Download Griffith`s Experiment

Chapters 12 and 13: DNA and the Language of Life
Mendel concluded that parents pass on factors to their
offspring.
 We now know these factors to be genes.
 How did scientists determine what genes were made
from?
Three Important Experiments
1. Griffith – 1920’s
Investigating what makes people sick.
Discovered two strains of pneumonia bacteria
 “Smooth” S-strain – forms smooth, round colonies
when grown in a Petri dish.
 “Rough” R-strain – forms rough-edged colonies
when grown in a Petri dish.
A
B
C
D
A. What happened when the S strain was injected into the
mouse?
Mice died
B. What happened when the R strain was injected into the
mouse?
Mice lived
C. What happened when the S strain was heat-killed and
then injected into the mouse?
Mice lived
Would a Petri dish inoculated with a sample from syringe
“C” show any bacterial growth?
If so, what would the colonies look like?
No
Growth
D. What happened when the killed S strain was mixed
with the R strain and injected into the mouse?
Mice died
Would a Petri dish inoculated with a sample from syringe
“D” show any bacterial growth?
If so, what would the colonies look like?
What conclusion can you draw from this experiment?
When heat-killed harmful bacteria were mixed with
harmless, some “factor” transformed the rough, harmless
bacteria into smooth, harmful bacteria.
2. Avery’s Experiment – 1940’s
Wanted to determine what the transformation factor was
from Griffith’s experiment.
Thought the transformation factor was a macromolecule.
 Carbohydrate
 Lipids
 Proteins
 Nucleic Acids
o RNA
o DNA
What happened when the heat-killed S strain was mixed
with an enzyme to destroy RNA and then mixed with the
R strain?
R and S colonies visible
What happened when the heat-killed S strain was mixed
with an enzyme to destroy Proteins and then mixed with
the R strain?
R and S colonies visible
What happened when the heat-killed S strain was mixed
with an enzyme to destroy DNA and then mixed with the
R strain?
Just R colonies
What happened when the heat-killed S strain was mixed
with an enzyme to destroy Lipids and then mixed with the
R strain?
R and S colonies visible
What happened when the heat-killed S strain was mixed
with an enzyme to destroy Carbohydrates and then
mixed with the R strain?
R and S colonies visible
Conclusion:
No transformation when DNA is destroyed. DNA must be
the transformation factor
3. Hershey and Chase’s Experiment – 1950’s
Used Escherichia coli (E. coli) bacteria and
bacteriophages
 Bacteriophages are viruses that attack bacteria
Why did Hershey and Chase use bacteriophages in their
experiment?
Made only of Proteins and DNA
What macromolecule contains sulfur?
Proteins
What macromolecule contains phosphorus?
DNA
Why were the samples agitated and placed into a centrifuge
before the radiation detector was used to locate the
radioactive isotopes?
Separate viruses from bacteria. Viruses suspended in the
liquid and bacteria in the pellet at the bottom.
Where were radioactive proteins found?
Where was radioactive DNA found?
Liquid
Pellet
What conclusion was Hershey and Case able to make
based on their experiment?
Viruses pass DNA, not Proteins, into bacteria
Why didn’t Hershey and Chase culture viruses in media
containing both radioactive sulfur and phosphorus?
They wouldn’t know what was actually being passed on.
The Role of DNA
1. Store Information – Information is stored in the
order and amount of nucleotides that make up the
DNA. The sequence of DNA that codes for a
particular trait is called a … Gene
2. Copy Information – During S of Interphase your
cells replicate the DNA.
3. Transmitting Information – Copies of all of your
genes (both sets) are passed to daughter cells at the
end of Mitosis, while only 1 set is passed to daughter
cells at the end of Meiosis.
Building Blocks of Nucleic Acids
DNA and RNA are Nucleic Acids
Nucleic Acids are polymers
 The monomer building blocks of Nucleic Acids are
Nucleotides
Nucleotides are made of three subunits:
 5-Carbon Sugar (Deoxyribose or Ribose)
 Phosphate Group
 Nitrogenous Base (1of 5)
There are 5 different Nitrogenous Bases
 Adenine (A)
 Guanine (G)
 Cytosine (C)
 Thymine (T)
 Uracil (U)
Purines
Pyrimidines
Each base makes a different Nucleotide
 4 of the 5 Nucleotides are found in DNA
 4 of the 5 Nucleotides are found in RNA
The sugar of one nucleotide is connected to the phosphate
group of the next nucleotide by covalent bonds forming the
“Sugar-Phosphate Backbone”
DNA Structure
Once DNA was determined to be the transformation factor,
many scientists raced to discover its structure.
 Rosalind Franklin/Maurice Wilkins
 Erwin Chargaff
 James Watson/Francis Crick
Rosalind Franklin and Maurice Wilkins
Used X-ray crystallography to take a picture of the
structure of DNA
 A similar picture is shown below
From this picture, Watson could tell the “strandedness” of
DNA.
Double stranded
Twisted – DNA had a uniform diameter
This meant there were four options for the structure
Options:
1.
AA
cc
GG
tt
tt
AA
2.
AG
ct
tc
GA
tc
AG
3.
At
cG
tA
Gc
cG
At
4.
Ac
Gt
Gt
cA
tG
Ac
Further analysis allowed Watson and Crick to rule out two
other options.
 The DNA helix had a uniform 2nm diameter.
o Which two could be ruled out?
Erwin Chargaff
Studied the percent of each nitrogenous base found in an
organism’s DNA.
Nitrogenous Base Make-Up of Different Organisms’ DNA (%)
Organism
Mycobacterium
tuberculosis
Yeast
Wheat
Sea Urchin
Marine Crab
Turtle
Rat
Human
A
15.1
G
34.9
T
14.6
C
35.4
31.3
27.3
32.8
47.3.
29.7
28.6
30.9
18.7
22.7
17.7
2.7
22.0
21.4
19.9
32.9
27.1
32.1
47.3
27.9
28.4
29.4
17.1
22.8
17.3
2.7
21.3
21.5
19.8
What observation can you make about the data? Is there a
pattern?
What does the data show about the make-up of DNA for
different species?
Based on the data, what do you think is Chargaff’s Rule?
A
T
and
G
C
With this data Watson and Crick “ruled-out” another option
and determined the structure of DNA.
Options:
1.
2.
3.
4.
AA
cc
GG
tt
tt
AA
AG
ct
tc
GA
tc
AG
At
cG
tA
Gc
cG
At
Ac
Gt
Gt
cA
tG
Ac
 Sugar and Phosphate Groups make-up the sides of the
ladder
o Sugar-Phosphate backbones run in opposite
directions
 “Rungs” of the ladder are made of two nitrogenous
bases called “Base Pairs”
 What makes each species unique?
o Amount and sequence of “base-pairs”
What type of bond connects the nucleotides in a chain
together? Covalent Are they strong or weak? Strong
Why important? Information is stored in nucleotide order
What bonds hold the two strands together? Hydrogen Are
they strong or weak? Weak Why is important?
Separate the strand for replication
Watson and Crick used the work from Franklin and
Chargaff to determine the structure of DNA.
 They did determine the mechanism of replication
Published their conclusions in April 1953.
 Won the Nobel Prize in 1962.
DNA Mistakes in Movies
Replication
The base-pairing rule established by Chargaff provided a
possible copying mechanism.
Template/Semiconservative Model:
 When: S-phase of Interphase
 Where (Eukaryotic cells): In the nucleus
 With What: DNA, DNA nucleotides, Enzymes
 How:
o Separate the two original strands of DNA.
o Each “old” strand becomes a template to make a
new complementary strand from free nucleotides
 Very fast: 50/sec in mammals; 500/sec in bacteria
 Very accurate: 1 nucleotide/billion wrong
Diagrams like the ones above make it look like the parent
DNA molecule is completely opened before replication
occurs, but that is not true.
There are many “origins of replication” along the length of
the parent DNA molecule.
 Helicase attaches to the DNA molecule and break the
hydrogen bonds holding the two strands together.
o Forms replication bubbles.
 What is beneficial about multiple origins?
One strand (Leading Strand) can be replicated in one piece.
Individual nucleotides attached by DNA Polymerase
Other strand (Lagging) is replicated in fragments by DNA
Polymerase (attaches individual nucleotides) and then the
fragments are bonded together by DNA Ligase
DNA polymerases also proofread the newly replicated
strand and remove incorrectly paired nucleotides
DNA polymerase and DNA Ligase also repair damage
done to DNA by exposure to radiation (UV and X-ray) and
toxic chemicals.
Replication Overview
http://www.youtube.com/watch?v=zdDkiRw1PdU
http://www.youtube.com/watch?v=gW3qZF9cLIA
3 Types of RNA
1. Messenger RNA (mRNA) – A single strand of RNA that
is a temporary (disposable) copy of a single gene.
2. Ribosomal RNA (rRNA) –
Ribosomes are made from rRNA
and proteins.
3. Transfer RNA (tRNA) –
Pick-up and carry amino acids to
the mRNA and Ribosome to
make proteins
DNA to Proteins
DNA - Long term storage of all information
Transcription – Location: Nucleus
mRNA - Short term storage of one gene’s information
Translation – Location: Cytoplasm
Protein
One sequence of nucleotides/“gene” codes for the
production of one polypeptide*.
 Multiple polypeptides can bond together to form more
complex proteins
Overview:
1. Copy part of the
DNA nucleotide
sequence into RNA
2. Read the mRNA
3. Attach amino acids
4. Release protein
Transcription
Transcription – 1 strand of DNA nucleotides (template
strand) is converted into a single strand of complementary
RNA nucleotides (mRNA).
Language Analogy:
Spoken English  Written English
 If you break your hand and can’t write, you might
need someone to transcribe (scribe) your verbal
answers on a test.
When: When a cell needs to produce a specific protein
Where: Nucleus (Eukaryotes)
With What: DNA, RNA nucleotides, RNA polymerase
How:
1. RNA polymerase binds to DNA in the nucleus and
separates the DNA strand for 1 gene.
2. RNA polymerase “reads” 1 strand of DNA to produce
a strand of messenger RNA (mRNA).
3. Complementary RNA nucleotides pair across from
the DNA nucleotides (A-U; G-C, C-G; T-A)
4. RNA polymerase links the nucleotides together.
5. The process continues until the end of the gene is
reached
http://www.youtube.com/watch?v=WsofH466lqk
http://www.youtube.com/watch?v=Kzgnl5-8WAk (3:40 – 5:30)
In eukaryotic cells, the mRNA sequence is further modified
before it leaves the nucleus through nuclear envelope pores.
RNA Splicing/Editing
o Eukaryotic mRNA contains non-coding regions
(Intervening sequence - Introns) that need to be
removed before translation
o Expressed sequences (Exons) are the coding
regions that are maintained.
 The final mRNA transcript (message)is
short than the original.
Analogy
TV Box Set or Movie
Hola, mi nombre es Kevin.
Translation
Translation – Single stranded mRNA is used to produce a
sequence of amino acids (polypeptide).
Language Analogy: Spanish  English
Written Spanish is translated into English with the help of
an interpreter.
mRNA language (nucleotides)  Protein language (amino acids)
When: After Transcription when a cell needs to produce a
specific protein
Where: Cytoplasm
With What: mRNA, ribosomes (rRNA + proteins), tRNA,
amino acids and enzymes.
How:
1. Transcribed, edited mRNA strand moves out of the
nucleus and into the cytoplasm.
2. mRNA binds to a ribosome in the cytoplasm or RER.
3. A ribosome begins reading the mRNA at a start codon
and continues to read the nucleotides in groups of
three (codon).
Why read in groups of three?
How many different amino acids?
20
How many amino acids could be coded for if you read 1
nucleotide at a time? 1  4 different amino acids
4 < 20
How many amino acids could be coded for if you read 2
nucleotides at a time? 2  4 x 4 = 16 amino acids
16 < 20
How many amino acids could be coded for if you read 3
nucleotides at a time? 3  4 x 4 x 4 = 64 different amino
acids
64 > 20
64 different nucleotide triplets
o 61 code for amino acids (1 also “start”)
o 3 code for stop
THEFATCATATETHERAT
THE-FAT-CAT-ATE-THE-RAT
Codon Chart
Redundant But Not Ambiguous
Multiple codons can code for the same amino acid,
but each codon can code for only1 specific amino acid.
4. Ribosomes can hold 2 codons at once in the “P” site
and “A” site.
5. Transfer RNA (tRNA) with the anticodon
complementary to the mRNA codon, bring specific
amino acids to the ribosome/mRNA complex.
6. Peptide bonds form between two amino acids and the
1st tRNA is released.
7. mRNA moves so the 2nd tRNA and growing amino
acid chain are now in the “P” site.
8. Process continues until a stop codon is reached.
http://www.youtube.com/watch?v=5bLEDd-PSTQ
http://www.youtube.com/watch?v=Kzgnl5-8WAk – 5:25
Overview: DNA  RNA  Protein
Mutations
Mutations – any mistake or change in the nucleotide
sequence of DNA.
 Chromosomal mutations – large changes to regions of
a chromosome.
o Deletion
o Duplication
o Inversion
o Translocation
 Gene (Point) mutations – change single gene
 Substitution
 Frameshift
Point Mutations – mutation that involves a single
nucleotide
1. Substitution – replace 1 nucleotide with another
a. Silent: A mutation that changes the DNA sequence of
nucleotides, but does not change the amino
acid sequence of a protein.
DNA: GAA – GGG – CCA
RNA: CUU – CCC – GGU
Amino Acids: LEU – PRO – GLY
DNA: GAA – GGT – CCA
RNA: CUU – CCA – GGU
Amino Acids: LEU – PRO – GLY
Mutation
b. Expressed: A mutation that changes the DNA
sequence of nucleotides and the
amino acid sequence of a protein
DNA: TTT – GTG – AGG
RNA: AAA – CAC – UCC
Amino Acids: LYS – HIS – SER
DNA: TTT – GTT – AGG
RNA: AAA – CAA – UCC
Amino Acids: LYS – GLN – SER
Mutation
2. Frameshift Mutations – changes in the DNA that
can change the rest of the amino acid sequence after
the mutation.
a. Insertion – add a nucleotide into DNA sequence
DNA: TAC – GCA – TTT
RNA: AUG – CGU – AAA
Amino Acids: MET – ARG – LYS
DNA: TAC – GGC- ATT –T
RNA: AUG – CCG – UAA –
Amino Acids: MET – PRO – STOP
Mutation
b. Deletion – remove a nucleotide from a DNA
sequence
DNA: TAC – GAG – GAT – AGC
RNA: AUG – CUC – CUA – UCG
Amino Acids: MET – LEU – LEU – SER
DNA: TAC – AGG – ATA – GC
RNA: AUG – UCC – UAU – CG
Amino Acids: MET – SER – TYR –
Mutation
Mutagenesis – the production of mutations
 Spontaneous - errors during DNA replication or
recombination
 Mutagens – physical/chemical agent that causes an error.
o High energy radiation (X-ray, UV)
o Toxic chemicals