1. Chapter 16 – The Molecular Basis of Inheritance

2. Searching for Genetic Material
Mendel: modes of heredity in pea plants (1850s)
Morgan: genes located on chromosomes (1910s)
Frederick Griffith: bacterial work; identified the concept of transformation – a change in genotype and phenotype due to assimilation of an external substance (DNA) by a cell (1928)
Oswald Avery: transformation agent was DNA (1944)
Before Avery, many scientists believed that protein was the genetic material and not DNA. Can you think of a reason for this theory?
Protein is more complex (20 AAs as compared to 4 nucleotides) and a simple DNA strand couldn’t possibly give rise the complexity of humans.

3. Griffith’s Experiment
2 strains of Streptococcus pneumoniae – 1 with a polysaccharide coat (smooth or S), one without (rough or R)
Conducted 4 experiments (fig. 16.2)
Injected mice with live S, the mice died, so encapsulated strain was pathogenic (disease causing)
Injected mice with live R, the mice stayed healthy, so the strain was non- pathogenic
Injected mice with heat-killed S, mice healthy, the polyscaaharide coat was found to not be the disease-causing agent because the coat was still intact in the heat-killed S bacteria
Heat killed S mixed with R and injected into mice, the mice died, the blood contained live S strain cells. The R strain “acquired” from S the ability to make a polysaccharide coat. These S cells were cultured and found to produce encapsulated daughter cells, concluded that newly acquired trait was inheritable.
The change in phenotype was due to an assimilation of external genetic material by a cell – transformation
Concluded that this could not be a protein because heat kills (denatures) proteins

4. Griffith’s Experiment

5. Hershey and Chase
1952 – discovered that DNA is the genetic material of a phage called T2
T2 phage can infect E.coli, which is simply DNA enclosed by a protein coat, and could quickly reprogram the E.coli to produce new T2 phages.
Conducted experiments to figure out if the DNA or the protein was responsible for this reprogramming phenomenon
Phage protein was tagged with 35S, phage DNA was tagged with 32P
Allowed phage to infect separate samples of non-radioactive E. coli cells.
Centrifuged the samples- the heavier bacteria would fall to the bottom while the light virus would be on top
Measured radioactivity
Tubes with protein-labeled T2 had radioactivity in supernatant (liquid)
Tubes with DNA-labeled T2 had radioactivity in the pellet (bacteria)
Phage proteins remained outside the bacterial cells during infection, while phage DNA entered the cells
When cultured, bacterial cells with radioactive phage DNA released new phages with some 32P DNA
Concluded that DNA is the hereditary material of T2, not protein

6. Hershey and Chase

7. DNA Structure
Edwin Chargaff (1947) – figured out ratio of nitrogen bases in a DNA molecule, approximate numbers of T=A and C=G, Chargaff’s rule
Rosalind Franklin (1950’s) – used X-ray crystallography to determine that DNA is in the form of a double helix

8. DNA Structure Watson and Crick (1953) – proposed a model for DNA
Used x-ray crystallography data from Franklin as well a Chargaff’s rule to propose their model:
The structure is a double helix with a sugar-phosphate backbone and nitrogen bases in the center
Their model was not widely accepted until they could demonstrate how DNA could be replicated (i.e. its functionality)
In 1954, Watson and Crick published a second paper outlining how the process occurs – demonstrated that DNA replication is semiconservative
Data supported by Maurice Wilkins in 1956
Watson, Crick, and Wilkins win the Nobel Prize in 1962 (Franklin not awarded prize – deceased, 1958)

9. DNA Structure

10. DNA structure (and RNA)
DNA structure animation

11. DNA Replication - semiconservative
Proposed by Watson and Crick - strands are complementary; nucleotides line up on template according to base pair rules
Each new double helix will have one “old or original” strand and one “new” strand

12. DNA Replication – semiconservative
Meselson and Stahl (late 1950s) designed an experiment to test the theory of semiconservative DNA replication

13. DNA Replication Bubble
Origins of replication – a replication bubble
Begins at specific sites where parental strands separate with helicase enzyme and form bubble, stabilized with single-strand binding proteins
Each end of the bubble is a replication fork
Bubble expands laterally, DNA replication occurs in both directions
Eventually bubbles fuse, replication complete

14. DNA Replication Nucleotide Addition
New nucleotides are added to the free 3’ end of the growing strand (DNA formed in the 5’ to 3’ direction)
Utilizes a variety of polymerase enzymes to compete this addition
DNA is antiparallel, so how replicate both sides, simultaneously, in the same direction?

15. Leading and Lagging Strands
DNA Polymerase III adds new nucleotides to 3’ end
Leading strand – continuous replication towards the replication fork
Lagging strand – discontinuous replication away from the replication fork, replicated in chunks called Okazaki fragments
Animation overview

16. Lagging Strand
All replication begins with primase adding RNA nucleotides to DNA
DNA poly III adds nucleotides to the RNA primer sequence
DNA poly III releases when reach next RNA primer
DNA poly I replaces the RNA with DNA adding to 3’ end
DNA ligase forms bond between these two Okazaki fragments to complete DNA chain

17. Replication Overview

18. Replication Overview
Overview

19. DNA Repair Enzymes proofread DNA for errors
1 in 10,000 initial errors, 1 in 1 billion end errors
Mismatch repair – occurs while being synthesized, uses DNA polymerase
Excision repair – corrects accidental changes to DNA, uses nuclease, polymerase, and ligase

20. DNA Shortening On end of DNA is an RNA primer
Since DNA poly can only add to 3’ end, this is a problem
Because of this the ends get shorter
Telomeres – noncoding regions of DNA at end of chain, in humans repeating chains of TTAGGG
Protects genes from being eroded from successive rounds of replication
Telomerase catalyzes the length of telomeric DNA restoring the DNA length in germ cells; usually inactive in adult somatic cells