleosome
(10 nm in diameter)
DNA double helix (2 nm in diameter) H1
Figure 16.21a Chromatin packing in a eukaryotic chromosome
Histone tail (modification)
Histones
DNA, the double helix
Histones
Nucleosomes, or “beads on a string” (10-nm fiber)

32. Nucleosome (30-nm fiber) Looped domains (300-nm fiber)
Chromatid
(700 nm)
30-nm fiber
Loops
Scaffold
300-nm fiber
Figure 16.21b Chromatin packing in a eukaryotic chromosome
Scaffold-associated (SAR) regions bind nonhistone proteins to form loops that spirally radiate out
Replicated chromosome (1,400 nm)
Nucleosome (30-nm fiber)
Looped domains (300-nm fiber)
Metaphase chromosome

33. Summary…
Histones
Are proteins responsible for the first level of DNA packing in chromatin. Chromatin is further organized into fibers
Histones undergo chemical modifications during the cell cycle, causing changes in chromatin organization (more or less condensation)
10-nm fiber
DNA winds around H2a, b, H3 and H4 histones to form nucleosome “beads”
Nucleosomes are strung together like beads on a string by linker DNA
30-nm fiber
Interactions between nucleosomes, due to H1 histones, cause the thin fiber to coil or fold into this thicker fiber
300-nm fiber
The 30-nm fiber forms looped domains that attach to nonhistone proteins which associate with nuclear scaffold
Metaphase chromosome
The looped domains coil further
The width of a chromatid is 700 nm

34. Euchromatin and Heterochromatin
Histones undergo chemical modifications during the cell cycle, causing changes in chromatin organization.
Most chromatin is loosely packed in the nucleus during interphase (so it can be replicated during S phase and be used as a template for protein synthesis). The most common form, euchromatin, is transient in its condensation and lacks repetitive DNA sequences
In contrast, heterochromatin remains condensed throughout the cell cycle. It replicates last and is transcriptionally inactive. There are 2 types:
Constitutive heterochromatin is conserved at the same sites between homologous chromosomes and contains repetitive sequences (i.e. centromeres and telomeres)
Facultative heterochromatin varies between cell types, stages of development or homologous chromosomes. It also contains condensed euchromatin (i.e. Barr body)

35. Centromeric and Telomeric DNA
Centromeres and telomeres are eukaryotic chromosomal regions with special functions
Centromeres:
Are located in the center of the chromosome
Sequences are similar (not identical) between chromosomes
They are the site of the kinetochore, which is where spindle fibers attach during mitosis and meiosis, facilitating accurate segregation of the chromosomes.
Telomeres:
Are located at the ends of the chromosome and interact with the nuclear envelope
Their DNA sequences are highly conserved tandem repeats, forming unique t- and d-structures (next slide…)
Are replicated by telomerase (more in next lecture)
Are needed for chromosomal replication and stability (protect from nuclease attack)

37. Unique-Sequence and Repetitive-Sequence DNA
Sequences vary widely in how often they occur within a genome
Unique-sequence DNA is present in one or a few copies. It includes most of the genes that encode mRNA for proteins
Repetitive DNA is present in a few to 107 copies
With exceptions for rRNA and tRNA genes, prokaryotes have mostly unique-sequence DNA
In contrast, eukaryotes have a mixture of unique (65%), dispersed and tandemly repetitive sequences
Dispersed repetitive sequences include LINEs and SINEs
- LINEs (long interspersed repeated sequences) with sequences of 1,000–7,000 bp or more. The common example in mammals is LINE-1, with sequences up to 7 kb in length, that can act as transposons
- SINEs (short interspersed repeated sequences) with sequences of 100–500 bp. An example is the Alu repeats found in some primates, including humans, where these repeats of 200–300 bp make up 9% of the genome
Tandemly repetitive sequences range from very short sequences (1–10 bp) to genes and even longer sequences. Includes centromere and telomere sequences as well as rRNA and tRNA genes

38. a word about C values…
The C value refers to the DNA content in a haploid cell
It does not correlate with genome complexity due to the inclusion of repetitive sequences.
Organisms bp/genome
E.coli X 106
Yeast X 107
Amoeba 2.9 X 1011
Fruit Fly 1.8 X 108
Frog X 1010
Humans 3.4 X 109
Corn X 109

40. Search for RNA as the genetic material
Tobacco Mosaic Virus: causes tobacco mosaic diseases in the tobacco plant
Purified RNA from the virus has the ability to cause the disease.
Treating RNA with RNase causes a loss in this infectious property.

41. The type of RNA in the virus determines the progeny type.
Figure 2.7 Demonstration that RNA is the genetic material in tobacco mosaic virus (TMV). A TMV particle consists of a helical RNA core surrounded by a helical arrangement of protein subunits. Hybrid particles were made from the protein subunits of one TMV strain and the RNA of a different TMV strain. Tobacco leaves were infected with the reconstituted hybrid viruses, and the progeny viruses isolated from the resulting leaf lesions were analyzed. The progeny viruses always had protein subunits specified by the RNA component; that is, the character of the protein coat cannot be transmitted from the hybrid particles to their progeny.

42. phage: chromosome structure varies at stages of lytic
infection of E. coli. To begin with the virus has two single-stranded, complementary (“sticky”) ends and the chromosome circularizes after infection
 Figure 2.18 chromosome structure varies at stages of lytic infection of E. coli. Parts of the  chromosome, showing the nucleotide sequence of the two single-stranded, complementary (“sticky”) ends and the chromosome circularizing after infection by pairing of the ends, with the single-stranded gaps filled in to produce a covalently closed circle.