Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
We now know…
DNA-genetic material that contains all of Mendel’s heritable factors and Morgan’s
genes on chromosomes
-true, but scientists in the pastÆchromosomesÆDNA and protein
-what actually contains all of the heritable info?
-DNA or protein?
By 1940’s scientist believed that it was the protein:
-very diverse in structure and function
-great heterogeneity and specificity of function
-little was known about nucleic acids
-physical and chemical properties of nucleic acids seemed too
uniform to account for the multitude of inherited traits
1928 Frederick Griffith (British medical officer)-experiment with bacteria
(microbes; simpler than pea plants, fruit flies or humans
-trying to find vaccine for Streptococcus pneumoniae –a bacteria that
causes pneumonia in mammals
He knew:
-2 strains of pneumococcus [smooth (S)-pathogenic, disease-causing;
capsule protects from mouse’s defenses and rough (R) colonies]
-cells of S strain are encapsulated with a polysaccharide coat and cells of
R aren’t
-alternate phenotypes (S & R) are inherited
With this info: he performed 4 experiments
1. injected live S: mice died
2. injected live R: mice lived
3. injected heat killed S: mice lived
4. injected heat killed S with live R: mice died and he found S cells in the
mouse (this surprised him)
Experiment 1
Experiment 2
Experiment 3
Experiment 4
R cells
injected
S cells
injected
Heat-killed
S cells injected
R cells and heatkilled S cells injected
Mouse lives
Mouse dies
Mouse lives
Mouse dies
Fig. 12-1, p. 261
-concluded that R cells had acquired (were converted) from dead S cells
the ability to make polysaccharide coats
-in other words, genetic material from S cells was picked up by R cells,
integrated and passed onto the offspring; therefore, live S cells
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
-provided some proof that proteins were not genetic material because heat
(killed S cells), denatures proteinsÆmaking useless; something is still
viable
-now called transformationÆ change in genotype and phenotype due to
assimilation of external genetic material (DNA) by a cell
What was the chemical nature of the transforming agent? (14 year search)
-American bacteriologist: Oswald Avery’s associates (Maclyn, McCarty,
Colin, MacLeod) performed test with the S and R strains
-they isolated different substances from S cells introduced to R cells
-only DNA enabled R cells to produce coat
-in 1944, announced that the transforming agent had to be DNA
-this announcement was met with skepticism
-how closely related are bacteria and mammals anyway?
-proteins are better candidates
-how could DNA carry genetic information? So little was known
about it
Additional evidence: 1952- Alfred Hershey and Martha Chase discovered that
DNA is the genetic material of a bacteriophage (bacteria eaters) or phages
-T2 (virus that infects bacteria)
-(viruses are DNA or RNA with a protein coat)
-infects E. coli (Escherichia coli)
-can quickly program E. coli to produce T2 phages and release the viruses
when the cell lyses
-was this a result from transformation of DNA or protein?
To determine this they tagged protein and DNA with different radioactive isotopes
-protein- 35S; proteins have sulfur
-DNA- 32P; no sulfur, but has phosphorus
35S
1
32 P
3
Bacterial viruses
grown in 35S to
label protein coat
or 32P to label DNA
Agitate cells
in blender
Agitate cells
in blender
4
Separate by
centrifugation
2
Separate by
centrifugation
Viruses infect bacteria
32 P
35S
5
Bacteria in pellet
contain 32Plabeled DNA
35S-labeled
protein in
supernatant
Fig. 12-2, p. 262
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Fig. 12-2, p. 262
Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
6
Viral reproduction
inside bacterial cells
from pellet
7
Cell lysis
32P
5
6
7
Fig. 12-2, p. 262
Æviruses-protein coat with DNA inside
-virus injects what? into E. coli bacterium
-blender shook the protein coats of viruses or virus parts off the bacteriumanything left outside the bacterium
-DNA remained inside
-phage/phage parts in supernatant (35S); bacteria in pellet (32P)
-found that DNA from virus was injected into bacteria and not proteins
-32P isotope was then passed down to offspring; therefore, it is nucleic
acids, not proteins that carry genetic information of the cell-at least for
viruses
Other evidence:
1. eukaryotic cell doubles its DNA prior to mitosis
2. during mitosis DNA is equally divided between 2 daughter cells
3. an organism’s diploid cells have twice the genetic material as haploid
cells
Further evidence:
1947-Erwin Chargaff (Biochemist-already knew DNA was N-base, deoxyribose,
and phosphate group) used paper chromatography to separate the different
bases (nitrogenous) of different species
-he found
1. DNA composition is species specific
-amount and ratios of bases vary from one species to another, but
is a characteristic ratio
-source of diversity made it more credible that DNA
is the genetic material
2. in every species, he found
-#A~#T
-#G~#C
In humans A = 30.9%; T = 29.4%; G = 19.9%; C = 19.8%
-A-T, G-C ratios later became known as Chargaff’s rules
-but why?
-better understood with Watson and Crick model of DNA
Watson-relatively unknown American and Crick-British Double Helix
(Linus Pauling in California thought it was triple stranded)
(Rosalind Franklin and Maurice Wilkins in England did x-ray crystallography)
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
Model of DNA
-by 1950s-DNA was accepted as the genetic material
-the arrangement of the nucleotide (phosphate, sugar, nitrogenous base)
and nucleic acid polymer was also known
-structure of DNA was yet to be discovered
Woman named Rosalind Franklin took an X-ray photo of DNA
-spots and smudges with mathematical formulae gave the shape of the molecule
Watson was familiar with types of patterns helical molecules produced. With a
glance, knew it was helical and deduced width of helix and spacing
Fig. 12-4b, p. 265
-with this Watson and Crick made some deductions:
a. DNA is a helix with a uniform width of 2 nm-suggests 2 strands.
b. Purine and pyrimidine bases are stacked 0.34 nm apart.
c. Helix makes one complete turn every 3.4 nm.
d. There are 10 layers of N-base pairs in each turn of the helix.
ModelsÆconcluded:
a. ladder-like structure twisted like a spiral with sugar-phosphate
backbones and N-bases as rungs
b. 2 sugar-phosphate backbones are anti-parallel-run opposite directions
c. N-bases are paired specifically
-because of 2 nm width, one purine (A or G) (2 C-ring) always had
to be bonded with pyrimidines (T or C) (1-C ring)
-pyrimidines together were too narrow
-purines together were too wide
-first thought A-A, C-C, etc.
-must be a purine with a pyrimidine
-found that A-T with 2 H-bonds
-found that G-C with 3 H-bonds
-puts hydrophobic N-bases in center away from aqueous medium,
but thought bases were on the outside for a while
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
Sugar–phosphate backbone
Minor groove
Major groove
3.4 nm
0.34 nm
2.0 nm
= hydrogen
= atoms in base pairs
= carbon
= oxygen
= phosphorus
Fig. 12-5, p. 266
1. Important because it explains Chargaff’s rules; since A-T and C-G, explains
why there are near equal amounts
2. suggests general mechanisms for DNA replication; if bases form specific pairs,
the info on one strand compliments that on the other
-weak H-bonds between bases stabilize DNA molecule
-van der Waals forces between stacked bases also stabilize DNA
-number of bases and pairing limited
-sequence is endless
They ended their classic paper with this wry statement: “It has not escaped our
notice that the specific pairing we have postulated immediately suggests a
possible copying mechanism for the genetic material.” April, 1953 Nature
1962-Wilkins, Crick, and Watson were given the Noble prize; Franklin had died at
38 years of age from cancer from the radiation; not given posthumously
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
Structure of a DNA polymer:
Adenine
Deoxyribose
Guanine
Deoxyribose
Fig. 12-6a, p. 267
Thymine
Deoxyribose
Cytosine
Deoxyribose
Fig. 12-6b, p. 267
-strands are anti-parallel
-one strand is arranged 5’Æ3’
-5’-phosphate group
-3’-OH where next Pi would attach
-other strand runs in 3’Æ5’ direction
RNA-also a nucleic acidÆpolymers of nucleotides
-differs from DNA
1. sugarÆribose
2. U (uracil), not A
3. single-stranded-no double helix
DNA Replication
-1953-Watson and Crick
-DNA model-double helix
-because of specific pairing of bases, suggested a “template” mechanism
for DNA replication
-have one strand of DNA, same complimentary strand will always be
created
Elaborated: 2nd paperÆ DNA Replication 1954 Proceedings of Royal Society
1. DNA strands separate.
2. Each strand is a template for assembly of 2nd strand.
3. Nucleotides line up singly in accordance with base pairing nucleotides;
A double H-bonds with T, G triple H-bonds with C
4. enzymes link nucleotides at their sugar-phosphate groups.
Considered to be a semiconservative model of DNA replication
-each daughter DNA molecule will have one old or “conserved” strand
from parent and one newly created strand.
Good Hypothesis-real proof:
Late 1950s-Meselson and Stahl provided the experimental evidence to support
semiconservative model.
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
Hypothesized:
-3 alternate models for pattern of DNA replication
1. SemiconservativeÆone old strand and one new strand
(a) Hypothesis 1: Semiconservative replication
Parental DNA
First generation
Second generation
Fig. 12-7a, p. 268
2. ConservativeÆparental double helix remains intact and 2nd DNA
molecule should be constructed as entirely new DNA
-new DNA merely copies the original DNA
(b) Hypothesis 2: Conservative replication
Parental DNA
First generation
Second generation
Fig. 12-7b, p. 268
3. DispersiveÆboth strands contain a mixture of old and new DNA
(c) Hypothesis 3: Dispersive replication
Parental DNA
First generation
Second generation
Fig. 12-7c, p. 268
Conducted an experiment:
-grew E. coli (single chromosome with approximately 5 million base pairs; can
copy in less than 1 hour) for many generations in a medium containing heavy
nitrogen N-15, not N-14
-bacteria, as make new material from surroundings, incorporated N-15 into their
nitrogenous bases
-transferred E. coli into medium of N-14 (regular N)
-when E. coli reproduced, they would have to duplicate DNA
-in order to duplicate DNA, they would need to incorporate materials from
the environment; therefore, they would incorporate N-14
-isolated and centrifuged E. coli after each replication (after each division)
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
Bacteria are grown in
15N (heavy) medium. All
DNA is heavy.
Some cells are
transferred to
14N (light)
medium.
Some cells
continue to
grow in 14N
medium.
First generation Second generation
Cesium
chloride
(CsCl)
High
Low
density density
DNA
DNA is mixed with CsCl
solution, placed in an
ultracentrifuge, and
centrifuged at very high
speed for about 48 hours.
The greater concentration
of CsCl at the bottom of
the tube is due to
sedimentation under
centrifigal force.
15N (heavy)
14N (light)
14N – 15N
DNA
DNA
hybrid DNA
DNA molecules move to positions
where their density equals that of
the CsCl solution.
Fig. 12-8a, p. 269
-Original E.coli:15N much heavier 1st generation saw 2nd generation saw
14N
(light)
DNA
14N
– 15N
hybrid DNA
14N
– 15N
hybrid DNA
15N
(heavy)
DNA
Before transfer
to 14N
One cell generation
after transfer to 14N
Two cell generations
after transfer to 14N
The location of DNA molecules within the centrifuge tube can be
determined by UV optics. DNA solutions absorb strongly at 260 nm.
Fig. 12-8b, p. 269
-gave concrete experimental evidence for Watson and Crick’s semiconservative
model.
Basic principle of replication is elegantly simple.
The actual process is complex biochemically.
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
DNA replication:
-complex: requires replication of 2 strands simultaneously-over a dozen enzymes
Base
Nucleotide joined to
growing chain by
DNA polymerase
Phosphates
released
Fig. 12-10, p. 271
-extremely rapid and accurate
-prokaryotes: 500 nucleotides/ second; humans: 50 nucleotides/ second
-only a few hours to copy the 6 billion bases (put these bases in this size
font in a book-will fill 900 books) of a single human cell
-is accurate-only 1 in a billion nucleotides is paired incorrectly; begins as 1
in 10,000, but there are lots of error inspectors
-Begins at a site called the origin of replication-only 1 origin in bacteria, but
hundreds in humans (specific sequence of nucleotides)
-DNA double helix unwinds because of enzyme helicase
-once unwound, enzymes allow breakage of H-bonds between the base
pairs
-eukaryotic cells have hundreds or thousands of origins of replication
-unzipping creates a replication forkÆY-shaped regions of DNA where
new strands are forming
-single stranded binding proteins keep separated strands apart and
stabilize the unwound DNA until the complimentary strands can be synthesized
-DNA polymerases catalyze synthesis of new DNA strands from nucleotides in
nucleoplasm
-according to base pairing rules, new nucleotides align along the
templates of old DNA strands
-DNA polymerase links nucleotides to the growing strand
-strands grow in the 5’Æ3’ direction
-new nucleotides are added only to 3’end
-added nucleotides are actually nucleoside triphosphates:
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
-sugar, base, 3 Ps (like ATP)
-adding of nucleotides is endergonic process
-when nucleotide is added, 2 extra Pis break off-provide the energy needed to
synthesize nucleotides
-when nucleotides are added, they can only be added in the 5’Æ3’ direction
Twist introduced into the
helix by unwinding
RNA primer
Single-strand
binding proteins
DNA polymerase
3’
5’
3’
DNA
helicase
3’
5’
3’
RNA primer
Direction of
replication
Fig. 12-11b, p. 272
-problem:
-2 strandsÆ first runs 3’Æ5’, new strand is made 5’Æ3’ (3’ is end on which
nucleotides can be added)
-this strand can be made continuously; therefore, new strand is leading
strand
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
-second strand runs 5’Æ3’Æ new strand will run 3’Æ5’; but, nucleotides can only
be added 5’Æ3’; what happens is that this strand is assembled in segments,
growing each time from the replication fork until it meets the next segment
-instead of on continuous strand, it makes many short fragments of
complimentary DNA called Okazaki segments
-Okazaki segments are later connected via DNA ligase, producing a single
complimentary strand; ligate means to bind
-this is called the lagging strand-takes more time to assemble
-Okazaki (Japanese) segments-1000-2000 nucleotides long in bacteria
and 100-200
nucleotides long in eukaryotes
3’
5’
Leading strand
DNA helix
RNA primer
3’
5’
3’
5’
DNA polymerase
Replication fork
3’
5’
Lagging strand
(first Okazaki fragment)
Direction of
replication
Fig. 12-12a, p. 273
3’
5’
Leading strand
3’
RNA
primers
3’
5’
5’
5’3’
3’
5’
Two Okazaki
fragments
Fig. 12-12b, p. 273
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
3’
5’
Leading strand
3’
3’
DNA ligase
5’ 3’
5’
5’
Third Okazaki
fragment
Lagging strand
3’
5’
Fig. 12-12c, p. 273
Primers:
-DNA nucleotides are not directly added to complimentary strand at beginning of
replication
-before each nucleotide is added, an RNA primer is first put into place
-therefore, DNA replication is actually initiated by a primosome
-a structure composed of RNA primase (enzyme that creates RNA
primers) and other proteins
-primosome initiates the leading strand and each Okazaki segment with
RNA nucleotides-not DNA
-DNA polymerase III continues to add DNA nucleotides to the RNA primer
-later, the RNA nucleotides are replaced with DNA nucleotides by another
DNA polymerase I
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
Summary of Replication
1. Helicase unwinds DNA-produces replication fork
-proteins (single-stranded binding proteins) hold fork open for replication
to begin
-topoisomerases help the DNA to not get tangled when unwound
- Topoisomerase I solves the problem caused by tension generated by
winding/unwinding of DNA. It wraps around DNA and makes a cut permitting the
helix to spin. Once DNA is relaxed, topoisomerase reconnects broken strands
2. Primosomes initiate DNA replication at special nucleotide sequences (origins
of replication with short segments of RNA nucleotides (RNA primers)
3. DNA polymerase attaches to RNA primers and begins elongation-adding of
DNA nucleotides to compliment strand
4. leading strandÆassembled 5’Æ3’ continuously (3’Æ5’ complimentary to)
5. lagging strandÆassembled 5’Æ3’ in short Okazaki fragments, which are joined
by DNA ligase
6. RNA primers are replaced by DNA nucleotides
DNA packaging in chromosomes:
-prokaryotesÆone circular chromosome~4-5 million base pairs
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
-1000X greater than size of cell itself
-eukaryotesÆmuch more-6 billion base pairs; varies between species
1. DNA-negatively charged
2. wraps around proteins, histones, positively charged
-not in all eukaryotic cells
Mistakes:
-replication is highly accurate, but errors do occur
-DNA polymerase (in bacteria) proofreads the pairing process-checks
nucleotides to be sure they are correct
-other enzymes also act when errors are missed by DNA polymerase
-initial pairing errors~1/10,000, while those that are not caught are 1/109
-DNA repair-when it is synthesized or after accidental changes in existing DNA
1. Mismatch Repair
Æcorrects mistakes after DNA is being synthesized
-DNA polymerase (bacteria)
-eukaryotesÆalso additional proteins (special enzymes)
-heredity defect in one of these proteins has been associated with a
form of colon cancer
-protein can’t fix mutation
S is an endonuclease that identifies the mismatch and cuts phosphodiester linkages. L is ligase
and it will repair the linkage. H is Helicase and it unwinds the DNA. Exonuclease cuts out the
bad segment and DNA polymerase III replaces the nucleotides. Ligase will join linkages again.
2. Excision Repair
-changes in DNA can result from exposure to mutagensÆreactive
chemicals, radiation, X-rays, U-V light, etc.
-100 types of repair enzymes in bacteria; 130 in humans that repair
damage
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
-damaged segments are excised and remaining gap is filled
-DNA polymerase and ligase
-ex: when 2 adjacent nucleotides bond to each other rather
than to complimentary base
-ex: thymine dimer-distorts DNA molecule
-usually caused by U-V radiation
-important to repair damage
-xeroderma pigmentosum
-inherited degect in nucleotide excision repair enzyme
-hypersensitive to sunlight
-mutations in skin from U-V light are left uncorrected
and cause skin cancer
-2 T nucleotides bond with each other, not to As
-excised and replaces
-if DNA error is not repaired- it becomes a mutation
Æany sequence of nucleotides in a DNA molecule that does not exactly
match the original DNA molecule from which it was copied
-can occur via:
1. substitutions
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
2. deletions
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
3. insertions
2 and 3 are especially dangerous, missing one nucleotide changes entire
sequence for which proteins DNA molecule encodes
1 isn’t so hot either!
Æcalled frameshift mutation
The end replication problem
DNA repair processes involve DNA polymerases
-can only add nucleotides to the 3’ end of a preexisting polynucleotide
-leads to problems
-no way to complete 5’ ends of daughter DNA strands
-result: repeated rounds of replication produce shorter and shorter DNA
molecules
-could lose essential genes
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
Prokaryotes-no problem
-have circular DNA
Eukaryotes
-have telomeres at ends
-do not contain genes
-contain repetitions of one short nucleotide sequence-TTAGGG-(repeated
100-1,000X)
-protects genes from being eroded through successive rounds of DNA
replication
Can restore shortened telomeres with telomerase
-special enzyme catalyzes lengthening of telomeres
-how? DNA template is lost
-has short molecule of RNA along with its protein
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Molecular Basis of Inheritance Notes
AP Biology
Mrs. Laux
-RNA contains nucleotide sequence that serves as template for new
telomere segments at 3’ end of telomere
Our cells do not contain
-may be a limiting factor in life span of certain tissues and/or organism as
a whole
-except for germ-line cells (give rise to gametes)
-produces long telomeres in these cells and consequently newborns
-telomerase found in cancerous somatic cells
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