15.1. The
Genetic Material
A. Genetic material must be:
1. able to store information used to control both the development and the
metabolic activities of cells;
2. stable so it can be replicated
accurately during cell division and be transmitted for generations; and
3. able to undergo mutations
providing genetic variability required for evolution.
B. Previous Knowledge About DNA
1. Knowing the chemistry of DNA was essential to discovery that DNA is genetic
material.
2. 1869, Swiss chemist Friedreich
Miescher removed nuclei from pus cells and isolated DNA "nuclein";
it was rich
in phosphorus and lacked sulfur.
3. Nuclein was analyzed by other
scientists who found that it contained an acid: nucleic acid.
4. Two types of nucleic acids were
discovered: DNA (deoxyribonucleic acid) and RNA
(ribonucleic acid).
5. Early in the twentieth century,
discovery that nucleic acids contain four types of nucleotides.
a. DNA was
composed of repeating units, each of which always had just one of each of four
different
nucleotide (A, T, G, or C).
b. In this
model, DNA could not vary between species and therefore could not be the
genetic material;
therefore some other protein component was expected to be the genetic material.
C. Transformation of Bacteria
1. In 1931, bacteriologist Frederick Griffith experimented with Streptococcus
pneumoniae (pneumococcus)
that causes
pneumonia in mammals.
2.
non-encapsulated (R) strain.
a. The S
strain is virulent (the mice died); it has a mucous capsule and forms shiny
colonies.
b. The R
strain is not virulent (the mice lived); it has no capsule and forms dull
colonies.
3. In an effort to determine if the
capsule alone was responsible for the virulence of the S strain, he injected
the mice
with heat-killed S strain bacteria; the mice lived.
4. Finally, he injected mice with a
mixture of heat-killed S strain and live R strain bacteria.
a. The mice
died and living S strain pneumococcus were recovered from their bodies.
b. Griffith
concluded some substance necessary to synthesis of the capsule and, therefore,
virulence must
pass from dead S strain bacteria to living R strain bacteria so the R strain
were transformed.
c.This
change in phenotype of the R strain bacteria must be due to a change in their
genotype, which
suggested that the transforming substance may have passed from S strain to R
strain.
D. DNA: The Transforming Substance
1. Oswald Avery and his coworkers reported that the transforming substance was
DNA.
2. Purified DNA is capable of
bringing about the transformation; their evidence included the following:
a. DNA from
S strain pneumococcus causes R strain bacteria to be transformed.
b. Enzymes
that degrade proteins cannot prevent transformations, nor did RNase, an enzymes
that digest RNA.
c. Digestion
of the transforming substance with enzyme that digests DNA prevents
transformation.
d. Molecular
weight of the transforming substance is great enough for some genetic
variability .
3. Their experimental results
demonstrated DNA is genetic material and DNA controls biosynthetic
properties
of a cell.
4. Modern experiments with bacteria
show some can take up DNA to gain penicillin resistance.
E. Reproduction of Viruses
1. Bacteriophage is a virus that infects bacteria; consists only
of a protein coat surrounding a nucleic acid core.
2. Bacteriophage T2 is
a virus that infects the bacterium Escherichia coli (E.
coli), a species of intensely
studied
bacteria that normally lives within the human gut.
3. In 1952, Hershey and Chase used
bacteriophage T2 in their experiments.
a. The
purpose of their experiments was to see which of the bacteriophage components
-- the protein coat
or the DNA -- entered bacteria cells and directed reproduction of the virus.
b. In two
separate experiments, they labeled the protein coat with 35S and
the DNA with 32P.
c. Viral
coats are sheared away from bacterial cells; they are separated by
centrifugation.
d. Results:
radioactive 32P alone is taken up by bacterial host and
incorporated in virus reproduction.
e. This
result reinforced the notion that DNA is the genetic material.
15.2. The
Structure of DNA
A. Nucleotide Data
1. In 1940's, Erwin Chargaff analyzed base content of DNA using new chemical
techniques.
2. It was known DNA contained four
different nucleotide:
a. two with
purine bases, adenine (A) and guanine (G); a purine
is a type of nitrogen-containing
base having a double-ring structure.
b. two with
pyrimidine bases, thymine (T) and cystosine (C); a pyrimidine
is a type of nitrogen-containing
base having a single-ring structure.
3. The results of his analysis
proved DNA does have the variability necessary to code genetic material.
4. Chargaff discovered that for a
species, DNA has the constancy required of genetic material.
5. This constancy is given in
Chargaff's rules:
a.The
amount of A, T, G, and C in DNA varies from species to species.
b. In
each species, the amount of A=T and the amount of G=C.
6. The tetranucleotide hypothesis
proposing DNA has repeating units of one of these four bases was disapproved.
B. Variation in Base Sequence
1. The variability is staggering; a human chromosome contains about 140 million
base pairs.
2. Since any of the four possible
nucleotide can be present at each nucleotide position, the total number
of possible
nucleotide sequence is 4 to 140,000,000.
C. Diffraction Data
1. Rosalind Franklin, a student at King's College, produced X-ray diffraction
photographs.
2. Franklin's work provided evidence
that DNA has the following features:
a. DNA is a
helix.
b. One part
of the helix is repeated.
D. The Watson and Crick Model
1. American James Watson joined with Francis H.C. Crick in England to work on
structure of DNA.
2. Watson and Crick received the
Nobel Prize in 1962 for their model of DNA.
3. Using information generated by
Chargaff and Franklin, Watson and Crick built a model of DNA as
double
helix; sugar-phosphate molecules on outside, paired bases on inside.
4. Their model was consistent with
both Chargaff's rules and dimensions of DNA polymer provided by
Franklin's
photograph of X-ray diffraction of DNA.
5. Complementary base pairing
is the paired relationship between purines and pyrimidines in DNA, such
that A is
hydrogen-bonded to T and G is hydrogen-bonded to C.
15.3.
Replication of DNA
A. DNA replication is the process of copying a DNA molecule.
1. Unwinding: old strands of the parent DNA molecule are unwound
as weak hydrogen bonds between
the paired
bases are unzipped and broken by the enzyme helicase.
2. Complementary base pairing:
free nucleotide present in nucleus bind with complementary bases on
unzipped
portions of the two strands of DNA; process is catalyzed by DNA
polymerase.
3. Joining:
complimentary nucleotide bond to each other to form new strands; each daughter
DNA molecule
contains and
old strand and a new strand; process is also catalyzed by DNA polymerase.
4. DNA replication must occur before
a cell can divide; in cancer, drugs with molecules similar to the four
nucleotide
are used to stop replication.
B. Replication is Semiconservative
1. DNA replication is semiconservative because each daughter double helix has
one parental strand
and one new
strand.
2. 1958, Matthew Meselson and
Franklin Stahl confirmed a model of DNA replication.
a. They grew
bacteria in medium with heavy nitrogen (15N), then switched to
light nitrogen (14N).
b. Density
of DNA following replication is intermediate as measure by centrifugation of
molecules.
c. After one
division, only hybrid DNA molecules were in the cells.
d. After two
divisions, half the DNA molecules were light and half were hybrid.
3. These were exactly the results to
be expected if DNA replication is semiconservative.
C. Prokaryotic Versus Eukaryotic Replication
1. Prokaryotic Replication
a. Bacteria
have a single loop of DNA that must replicate before the cell divides.
b.
Replication in prokaryotes may be bidirectional from one point of origin or in
only one direction.
c.
Replication only proceeds in one direction, from 5' to 3'.
d. Bacterial
cells are able to replicate their DNA at a rate of about 106 base pairs per
minute.
e. Bacterial
cells can complete DNA replication in 40 minutes; eukaryotes take hours.
2. Eukaryotic Replication
a.
Replication in eukaryotes starts at many points of origin and spreads with may
replication bubbles
-- places where the DNA strands are separaating and replication is occurring.
b. Replication
forks are the V-shaped ends of the replication bubbles; the sites of
DNA replication.
c.
Eukaryotes replicate their DNA at a slower 500 - 5,000 base pairs per minute.
d.
Eukaryotes take hours to complete DNA replication.
D. Replication Errors
1. A genetic mutation is a permanent change in the sequence of bases.
2. Base changes during replication
are one way mutations occur.
3. A mismatched nucleotide may occur
one per 100,000 base pairs, causing a pause in replication.
4. DNA repair enzymes perform a
proofreading function and reduce the error rate to one per billion.
5. Incorrect base pairs that survive
the proofreading process contribute to gene mutations.