1. DNA as the Model of Inheritance
Molecular Genetics
DNA as the Model of Inheritance

3. Components of DNA Nucleotides Sugar Phosphate Groups
ADENINE
GUANINE
CYTOSINE
THYMINE
Sugar
DEOXYRIBOSE
Phosphate Groups
Adenine and Guanine are PURINES
Cytosine and Thymine are PYRIMIDINES

4. LE 16-5 Sugar–phosphate backbone Nitrogenous bases 5 end Thymine (T)
Adenine (A)
Cytosine (C)
Phosphate
DNA nucleotide
Sugar (deoxyribose)
3 end
Guanine (G)

5. Antiparallel Arrangement
Complimentary Arrangement
Adenine always pairs with Thymine
Cytosine always pairs with Guanine
5’ end and 3’ end
3’ end has a terminal –OH
5’ has a terminal Phosphate Group

6. LE 16-7 5 end Hydrogen bond 3 end 1 nm 3.4 nm 3 end 0.34 nm 5 end
Key features of DNA structure
Partial chemical structure
Space-filling model

7. Watson and Crick
Presented the double helical model in April 1953 (1 page paper in Nature).
Transcended the scientific world
Won Nobel Prize in 1962 along with Maurice Wilkins
Their model explained Chargoff’s rules.

9. What about Rosalind??
Around 1950, Wilkins and his colleague, Rosalind Franklin, used a technique called x-ray crystallography to try to determine the structure of DNA.
Watson and Crick used their data to develop their theory. Rosalind found the sugar-phosphate backbone
But, before the Nobel Prize was awarded, Rosalind died, and therefore did not receive the Nobel recognition.
But, would the committee have recognized Rosalind’s contributions if she had lived? She was a woman you know…

10. LE 16-6 Rosalind Franklin Franklin’s X-ray diffraction
photograph of DNA

11. Watson & Crick – part 2
In their 2nd paper, Watson and Crick proposed the semiconservative model of DNA replication.
It states that when DNA replicates, each of the 2 daughter molecules will have 1 old strand of from the parent, and 1 newly formed strand.

12. LE 16-9_1 The parent molecule has two complementary
strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.

13. LE 16-9_2 The parent molecule has
two complementary
strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.
The first step in replication is separation of the two DNA strands.

14. LE 16-9_3 The parent molecule has two complementary
strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.
The first step in replication is separation of the two DNA strands.
Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.

15. LE 16-9_4 The parent molecule has two complementary
strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.
The first step in replication is separation of the two DNA strands.
Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.
The nucleotides are connected to form the sugar-phosphate back-
bones of the new strands. Each “daughter” DNA
molecule consists of one parental strand and one new strand.

16. If not Semiconservative, then what?
Conservative – parent emerges from the replication intact.
Dispersive – all 4 strands formed are a mixture of old and new.

17. LE 16-10 First Second replication replication Parent cell
Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix.
Semiconservative model. The two strands of the parental
molecule
separate, and each functions as a template for synthesis of a new, comple-mentary strand.
Dispersive model. Each strand of both daughter molecules contains
a mixture of
old and newly synthesized
DNA.

18. Meselson and Stahl
In late 1950’s Matthew Melelson and Franklin Stahl devised experiments to test the 3 models of replication.
Their findings supported Watson and Crick that replication was semiconservative.

19. How did they do it?
Cultured E. coli bacteria for several generations in a medium containing a heavy isotope of nitrogen (15N)
The bacteria incorporated the isotope into their DNA
Then, the bacteria was transferred to a medium containing a lighter isotope of nitrogen (14N)

20. Then they…
Observed the bacteria. Any new DNA that the bacteria synthesized would be lighter than the “old” DNA (made in the 15N medium).
Would centrifuge and extract DNA strands and mass them.

21. Well, what did they find?
The 1st replication in the 14N medium produced a band of hybrid (15N-14N) DNA.
This eliminated the conservative model.
The 2nd replication produced both light and hybrid DNA.
This eliminated dispersive and supported semi-conservative.

22. LE 16-11 Bacteria cultured in medium containing 15N Bacteria
transferred to
medium
containing
14N
DNA sample
centrifuged
after 20 min
(after first
replication)
DNA sample
centrifuged
after 40 min
(after second
replication)
Less
dense
More
dense
First replication
Second replication
Conservative
model
Semiconservative
model
Dispersive
model

23. Hershey and Chase
In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage (virus) called T2.
T2 infects E. coli
Knew T2 could make a normal cell produce viruses, but wanted to know what it a protein or DNA?

24. LE 16-3
Phage
head
Tail
Tail fiber
DNA
100 nm
Bacterial
cell

25. How they did it… 2 Batches of Broth
One contained T2 with E. coli in the presence of radioactive sulfur.
The other contained T2 with E. coli in the presence of radioactive phosphorus.
Radioactive Sulfur is only incorporated into the protein coat of phage.
Radioactive Phosphorus is only incorporated into the DNA of the phage.

26. Next they…
Agitated each broth in a blender separate phages outside the bacteria from the cells in their contents.
Then they centrifuged the mixture so that the bacteria formed a pellet at the bottom of the test tube.
Measured the radioactivity of the pellet and the liquid.

27. Well, what did they find?
In the broth with the sulfur, radioactivity was only measured in the liquid, which indicated that the protein was not incorporated into the bacterium.
In the phosphorus broth, radioavtivity was measured in the pellet (bacteria), which indicated that the viral DNA was incorporated into the bacterium.

28. LE 16-4 Empty Radioactive protein shell Radioactivity Phage protein
in liquid
Phage
Bacterial cell
Batch 1:
Sulfur (35S)
DNA
Phage
DNA
Centrifuge
Radioactive
DNA
Pellet (bacterial
cells and contents)
Batch 2:
Phosphorus (32P)
Centrifuge
Radioactivity
(phage DNA)
in pellet
Pellet

29. DNA Replication
It takes a human cell only a few hours to complete replication.
Use proteins and enzymes

30. Origins of Replication
Special site where replication begins
Specific sequence of nucleotides
2 parental strands will separate to form replication bubbles.
At the end of each replication bubble is a replication fork
The bubbles expand laterally, and DNA replication proceeds in both directions until replication is complete.

31. DNA Polymerases
Elongation of new DNA at a replication fork is catalyzed by DNA Polymerases.
Polymerase adds the new base pairs to growing DNA strand.
Rate of expansion is 500 nucleotides per second in bacteria and 50 per second in human cells.

32. Energy for Replication
Comes from nucleoside triphosphates, which are nucleotides with 3 phosphates.
Similar to ATP; only ATP has ribose, and NTP has deoxyribose as sugar.
As each monomer is added to growing DNA strand, it loses 2 phosphate groups as a molecule of pyrophosphate.
Hydrolysis of pryophosphate into 2 phosphates is the exergonic reaction that releases the energy for polymerization.

33. Antiparallel Arrangement of DNA
The sugar-phosphate backbones run in opposite directions.
The 5 carbons of one deoxyribose sugar are numbered 1’ to 5’.
One strand has the first phosphate attached at 5’.
The other strand does not have a phosphate attached at the 3’ end.

34. 5’ to 3’ Replication always proceeds in a 5’ to 3’ direction.
DNA polymerase only adds nucleotides to the 3’ end.
This creates 2 newly forming strands, the leading strand and the lagging strand.

35. Leading Strand Parental Strand runs 5’ to 3’ Continually elongates…
1 continuous strand
“Top” strand

36. Lagging Strand Parental Strand runs 3’ to 5’ But still grows 5’ to 3’
Grows in small segments, called Okazaki fragments.
DNA Ligase connects the Okazaki fragments.

37. Priming DNA Synthesis
None of DNA polymerases actually initiate the synthesis of a polynucleotide.
They only add nucleotides to the existing chain.
The start of the new DNA chain is called the primer, which is a short stretch of RNA.
Only 1 primer is required to begin replication of new DNA strand.
Primase is the enzyme which makes the primer.

38. Other Proteins Involved in Replication
Helicase – enzyme that untwists the original DNA at the replication fork.
Other proteins called single strand binding protein line up along the unpaired DNA, and hold the strands apart so that replication can proceed.

39. Damaged DNA Sometimes nucleotides are mispaired in the DNA molecule.
Must undergo mismatch repair
Sometimes mismatches are caused by chemicals, radioactive emissions, X-rays, UV light
Damaged part is removed by nuclease.
Repair is carried out by a DNA polymerase and ligase

40. End of DNA molecule replication
The ends of DNA contain nucleotide sequences called telomeres.
Telomeres do not contain genes.
Telomerase – enzyme which synthesizes telomers.

41. From Gene to Protein One Gene, One Polypeptide Hypothesis
Each gene will code for one enzyme or one polypeptide.
Work of Garrod and Beadle and Tatum led to this important discovery.

42. Garrod
In 1902, a British physician named Archibald Garrod noticed that a disorder called alkaptonuria seemed to run in families.
Suggested that the disease was result of Mendelian inheritance.
Disease was caused by the lack of an enzyme which breaks down an acid found in urine.
Without the enzyme, the acid oxidized rapidly when exposed to air, and turned the urine BLACK!

43. Beadle and Tatum
In 1941, confirmed Garrod’s suspicions that DNA sequences code for enzymes.
They were Americans! Conducted their experiments at Stanford University.
Studied genetic mutations.

44. What did they do?
Exposed bread mold to x-rays expecting them to damage some of the DNA.
Mutations
Concluded that mutations affected the growth rate of mutants and their ability to metabolize sugars.

45. Mutations What is a mutation? What is it caused by?
Name several different types of mutations.