DNA: The Molecular Basis of Inheritance
Hbio
Ms. Pagodin
Do Now:
Happy Pi Day! Grab your clickers Talk to your classmates and find out who
read the same article as you! In your article group, discuss the experiment
and conclusion!
Nuclear Composition?
1868- Johann Miescher Collected pus & fish sperm
Isolated and identified acidic compound with nitrogen and phosphorus…. Today we know it as Deoxyribonucleic acid
Molecule of Heredity
Is it Proteins or Nucleic Acid??
What makes up proteins?A. Nucleotides
B. Amino acids
C. Monosaccharides
Nucleotides
Amino acids
Monosa
ccharid
es
33% 33%33%
What makes up nucleic acids?A. Nucleotides
B. Amino acids
C. Monosaccharides
Nucleotides
Amino acids
Monosa
ccharid
es
33% 33%33%
How many amino acids are there?A. 4
B. 16
C. 20
D. 60
4 16 20 60
25% 25%25%25%
How many different DNA nucleotides are there?A. 4
B. 16
C. 20
D. 60
4 16 20 60
25% 25%25%25%
Identifying the Genetic Material 1928 Fredrick Griffith (English Bacteriologist)
Trying to find a vaccine for pneumonia Vaccine: prepared from killed/weakened microorganisms
introduced into the body to produce immunity Griffith worked with 2 strains of Streptococcus pneumoniae
bacteria S strain
Polysaccharide Capsule “Smooth” edged colonies Virulent – able to cause disease
R strain No Capsule “Rough” edged colonies Nonvirulent - does not cause disease
Griffith’s Experiment
Griffith’s Conclusion: Something had passed from heat killed bacteria to the nonvirulent R strain making them virulent… he called this the “transforming principal”
Griffith did not know what it was, but many scientists thought it was proteins
Today we know…
Transformation – cells take up foreign genetic material, changing their own genes (used for genetic engineering)
Heat killed S bacteria – enzymes were denatured therefore the DNA could not be copied
Proteins are denatured at 600C and DNA is denatured at 900C
DNA of heat killed S bacteria survived and transformed DNA of R bacteria
Virulent strainsA. Have a capsuleB. Cause diseaseC. Do not have a
capsuleD. Do not cause
diseaseE. A&BF. C&D
Have a capsu
le
Cause dise
ase
Do not have a ca
psule
Do not cause
disease A&B
C&D
17% 17% 17%17%17%17%
Transformation is the addition of genes to another organisms genome
A. True
B. False
True
False
50%50%
The Search for what caused the Transformation…
1944 – Oswald Avery, MacLeod, & McCarty (American Bacteriologists)
Experiment:1. Added protease to “R and heat-killed S” mixture
Result Mice died
2. Added DNAase to “R and heat-killed S” mixture Result Mice Lived
Conclusion: DNA, not protein, is the transforming factor in
Griffith’s experiment
More Evidence that DNA is the Genetic Material…
1952 – Alfred Hershey & Martha Chase (NY) Used T2 bacteriophages (phage) – virus
that infects bacteria Composed of nucleic acid surrounded by a
protein coat Viruses infect specific host Viruses are not living
Not composed of cells Cannot reproduce on their own Do not grow and develop
Background Info on Viruses
Which type of virus is chicken pox?A. Lytic
B. lysogenic
Lytic
lysoge
nic
50%50%
Which type of virus is the flu?A. Lytic
B. lysogenic
Lytic
lysoge
nic
50%50%
Hershey & Chase Experiment Experiment:
1. Grew T2 w/radioactive Sulfur 35S (protein coat takes in 35S)2. Grew another group of T2 w/ radioactive Phosphorus 32P (DNA
takes in the 32P)3. 35S-labeled and 32P–labeled phages were used to infect E.Coli
bacteria4. Separated phages from bacteria using a blender and a
centrifuge… the bacterial cells at bottom and viral parts at the top
Results: 35S-labels still in viral parts 32P-labels mostly in the bacterial cells, and new phages also
contained 32P DNA Conclusion:
Viral DNA (not protein) enters bacteria and carries instructions on how to make more phages
Without a doubt, DNA is the hereditary material!
Hershey & Chase Experiment
A bacteriophageA. Is a virus that
infects bacteria
B. Is a virulent bacteria
C. Cannot be used for experiments
Is a vi
rus t
hat infects
bac...
Is a vi
rulent b
acteria
Cannot be use
d for e
xp...
33% 33%33%
Structure of DNA?
Linus Pauling Nobel prize for deducing structure of protein
Collagen
If protein structure could be determined and modeled, why not DNA?
Structure of DNA By 1950’s most scientists were convinced that
Chromosomes carry genetic material Genes are on chromosomes Genes are made of DNA
Basic Structure of DNA Composed of nucleotides Nucleotides made of 3 parts deoxyribose, phosphate, N base 2 types nitrogen bases:
Purines – double ring of C and N Adenine Guanine
Pyrimidines – single ring of C and N Cytosine Thymine
Discovering DNA’s Structure Erwin Chargaff (NYC)
1947 – DNA composition varies among different species
1949 -Chargaff’s Rules- Discovered regularity of ratios: # Adenines = # Thymines
(ie. Humans A =30%, T=30%) # Guanines = # Cytosines
(ie. Humans G = 20%, C = 20%)
1952 Rosalind Franklin & Maurice Wilkins (England) Developed X-ray crystallography photographs of
DNA Suggested “helix” shape of 2-3 chains of
nucleotides
April 25th, 1953 James Watson & Francis Crick
(England) Built the 1st accurate 3D (tin
and wire) model of DNA “Double Helix” – spiral
staircase Purine is always linked by h-
bond to a pyrimidine 2 strands of DNA are
complimentary to each other 2 strands are anti-parallel 5’(phosphate end)
3’(deoxyribose end) 1962 Awarded the Nobel Prize
More on DNA
Ex. If the sequence of bases on one strand is AATGCGCAT, than the complimentary strand will be: ________________
Human DNA has 3 billion base pairs.. Less than 1% of our DNA makes us different from one another!
Which seems most likely?
Models of DNA Synthesis
Semiconservative ea/ daughter molecule
will have 1 new strand and 1old strand
Conservative Parent molecule reforms
Dispersive All 4 strands have a
combination of old and new strands
Assignment:
Propose an experiment to determine how DNA replication occurs
1950’s Meselson & Stahl
Cultured Ecoli on medium labeled w/ 15N nt
Transferred EColi to medium labeled w/ 14N nt
Centrifuge after each replication and analyze
Origin of Replication Prokaryotic Cell – single origin of replication where proteins
separate the 2 strands and create a replication bubble, replication proceeds in both directions from the replication fork
Eukaryotic Cells – hundreds or thousands of replication bubbles form to speed up the copying process, replication proceeds in both directions from the replication fork
http://sites.fas.harvard.edu/~biotext/animations/replication1.swf
DNA Replication Watson and Crick proposed that the complimentary strand of DNA serves
as a template for which the other strand is built…experiments confirmed this 5 years later
DNA Replication: Process of Synthesizing new molecules of DNA1. Helicases catalyze the breaking of H-bonds (driven by ATP) and
opens up the double helix forming replication forks (point at which DNA separates into single strands)
2. Topoisomerase temporarily bind to relieve strain ahead of replication fork
3. Single-strand binding protein – binds to unpaired DNA strands until they serve as templates for new complimentary strand
Elongation DNA pol adds 50 nt/sec in Euk cells Each nt is a nucleoside triphosphate
1. At the replication fork, DNA Polymerase III continuously adds complimentary nucleotides to exposed bases on 3’ end of new strand, this is called the leading strand
2. DNA polymerase III must work away from the replication fork on the other strand, the lagging strand, to follow the 5’-3’ direction creating short segments of DNA called Okazaki fragments. DNA Ligase joins the Okazaki fragments together.
3. Process continues until all DNA has been copied, end result is 2 new molecules of DNA each identical to the original and composed of one new and one old strand
Priming DNA Synthesis
DNA pol can not initiate – only add nt to 3’ end of existing chain
Primer – short chain (5-10nt) of RNA Primase – enzyme starts RNA chain from scratch
Leading strand – 1 primer needed
Lagging strand – 1 primer needed for ea/Okazaki fragment
DNA pol I replaces RNA nt of primers w/DNA versions
DNA Synthesis
http://www.dnai.org/a/index.html
Proofreading
DNA polymerase only moves to the next nucleotide if the previous nucleotide was a correct match
If mismatched, DNA Polymerase backs up, removes the mismatched nucleotide(s) and replaces it with the correct one(s).
Repair enzymes can recognize and repair damaged sites too
Only 1 error per 1 billion nucleotides!
DNA Replication & Aging Every time DNA is copied,
DNA polymerase cannot complete replication on the ends
Eukaryotic DNA has a non-coding, repeating nucleotide sequence on the ends called telomeres that protects genes from being eroded over successive replications
It is believed that telomeres are directly related to the aging process