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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…
Authored by Peter J. Russell
CHAPTER 2
DNA: The Genetic Material
Of note: Microbiologists
commonly refer to
bacterium as ‘bugs’

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!)
Conclusion: Something was passed from the dead smooth bugs
to the living rough ones, allowing them to transform their surface
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.
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…
Animation: Phage T2 Reproductive Cycle
EXPERIMENT
Phage
Empty
Radioactive protein
shell
protein
Radioactivity
(phage
protein)
in liquid
Bacterial cell
Batch 1:
radioactive
sulfur (35S)
DNA
Phage
DNA
Centrifuge
Pellet (bacterial
cells and contents)
Radioactive
DNA
Batch 2:
radioactive
phosphorus (32P)
Centrifuge
Pellet
Animation: Hershery-Chase Experiment
Radioactivity
(phage DNA)
in pellet
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
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.
2 Classes of Nitrogenous Bases


Purines: double-ringed, includes A and G
Pyrimidines: single-ring, includes C, U and T
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...


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)

Maurice Wilkins
and Rosalind
Franklin used a
technique called
X-ray
crystallography to
study and
visualize the
molecular
structure of DNA




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
Animation: DNA Double Helix



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


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


Base pairing
Two hydrogen
bonds between A
and T
Three hydrogen
bonds between C
and G
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
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
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
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

Viral Chromosomes

The viral genome is
highly variable
 DNA or RNA
 Double or single
stranded
 Linear or circular
 Single or
segmented
molecule
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
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!)
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

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…
Changes in Chromatin Structure
Nucleosome
(10 nm in diameter)
DNA
double helix
(2 nm in diameter)
H1
Histones
DNA, the double helix
Histones
Histone tail
(modification)
Nucleosomes, or “beads
on a string” (10-nm fiber)
Chromatid
(700 nm)
30-nm fiber
Loops
Scaffold
300-nm fiber

Scaffold-associated (SAR)
regions bind nonhistone
proteins to form loops that
spirally radiate out
Nucleosome
(30-nm fiber)
Looped domains
(300-nm fiber)
Replicated
chromosome
(1,400 nm)
Metaphase
chromosome
Summary…

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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
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)
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)
Unique-Sequence and Repetitive-Sequence DNA

Sequences vary widely in how often they occur within a genome



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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
a word about C values…


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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
4.6 X 106
Yeast
1.3 X 107
Amoeba
2.9 X 1011
Fruit Fly
1.8 X 108
Frog
2.3 X 1010
Humans
3.4 X 109
Corn
6.6 X 109
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.
The type of RNA in the virus determines the progeny type.
 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