Frederick Griffith
Observe the following data – explain what is
going on
1928
Conclusion: heat-killed, virulent bacteria must have
released genetic material  transferred to R (non
virulent strain) cells
Transformation – DNA from dead cells cut into
fragments & exits cell → healthy cells pick up free
floating DNA and integrate
chromosomes via
recombination
Oswald Avery
Colin MacLeod
Maclyn McCarty

Avery, McCarty & MacLeod
◦ purified DNA & proteins separately from
Streptococcus pneumonia bacteria
 Experimental Question: which will
transform non-pathogenic bacteria?
◦ 1. injected protein into bacteria
 Mice lived!
◦ 2. injected DNA into bacteria
 transformed bacteria
 Mice died!
1952 | 1969
Martha Chase
Alfred Hershey
Protein coat labeled
DNA labeled with 32P
35
with S
T2 bacteriophages
are labeled with
Which
radioactive isotopes
radioactive
S vs. P
marker is
found inside
the cell?
This will be the
molecule
containing
genetic info!
35S
bacteriophages infect
bacterial cells
bacterial cells are agitated
to remove viral protein coats
from bacteria cell
radioactivity
found in the medium of
protein coat
32P
radioactivity found
in the bacterial cells
Watson and Crick
1952
1947

DNA composition: “Chargaff’s rules”
◦ varies from species to species
◦ all 4 bases not in equal quantity
◦ bases present in characteristic ratio
 humans:
A = 30.9%
Rules
T = 29.4%
A=T
C=G
G = 19.9%
C = 19.8%
purine
pyrimidine
http://academic.brooklyn.cuny.edu/biology/bio4fv/page/
molecular%20biology/dna-structure.html

How do your techniques compare with those used
by all of our important researchers visited today?
How have advances in technology changed our
way of thinking and approach to a scientific
question? Given the times, do you think the
experiments we reviewed today were
scientifically valid then? How about today? Is
there anything you would change about their
experiments to make them more scientifically
sound in today’s world? Identify those changes
and explain.
◦ base pairing suggests that
each side can serve as a
template for a new strand
“It has not escaped our notice that the specific pairing we have
postulated immediately suggests a possible copying mechanism for
the genetic material.”
— Watson & Crick


Bacterial DNA is circular
Eukaryotic DNA is linear
◦ Can you think of any problems this may pose in the
successful completion of replication?
◦ Cells can lose 30-200 base pairs each time a cell divides.
◦ Telomerase enzyme adds the TTAGGG sequence to the 3’
end of the DNA at the telomere regions
Animation
http://highered.mcgrawhill.com/novella/MixQuizProcessingServle
http://articles.mercola.com/sites/articles/archive/2010/02/23/scien
ce-finally-reveals-how-you-can-actually-revese-aging.aspx
utechdmd2015.wikispaces.com/file/vi
ew/unit_2_Replication_r_1_.ppt

(1957) Mathew Meselson and Franklin Stahl grew
the bacterium Escherichia coli on medium that
contained 15N in the form of ammonium chloride.

The 15N became incorporated into DNA
(nitrogenous bases).

The resulting heavy nitrogen-containing DNA
molecules were extracted from some of the cells.
DNA Replication is semiconservative experimental proof

When subject to density gradient centrifugation,
they accumulated in the high-density region of the
gradient.

The rest of the bacteria were transferred to a new
growth medium in which ammonium chloride
contained the naturally abundant, lighter 14N
isotope.
utechdmd2015.wikispaces.com/file/view
/unit_2_Replication_r_1_.ppt
In the experiments by Meselson and Stahl that
demonstrated semiconservative replication of DNA,
the researchers cultivated bacteria in a medium
containing a heavy isotope of nitrogen, 15N. They
then moved the bacteria to a medium containing 14N,
the lighter, more common isotope. After each round
of replication, the researchers extracted the DNA and
centrifuged the solution to separate the DNA bands
by density. The test tubes below illustrate the
possible banding pattern found after two bacterial
generations (two rounds of replication). Which test
tube best illustrates the bands predicted by the
semiconservative replication model of DNA
replication?
The test tubes below illustrate the possible banding pattern
found after two bacterial generations (two rounds of
replication). Which test tube best illustrates the bands
predicted by the semiconservative replication model of
DNA replication?
A scientist is using an ampicillin-sensitive strain of bacteria
that cannot use lactose because it has a nonfunctional gene
in the lac operon. She has two plasmids. One contains a
functional copy of the affected gene of the lac operon, and
the other contains the gene for ampicillin resistance. Using
restriction enzymes and DNA ligase, she forms a
recombinant plasmid containing both genes. She then adds
a high concentration of the plasmid to a tube of the bacteria
in a medium for bacterial growth that contains glucose as the
only energy source. This tube (+) and a control tube (-) with
similar bacteria but no plasmid are both incubated under the
appropriate conditions for growth and plasmid uptake. The
scientist then spreads a sample of each bacterial culture (+
and -) on each of the three types of plates indicated below.
If no new mutations occur, it
would be most reasonable to
expect bacterial growth on
which of the following plates?
a. 1 and 2 only
b. 3 and 4 only
c. 5 and 6 only
d. 4, 5, and 6 only
e. 1, 2, 3, and 4 only

DNA
How does DNA code for cells &
bodies?
proteins
cells
bodies

The county fair is coming to town and every year
there is the famous Cookie Contest. You’ve been
experimenting in the kitchen and have come up with
the MOST delicious cookies you can think of (mmm
chocolate chip). Just to make sure it’s perfect, you
want your best friend to taste them. One problem:
your best friend lives in California. You need to get
the recipe to your friend. Do you send your original
recipe information? Why or why not? If not, what
steps do you take to pass on the information?
Remember, your success lies in the perfect execution
of this recipe!

Flow of genetic information in a cell
◦ DNA to proteins?
DNA
replication
RNA
protein
trait

suggest genes code for enzymes
◦ Disruptions in pathways result in
 lack of an enzyme
 disease
 variation of phenotype
metabolic pathway
A
disease
B
disease
C
disease
D
disease
E
Make a model!
Steps
o Structures
o


ribose sugar
N-bases
◦ uracil instead of thymine
◦ U:A
◦ C:G


single stranded
many RNAs
◦ mRNA, tRNA, rRNA, siRNA…
DNA
transcription
RNA

Making mRNA
◦ transcribed DNA strand = template strand
◦ untranscribed DNA strand = coding strand
◦ synthesize complementary RNA strand
 transcription bubble
◦ Enzymes involved
 RNA polymerase
coding strand
 Helicase
5
DNA
A
G
T
A T C
T A
3
mRNA
build RNA 53
5
G
C
A G C
A
T
C G T
T
G C A U C G U
C
G T A G C A
A
T
3 A
T
RNA polymerase
A
C
T
A G
C T
G
A
T
3
5
template strand

3 types of RNA polymerases
1. RNA polymerase 1
 transcribe rRNA genes ONLY
 makes ribosomes
2. RNA polymerase 2
 transcribe genes into mRNA
3. RNA polymerase 3
 transcribe tRNA genes ONLY
**each has a specific promoter sequence it recognizes**

Promoter region - site marking the start of gene
◦ TATA box binding site
◦ transcription factors (ie. proteins, hormones?) - on/off
switch; trigger binding of RNA pol
◦ RNA polymerase

Enhancer region
◦ binding site far upstream
◦ turns transcription on
HIGH

Eukaryotic genes contain “fluff” – spliced
◦ exons = expressed / coding DNA
◦ introns = the junk; inbetween sequence; now
thought to be involved in switches

5’ Cap & PolyA tail added (modified in the nucleus)
intron = noncoding (inbetween) sequence
~10,000 bases
eukaryotic DNA
exon = coding (expressed) sequence
primary mRNA
transcript
mature mRNA
transcript
pre-mRNA
~1,000 bases
spliced mRNA


snRNPs “snurps”
◦ small nuclear RNA
exon
◦ Proteins
◦ Done in nucleus before 5'
leaving nucleus
snRNPs
snRNA
intron
exon
3'
Spliceosome
◦ several snRNPs
◦ recognize splice site
sequence
spliceosome
5'
3'
 cut & paste gene
 Introns have specific 2
base codes in front
and end of intron to
5'
identify them
mature mRNA
exon
5'
lariat
3'
exon
3'
excised
intron

Enzymes in cytoplasm attack mRNA –
protection is needed
 add 5 GTP cap
 add poly-A tail
 longer the tail, longer mRNA lasts:
produces more protein
3'
mRNA
5'
P
G P
P
A
Transcription
 mRNA processing
 mRNA splicing

Make a model!
Steps
o Structures
o
DNA
4ATCG
TAC GCA CAT TTA CGT ACG CGG
mRNA AUG CGU GUA AAU GCA UGC GCC
4AUCG
?
protein
20
Met Arg Val Asn Ala Cys Ala
How can you code for 20 amino acids with
only 4 nucleotide bases (A,U,G,C)?



20 different amino acids
aa’s coded for by THREE nucleotides
–codons
4 bases, 3 per codon: 43 = 64 total
possible combinations
WOBBLE

Code is redundant
◦ several codons for
each amino acid
◦ 3rd base “wobble”
◦ Most codons = aa’s


Start codon
 AUG
 methionine
Stop codons
 UGA, UAA, UAG

“Clover leaf” structure
◦ anticodon on “clover leaf” end
◦ amino acid attached to 3 end

Aminoacyl tRNA synthetase - enzyme bonds aa’s to
tRNA
◦ requires energy
 ATP  AMP
 bond is unstable
 can easily release amino acid at ribosome
Trp
C=O
OH
OH
Trp
C=O
O
H 2O
activating
enzyme
tRNATrp
anticodon
tryptophan attached
to tRNATrp
Trp
O
mRNA
tRNATrp binds to UGG
codon of mRNA


RIBOSOMES!!!
Facilitate binding of
tRNA anticodon to
mRNA codon
Organelle or enzyme??
Structure
◦ rRNA & proteins
◦ 2 subunits
 large
 small
◦ 3 sites

A site (aminoacyl-tRNA site)
◦ tRNA carrying next aa to be added to chain binds
here

P site (peptidyl-tRNA site)
◦ holds tRNA carrying growing
polypeptide chain

Met
Met
E site (exit site)
◦ empty tRNA
leaves ribosome
from exit site
5'
UU AA CC
AA UU GG
E
E
PP
AA
3'

Initiation
◦ brings together mRNA,
ribosomal subunits,
initiator tRNA (amino acid
methionine)

Elongation
◦ adding amino acids based
on codon sequence

Termination
◦ end codon
3 2 1

Transcription & translation simultaneous in bacteria
◦ DNA in cytoplasm
◦ no mRNA editing
◦ ribosomes read
mRNA as transcribed
◦ Faster than in
eukaryotes (DNA to
protein ~1hr)

Prokaryotes
◦ DNA in cytoplasm
◦ circular
chromosome
◦ naked DNA
◦ no introns
◦ continuous
process

Eukaryotes
◦ DNA in nucleus
◦ linear
chromosomes
◦ DNA wound on
histone proteins
◦ introns vs. exons
◦ mRNA processing

Translation Animation
 Protein Synthesis