1. Chapter 16 DNA The Molecular Basis of Inheritance

2. molecule responsible for all cell activities
DNA
molecule responsible for all cell activities
and contains the genetic code
Genetic Code
method cells use to store the program that is passed from one generation to another

3. DISCOVERY OF THE GENETIC CODE
1928: Frederick Griffith
Transformation

4. 1928: Frederick Griffith Experiment 1
1. Grew 2 strains of bacteria on plates
- smooth colonies- caused disease (virulent)
- rough edge colonies-did not
cause disease (avirulent)
2. Injected into mice
Results:
- smooth colonies: died
- rought colonies: lived
Conclusion:
bacteria didn’t produce a toxin to kill mice
Experiment 1

5. Experiment 2 1. Injected mice with heat killed virulent strain
non -virulent strain + heat killed virulent strain
Results:
- heat killed: lived
- mixed strains: mice developed pneumonia
Conclusion:
heat killed virulent strain passed disease causing abilities to non virulent strain

6. Cultured bacteria from dead mice and they grew virulent strain.
After Experiment
Cultured bacteria from dead mice and they grew virulent strain.
Griffith hypothesized that a factor was transferred from heat killed cells
to live cells .
TRANSFORMATION

7. DNA was transforming factor
1944: Avery (et al)
1. Repeated Griffith’s experiment with same results.
- result: transformation occurred
2. Did a second experiment using enzymes that would
destroy RNA.
3. Did third experiment using enzymes that would destroy DNA.
- result: no transformation
CONCLUSION
DNA was transforming factor

8. Virus composed of DNA core and protein coat that infect bacteria
1952: Hershey / Chase
- studied how viruses (bacteriophage) affect bacteria.
Bacteriophage
Virus composed of DNA core and protein coat that infect bacteria

9. Hershey Chase Experiment
1. They labeled virus protein coat with radioactive sulfur
2. They labeled virus DNA with radioactive phosphorous
Result
observed that bacteria had
phosphorous
*** virus injected bacterial cells with its phosphorous labeled DNA***
Conclusion
DNA carried genetic code
since bacteria made new
DNA animation

10. DISCOVERY OF STRUCTURE OF DNA

11. x ray crystallography evidence:
Early 1950’s: Rosalind Franklin (English)
x ray crystallography evidence:
X pattern showed that fibers of DNA twisted and molecules are spaced at regular intevals on length fiber.
Maurice Wilkins: x ray diffraction, worked with Franklin

12. Chargaff (American biochemist)
Same time period:
Chargaff (American biochemist)
Chargaff’s Rule:

13. 1953 Watson (American) & Crick (English)
**double helix model**
won Nobel prize in 1962
Discovered the double helix by building models to conform to Franklin’s X-ray data and Chargaff’s Rules

14. DNA A – T - may have 1000’s of nucleotides in 1 strand
- double strand of nucleotides
- may have 1000’s of nucleotides in 1 strand
(very long molecule)
- bases join up in specific (complementary) pairs:
complementary pairs (base pairing rules)
1 purine bonds with 1 pyrimidine on one rung of the ladder connected by a weak H bond
C - G
A – T
Order of nucleotides not important,
proper complementary bases must be paired.

15. STRUCTURE OF DNA Backbone Phosphate + Deoxyribose sugar (5 C) Rungs
4 Nitrogenous bases
- Purines Adenine A
Guanine G
- Pyrimidines Thymine T
Cytosine C
D bases attached to sugar
E. bases attached to each other by weak H bond

16. Nucleotide Structure
Purines Pyrimidines
Sugar
Base
Phosphate

17. DNA makes up Chromosomes (chromatin packing)

18. DNA Comparison Prokaryotic DNA Double-stranded Circular One chromosome
In cytoplasm
No histones
Supercoiled DNA
Eukaryotic DNA
Double-stranded
Linear
Usually 1+ chromosomes
In nucleus
DNA wrapped around histones (proteins)
Forms chromatin

19. REPLICATION OF DNA Process of duplication of DNA
Before cell can divide a new copy of DNA
must be made for the new cell
- Semiconservative replication:
each strand acts as a template (pattern) for new strand to be made
End Result:
one old strand, one new daughter strand

20. DNA REPLICATION

21. Models of DNA Replication

22. Discovery of Replication Model
Meselson and Stahl
Cultured E coli with heavy
isotope 15N for many
generations.
Transferred to light isotope 14N
Sampled after first and second replication
Removed bacteria and extracted DNA
Centrifuged to separate different density DNA

23. Meselson and Stahl Compared results to models of replication
1st replication:
Hybrid DNA (14 and 15)
-Eliminated conservative model
2nd replication:
Light and hybrid DNA
- Eliminated dispersive
model
- Supported semi-conservative model of replicaiton.

24. Steps of Replication
Enzyme DNA helicase attaches to DNA molecule at origins of replication and breaks H bonds so strand unwinds
- single strand binding proteins bind to unpaired bases to keep them from re-binding
- topoisomerase relieves additional strain on forward DNA by breaking and rejoining DNA strands
- replication forks: two areas on either end of the DNA where double helix separates
- forms replication bubble: “bubble” under electron microscope

25. 2. enzyme RNA Primase lays down an RNA primer on DNA strand
- RNA primer segment signals beginning of replication
3. Enzyme DNA polymerase III moves along each of DNA strand and adds complementary bases of nucleotides floating freely in nucleus
A. DNA polymerase III begins synthesis at RNA primer segment
B. DNA polymerase I replaces
RNA primers with DNA nucleotides -

26. DNA Directionality antiparallel strands3’ 5’ 5’ 3’

27. - Directionality: DNA polymerase III reads the template in the 3’ to 5’ direction
Daughter DNA strand (since it is complementary) must be synthesized in the 5’ to 3’ direction
Strands are antiparallel.

28. But if there exist no DNA polymerases
capable of polymerizing DNA in the
3' to 5' direction, how could this be?

29. Discontinuous synthesis
- synthesis only occurs when a
large amount of single strand
DNA is present
- daughter DNA is then synthesized in 5’ to 3’ direction
- leading and lagging strands:
- leading strand – continuously synthesized DNA strand - lagging strand - delayed, fragmented, daughter DNA
- Okazaki fragments discontinuous fragmented DNA segments

30. D. DNA ligase stitches
together Okazaki
fragments into a
single, unfragmented
daughter molecule
E. enzyme chops off
RNA primer and
replaces it with DNA

31. Okazaki fragment animation
DNA polymerase III catalyzes formation of H bonds between nucleotides of template and newly arriving nucleotides which will form daughter DNA
Once all DNA is copied, daughter DNA detaches
Animation
DNA Replication Fork
Okazaki fragment animation

33. End Replication Problem
On one end, RNA primer cannot be replaced with DNA because it is a 5’
(DNA polymerase III can only read from 3’ to 5’)
Causes daughter DNA’s to be shorter with each
replication (cell division)

34. Solution to End Replication Problem
telomeres: repeated units of non-coding short nucleotide sequences (TTAGGG) at ends of DNA
- become shorter with repeated cell divisions
- once telomeres are gone, coding sections of chrom are lost and cell does not have enough DNA to function
***telomere theory of aging***
- telomerase: special enzyme that contains an RNA template molecule so that telomeres can be added back on to DNA
(rebuilds telomeres)
** found in:
Cancer cells - immortal in culture
Stem cells
** not found in most differentiated cells

35. Telomeres and Telomerase

36. Speed of Replication
Multiple replication forks- replication occurs simultaneously on many points of the DNA molecule
Would take 16 days to replicate 1 strand from one end to the other on a fruit fly DNA without multiple forks
Actually takes ~ 3 minutes / sites replicate at one time
Human chromosome replicated in about 8 hours with multiple replication forks working together

37. Accuracy and Repair DNA polymerases proofread as bases are added
- can remove damaged nucleotides and replace with new ones for accurate replication
Mismatch repair: special enzymes fix incorrect pairings
Nucleotide excision repair:
Nucleases cut damaged DNA
DNA polymerase and ligase fill in gaps
RNA does not have this ability-
reason RNA viruses mutate so much

38. Nucleotide Excision Repair
Errors:
Pairing errors: 1 in 100,000 nucleotides
Complete DNA: 1 in 10 billion nucleotides

39. Importance of DNA
Controls formation of all substances in the cell by the genetic code
Directs the synthesis of specific strands of m RNA to make proteins
RNA (Ribonucleic acid)
Another nucleic acid takes orders from DNA Used in protein synthesis