Chargaff.html: 10_T03-Chargaff.jpg
Chargaff used paper chromatography
to compile data on the base compositions in DNA of many species.
For each species, the amount of A is proportional to the amount of T,
and the amount of G is proportional to the amount of C.
There is a 1:1 ratio of
purine (A + G) and
pyrimidine (C + T)
bases,
but not for (A + T) and (C + G),
refuting the tetranucleotide
hypothesis.
DNA.html: 10_14a-double_helix.jpg
DNA_content.html: 10_T02-DNA_content.jpg
In eukaryotes, it was known that chromosomes contain the genetic material.
Thus, if DNA were the genetic material,
there should be a correlation between the amount of DNA and the ploidy of the cell.
Experimental data for DNA in haploid sperm and diploid somatic cells show this correlation,
while data for proteins do not.
DNA_denaturation.html: 10_20-DNA_denaturation.jpg
Double-stranded DNA can be unwound (denatured)
by applying heat to break the hydrogen bonds holding the bases together.
The denatured DNA exhibits stronger UV absorption, showing a hyperchromic shift during "melting".
hydrogen bonds while
AT
pairs have 2 hydrogen bonds.
DNA_forms.html: 10_17a-DNA_forms.jpg
A-DNA is slightly more compact than B-DNA.
Other forms of DNA can also exist under laboratory conditions.
C-DNA, D-DNA, and E-DNA are also right-handed forms of DNA that are less compact than B-DNA.
Z-DNA forms a left-handed double helix.
Diplococcus.html: 10_T01-Diplococcus.jpg
Virulent strains of Diplococcus pneumoniae
can be easily distinguished from avirulent strains by the morphology formed by colonies in culture,
due to the presence of a polysaccharide capsule in virulent bacteria that also enables them
to survive a host animal's immune system.
FISH.html: 10_22-FISH.jpg
Fluorescent in situ hybridization (FISH).
Single-stranded DNA or RNA complementary to specific segments
is added to metaphase chromosomes fixed to slides and allowed to hybridize.
The hybridized segment is then localized with a fluorescent molecule.
NDP-NTP.html: 10_11-NDP-NTP.jpg
A nucleotide is a nucleoside monophosphate (NMP),
with one phosphate group attached to the 5' carbon.
Two phosphate groups yield a diphosphate (NDP),
while three phosphate groups yield a triphosphate (NTP).
The molecule at right is adenosine triphosphate (ATP), an important energy molecule.
Hydrolysis
of ATP to ADP yields energy to power cellular processes.
RNA.html: 10_T04-RNA.jpg
In RNA, the pentose sugar in the nucleotide is ribose,
and uracil (U) instead of thymine (T) base-pairs with cytosine (C).
The 3 classes of RNA are
messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA).
T-even-phage_life_cycle.html: 10_05-T-even-phage_life_cycle.jpg
A T-even phage
such as T2 or T4
adsorbs to the cell wall of its host and injects its genetic material into the cell.
This material directs the reproduction of more phages,
eventually lysing the host and releasing its progeny in a lytic cycle.
T2-phage_infection.html: 10_06-T2-phage_infection.jpg
Hershey (Nobel 1969) and Chase used radioisotopes to label T2 phages.
Nucleotides contain phosphorus (P), so 32P labels the DNA core of the phage.
Some
amino
acids
contain sulfur (S),
so 35S labels the protein coat.
Differently labeled phages are used to infect unlabeled bacteria.
T2-phage_infection2.html: 10_06-T2-phage_infection2.jpg
After infection,
a blender
was used to strip infecting phages from bacteria.
Then centrifugation isolated both components.
The 35S-labeled protein remained outside the cells as phage coats ("ghosts").
The 32P-labeled DNA
were found with the cells, which eventually lysed and produced
progeny 32P-labeled phages.
TMV_hybrid.html: 10_08-TMV_hybrid.jpg
The RNA core and the protein coat from
tobacco mosaic virus (TMV) and
Holmes ribgrass virus (HR) can be isolated and reconstructed into "hybrids".
These hybrids produce leaf lesions characteristic of the RNA donor, and progeny virus also inherit that phenotype.
Thus, RNA serves as the genetic material in these viruses.
UV_spectra.html: 10_07-UV_spectra.jpg
Ultraviolet (UV)
light can induce mutations in the genetic material of bacteria, and is most mutagenic at 260 nm.
Absorption spectra measurements show that both DNA and RNA
absorb UV light most strongly at 260 nm, while protein absorbs most strongly at 280 nm.
This indicates a nucleic acid is the genetic material.
X-ray_diffraction.html: 10_13-X-ray_diffraction.jpg
X-ray diffraction "photo 51" of the B
form
of DNA made by Rosalind
Franklin,
who was working with Wilkins,
provided clues for Watson and Crick (Nobel 1962) to discover the structure of
DNA.
base_composition.html: 10_19-sedimentation_equilibrium_centrifugation.jpg
Since G-C base pairs in DNA are denser than A-T pairs,
the percentage of G-C pairs is directly proportional to the molecule's buoyant density.
base_pairing-A-T.html: 10_16-base_pairing-A-T.jpg
A-T base pairs form 2 hydrogen bonds.
base_pairing-G-C.html: 10_16-base_pairing-G-C.jpg
G-C base pairs form 3 hydrogen bonds.
central_dogma.html: 10_01-central_dogma.jpg
Central Dogma of Molecular Genetics.
dinucleotide.html: 10_12a-dinucleotide.jpg
Linkage of two nucleotides by the formation of a 3'-5' phosphodiester bond,
producing a dinucleotide.
Multiple phosphodiester bonds form a polynucleotide chain.
double_helix-antiparallel.html: 10_14c-double_helix.jpg
The 2 DNA strands run in opposite (antiparallel) directions;
one in the 3' - 5' direction, the other 5' - 3'.
The A-T
and
G-C
base pairing
provides complementarity of the two strands.
Thus, DNA follows these base-pairing rules:
A always pairs with T, and
G always pairs with C.
double_helix-right-handed.html: 10_15-double_helix-right-handed.jpg
Under normal biological conditions, DNA exists in the B form as a right-handed double helix.
double_helix-turn.html: 10_14b-double_helix.jpg
The 2 strands have antiparallel
sugar-phosphate backbones, connected by
rungs of nitrogenous base pairs, of which there are 10 per turn.
These phosphodiester bonds
are covalent bonds
that are stronger
than the hydrogen bonds
between the bases.
On the inside, the nitrogenous bases are joined by hydrogen bonds:
2 hydrogen bonds between A-T pairs,
and
3 hydrogen bonds between G-C pairs.
double_helix.html: 10_14a-double_helix.jpg
Watson and Crick (Nobel 1962)
Watson and Crick proposed the double-helix structure of DNA in 1953.
Each turn of the helix contains 10 nucleotides*, so the
3.4 angstrom (0.34 nm) internucleotide
distance
add up to 34 angstroms per
turn,
twisting around major and minor grooves.
*Current calculations show that there are 10.4 nucleotides per turn.
electrophoresis.html: 10_27-electrophoresis.jpg
Electrophoresis
A sample is placed on a porous medium (filter paper or semisolid gel) immersed in a solution that conducts electricity.
When current is applied, the negatively charged DNA fragments
will migrate toward the positive electrode (anode).
The fragments move at different rates, based mostly on fragment size: smaller molecules migrate at a faster rate through the pores of the medium than larger ones.
The bands representing the fragments can be visualized by applying dyes or by autoradiography (exposing a radioactive molecule to photographic film).
This diagram illustrates an agarose gel
as the medium for
electrophoresis.
gradient_centrifugation.html: 10_18-gradient_centrifugation.jpg
A mixture of molecules is loaded on top of a solution, forming a concentration gradient.
The tubes are spun in an ultracentrifuge for a given amount of time and
stopped.
molecular_hybridization.html: 10_21-molecular_hybridization.jpg
Double-stranded DNA is transcribed to form a single-stranded RNA transcript
complementary to one of the 2 DNA strands.
The DNA is heated to denature it, then slowly cooled
together with the RNA.
molecular_hybridization2.html: 10_21-molecular_hybridization2.jpg
Some of the RNA will find its single-stranded DNA complement and renature in a process called
molecular hybridization, creating a DNA:RNA hybrid duplex.
nitrogenous_bases.html: 10_09-nitrogenous_bases.jpg
Each atom in the ring of purines and pyrimidines is assigned an unprimed number.
The double-ring purines are adenine (A) and guanine (G).
The single-ring pyrimidines are cytosine (C), thymine (T), and uracil (U).
Thymine is 5-methyl uracil.
Both DNA and RNA contain A, C, and G; only DNA contains T, while only RNA contains U.
video
nucleotide.html: 10_10-nucleotide.jpg
A ribose or deoxyribose sugar with a purine or pyrimidine base attached to the 1'
carbon is called a nucleoside.
A nucleoside with a phosphate group at the 5'
carbon is called a
nucleotide.
Nucleosides and nucleotides are named according to the specific nitrogenous base
(A, T, G, C, and U) that is part of the building block.
pentose_sugar.html: 10_10-nucleotide.jpg
Each carbon atom in the pentose sugar is assigned a number with a prime sign (').
Ribonucleic acids (RNA) contain ribose, a 5-carbon sugar.
Deoxyribonucleic acids (DNA) contain deoxyribose,
which has a hydrogen atom at the C-2' position rather than a hydroxyl group.
reassociation.html: 10_23-reassociation.jpg
Denatured, single-stranded DNA fragments can reassociate
into complementary double strands.
The single-stranded DNA concentration (C) can be plotted against a logarithmic scale of the product of
C0t (initial concentration of DNA single strands), and t (time).
The half-reaction time C0t1/2 increases as the fragment size increases,
as shown in the genomes of viruses and bacteria
and some eukaryotes.
Repetitive DNA sequences in some eukaryotic DNA allow them to have much shorter
C0t1/2.
sedimentation_equilibrium_centrifugation.html: 10_18-gradient_centrifugation2.jpg
In sedimentation equilibrium centrifugation
(or density gradient centrifugation),
different molecules in the mixture will settle in bands of different buoyant densities where
the centrifugal force is equal and opposite to the upward diffusion force.
The gradient is eluted from the tube in fractions, which can then be measured for UV absorption at 260 nm.
This technique can be used to analyze base composition of double-stranded DNA.
sedimentation_velocity_centrifugation.html: Analytical_centrifuge.jpg
In sedimentation velocity centrifugation,
an analytical centrifuge is used to measure the speed at which molecules migrate down the tube by
monitoring with UV absorption optics while spinning.
This velocity is measured in Svedberg
coefficients.
tetranucleotide.html: 10_02-tetranucleotide.jpg
About 1910, Levene proposed the tetranucleotide model:
nucleic acids (DNA and RNA) were composed of repeating four-nucleotide units;
he assumed a 1:1:1:1 ratio of these 4 nucleotides.
The 4 nucleotide building blocks are
adenine (A),
thymine (T),
guanine (G), and
cytosine (C).
transformation-DNA.html: 10_04-transformation-DNA.jpg
Avery,
MacLeod, and McCarty isolated the transforming principle by
centrifugation
to collect the cells, followed by heat-killing and extraction of macromolecules.
This filtrate is capable of transformation.
Inactivating or removing the polysaccharides, proteins and RNA
left the filtrate still capable of transformation.
Inactivating DNA in the filtrate with DNase also inactivated transformation,
proving that the "active factor" is DNA.
transformation.html: 10_03-transformation.jpg
Virulent strains of Diplococcus pneumoniae possess a capsule that makes them
resistant to a hosr animal's immune system, causing fatal pneumonia in the host.
The virulent strain forms smooth (S) colonies in
culture,
and can be distinguished from avirulent bacteria which form rough (R)
colonies.
transformation2.html: 10_03-transformation2.jpg
Griffith showed that when mice were injected with a mixture of
living avirulent bacteria are mixed with heat-killed virulent cells,
the mice developed pneumonia and died, and living virulent bacteria were recovered from the blood.
The avirulent bacteria had been transformed into virulent ones.