Molecular Genetics: Transformation & Experiments

Transformation

• Griffith discovered that genetic material could be transferred from one bacteria to another.
• He observed a transfer of virulence from dead S strain bacteria to living R strain of bacteria.
• He called this transformation.
• He did not know the mechanism.
• Transformation involves the physical transfer of genetic material between cells.

Avery, MacLeod, & McCarty

• Repeated Griffith’s experiment using purified cell extracts.
• Used protein digesting enzymes and DNA digesting enzymes.
• Removal of all protein from the transforming material did not destroy its ability to transform R strain cells
• DNA-digesting enzymes destroyed all transforming ability.
• DNA is the genetic material, at least in bacteria.

Hershey & Chase

• Used bacteriophages to investigate genetic material.
• Bacteriophages infect bacteria.
• Composed of only DNA and protein.
• Wanted to determine what molecule is injected into bacteria.
• Radioactive phosphorus (32P) was used to label bacteriophage DNA.
• Radioactive sulfur (35S) was used to label bacteriophage protein.
• Only the bacteriophage DNA entered the bacteria – DNA is the genetic material.

James Watson and Francis Crick

• Determined the structure of DNA using evidence from Chargoff, Franklin, and others.
• Not themselves involved in any experimentation.
• Each DNA molecule was made up of two intertwined chains of nucleotides - a double helix.

Structure of DNA

• Each strand of DNA is made up of a phosphodiester backbone.
• The backbone is a repeating sugar and phosphate units joined by phosphodiester bonds.
• A single strand of DNA extends in a 5’ to 3’ direction.

Double Helix of DNA

• Two strands of DNA are arranged as a double helix.
• This forms two grooves: the larger major groove, and the smaller minor groove.
• Strands are connected via hydrogen bonds between bases on opposite strands.
• Specific base-pairing: A-T, and G-C.
• The helix has a consistent diameter and is stable due to the additive property of thousands of low-energy hydrogen bonds.

Base pairing

• Base pairing is complementary between A and T, and G and C.
• A forms two hydrogen bonds with T.
• G forms three hydrogen bonds with C.
• Two strands of DNA are not identical, but are complementary.

Antiparallel configuration

• Each phosphodiester strand has an inherent polarity.
• One end terminates in 3′ OH.
• One end terminates in 5′ PO4.
• The two strands of a single DNA molecule have opposite polarity to one another.

DNA replication

• Requires a parental DNA molecule to copy.
• Requires enzymes to do the copying (replication).
• Replication is the process by which a DNA molecule is copied.

Three possible DNA replication models

• Conservative model: parental strands remain intact; new DNA copies consist of all new molecules.
• Semiconservative model: daughter strands each consist of one parental strand and one new strand.
• Dispersive model: new DNA is dispersed throughout each strand of both daughter molecules after replication.

Meselson and Stahl

• Performed an experiment to determine the correct model of DNA replication.
• Grew bacteria in a heavy isotope of nitrogen 15N so that the DNA was denser than normal DNA.
• Switched to media containing lighter 14N.
• Extracted DNA from bacteria at various time intervals and centrifuged to separate by weight.
• Supported the semiconservative model of DNA replication.

Eukaryotic Replication

• More complex than prokaryotic replication.
• Larger amount of DNA in multiple chromosomes.
• Linear structure (versus circular chromosomes).

Eukaryotic Replication uses multiple origins

• Multiple replicons – multiple origins of replications for each chromosome.
• Not sequence specific; can be adjusted for example early in development.

Eukaryotic Replication fork is more complex

• Before S phase, helicases are loaded onto possible replication origins, but not activated.
• During S phase, a subset of these are activated, and the rest of the replisome assembled.
• Priming uses a complex of both DNA polymerase α and primase.
• DNA polymerase epsilon (Pol ε) synthesizes leading strand.
• DNA polymerase delta (Pol δ) synthesizes lagging strand.

Archaeal and Eukaryotic replication proteins are evolutionarily related

• Enzymes that are similar between eukaryotes and archaea, but different from those in prokaryotes:
  • DNA polymerases
  • Replicative helicases
  • Primases

Linear chromosomes have specialized ends

• Telomeres are specialized structures found on the ends of eukaryotic chromosomes.
• Composed of specific repeat sequences.
• Protect ends of chromosomes from nucleases.
• Maintain the integrity of linear chromosomes.
• Not made by the replication complex.