1. DNA! The stuff of Life

2. Observe the following data – explain what is going on
Frederick Griffith
1928
Observe the following data – explain what is going on

3. 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

4. Avery, McCarty & MacLeod - 1944
Oswald Avery
Colin MacLeod
Maclyn McCarty

5. Experimental Question: which will transform non-pathogenic bacteria?
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!

6. Hershey & Chase
1952 | 1969
Martha Chase
Alfred Hershey

7. Which radioactive marker is found inside the cell?
Protein coat labeled
with 35S
DNA labeled with 32P
T2 bacteriophages
are labeled with
radioactive isotopes
S vs. P
Which radioactive marker is found inside the cell?
bacteriophages infect
bacterial cells
This will be the molecule containing genetic info!
bacterial cells are agitated
to remove viral protein coats from bacteria cell
35S radioactivity
found in the medium of
protein coat
32P radioactivity found in the bacterial cells

8. Watson and Crick
1952

10. Rosalind Franklin & Maurice Wilkins

11. Chargaff 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%
T = 29.4%
G = 19.9%
C = 19.8%
Rules A = T
C = G

12. Antiparallel strands purine pyrimidine

13. Post Nature article…
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.

14. DNA Replication
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

15. http://highered.mcgraw- hill.com/novella/MixQuizProcessingServle
Telomeres: Repeating sequences of TTAGGG (protect our genetic instructions, can be as many as 8,000 bases long at birth).
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 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
hill.com/novella/MixQuizProcessingServle

16. http://articles. mercola

17. Bacterial DNA Replication

18. utechdmd2015.wikispaces.com/file/view/unit_2_Replication_r_1_.ppt

19. DNA Replication is semi-conservative experimental proof
(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.

20. DNA Replication is semi-conservative 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.

21. utechdmd2015.wikispaces.com/file/view/unit_2_Replication_r_1_.ppt

22. 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?

23. 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?

24. 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.

25. 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

26. The Cell Cycle

27. From Gene to Protein sections of DNA that code for
How does DNA code for cells & bodies?
DNA
proteins
cells
bodies

28. Think….
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!

29. The “Central Dogma” Flow of genetic information in a cell
DNA to proteins?
transcription
translation
DNA
RNA
protein
trait
replication

30. Metabolic Pathways… A B C D E suggest genes code for enzymes
Disruptions in pathways result in
lack of an enzyme
disease
variation of phenotype
metabolic pathway
disease
disease
disease
disease A B C D E

31. Transcription in short…
Make a model!
Steps
Structures

32. RNA review ribose sugar N-bases single stranded many RNAs
uracil instead of thymine
U : A
C : G
single stranded
many RNAs
mRNA, tRNA, rRNA, siRNA…
transcription
DNA
RNA

33. transcribed DNA strand = template strand
Making mRNA
transcribed DNA strand = template strand
untranscribed DNA strand = coding strand
synthesize complementary RNA strand
transcription bubble
Enzymes involved
RNA polymerase
Helicase
coding strand 3 A G C A T C G T 5 A G A A C A G T T T T C A T A C G
DNA T 3 C T A A T 5 G G C A U C G U T 3 C G T A G C A
mRNA
RNA polymerase
template strand 5
build RNA 53

34. RNA polymerases 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**

35. “What is a promoter?” you may ask
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

36. mRNA Processing 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
pre-mRNA
primary mRNA
transcript
~1,000 bases
mature mRNA
transcript
spliced mRNA

37. The splicing process… snRNPs “snurps” Spliceosome several snRNPs
exon
intron
snRNA 5' 3'
snRNPs “snurps”
small nuclear RNA
Proteins
Done in nucleus before leaving nucleus
Spliceosome
several snRNPs
recognize splice site sequence
cut & paste gene
Introns have specific 2 base codes in front and end of intron to identify them
spliceosome
exon
excised
intron 5' 3'
lariat
mature mRNA

38. The fancy cap & tail…
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 A
3' poly-A tail
mRNA 5'
5' cap 3' G P
A’s

39. Review Videos
Transcription
mRNA processing
mRNA splicing

40. Translation
Make a model!
Steps
Structures

41. How does mRNA code for proteins?
TAC GCA CAT TTA CGT ACG CGG
DNA 4
ATCG
AUG CGU GUA AAU GCA UGC GCC
mRNA 4
AUCG ?
Met Arg Val Asn Ala Cys Ala
protein 20
How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)?

42. 20 different amino acids
aa’s coded for by THREE nucleotides –codons
4 bases, 3 per codon: 43 = 64 total possible combinations

43. Why don’t these numbers match? 20 amino acids, 64 options??
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

44. How are codons matched to amino acids?

45. A little more on tRNA “Clover leaf” structure
anticodon on “clover leaf” end
amino acid attached to 3 end

46. Loading “naked” tRNA’s
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
Trp
Trp
C=O OH
H2O OH O
C=O O
activating
enzyme
tRNATrp
mRNA
anticodon
tryptophan attached to tRNATrp
tRNATrp binds to UGG codon of mRNA

47. So where’s the protein making factory?
RIBOSOMES!!!
Facilitate binding of tRNA anticodon to mRNA codon
Organelle or enzyme??
Structure
rRNA & proteins
2 subunits
large
small
3 sites

48. 3 ribosomal 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
E site (exit site)
empty tRNA leaves ribosome from exit site
Met
Met 5' U A C U A C U G A U G A 3' E P A E P A

49. Let’s translate… Initiation Elongation Termination1 2 3
Initiation
brings together mRNA, ribosomal subunits, initiator tRNA (amino acid methionine)
Elongation
adding amino acids based on codon sequence
Termination
end codon

50. Full circle: metabolic pathways

51. Translation in Prokaryotes
Transcription & translation simultaneous in bacteria
DNA in cytoplasm
no mRNA editing
ribosomes read mRNA as transcribed
Faster than in eukaryotes (DNA to protein ~1hr)

52. Do you think this process is the same for prokaryotes & eukaryotes
Do you think this process is the same for prokaryotes & eukaryotes? Explain your ideas.
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

53. Translation Animation
Let’s review
Translation Animation
Protein Synthesis