DNA: Prince of the Chemicals Timothy G. Standish, Ph. D.

Transformation Of Bacteria Two Strains Of Streptococcus Rough Strain (Harmless) Capsules Smooth Strain (Virulent)

Transformation Of Bacteria The Griffith Experiment OUCH! + Control - Control - Control Experimental

Avery, MacLeod and McCarty 1944 Avery, MacLeod and McCarty decided to repeat Griffith’s 1928 experiment and try to discover the “transforming factor” They did this by using extracts from the heat- killed cells and digesting specific classes of molecules with enzymes Transformation? Enzyme Lippase Yes Protease Yes Saccharase Yes Nuclease No

The Hershey-Chase Experiment The Hershey-Chase experiment showed definitively that DNA is the genetic material Hershey and Chase took advantage of the fact that T2 phage is made of only two things: Protein and DNA H OH P O HO NH2 Nucleotides contain phosphorous, thus DNA contains phosphorous, but not sulfur. H OH O H2N C CH2 SH CH3 S Some amino acids contain sulfur, thus proteins contain sulfur, but not phosphorous. Cysteine Methionine

Using S35 Did protein enter the bacteria? T2 grown in S35 containing media incorporate S35 into their proteins Bacteria grown in normal non-radioactive media T2 attach to bacteria and inject genetic material When centrifuged, phage protein coats remain in the supernatant while bacteria form a pellet Mix-O-Matic The supernatant is radioactive, but the pellet is not. Blending causes phage protein coat to fall off Did protein enter the bacteria? Is protein the genetic material?

Using P32 Did DNA enter the bacteria? Is DNA the genetic material? T2 grown in P32 containing media incorporate P32 into their DNA Bacteria grown in normal non-radioactive media T2 attach to bacteria and inject genetic material When centrifuged, phage protein coats remain in the supernatant while bacteria form a pellet Mix-O-Matic The pellet is radioactive, but the supernatant is not. Blending causes phage protein coat to fall off Did DNA enter the bacteria? Is DNA the genetic material?

A Nucleotide Adenosine Mono Phosphate (AMP) OH HO Phosphate NH2 N Base - H+ OH O CH2 Sugar H 2’ 3’ 4’ 5’ 1’ Nucleotide Nucleoside OH

Purines Pyrimidines Uracil CH3 N O NH Thymine N Adenine NH2 NH2 O N NH (RNA) CH3 N O NH Thymine (DNA) N Adenine NH2 NH2 O N NH Guanine N O NH2 Cytosine

Base Pairing Guanine And Cytosine H O N Guanine - N O H Cytosine + + - - +

Base Pairing Adenine And Thymine - + Adenine CH3 N O H + - Thymine

Base Pairing Adenine And Cytosine H Cytosine - + N H - + Adenine

Base Pairing Guanine And Thymine CH3 N O H + - Thymine H O N Guanine + -

D N A B A S E S SUGAR-PHOSPHATE BACKBONE H2O H2O H P O HO CH2 OH NH2 N

The Watson - Crick Model Of DNA T A G C C G G C T A 3.4 nm 1 nm Minor groove Major groove 0.34 nm

Large Linear Chromosomes Have Many Origins Of Replication 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’

Extension - The Replication Fork 5’ 3’ Primase - Makes RNA primers Single-strand binding proteins - Prevent DNA from re-annealing Lagging Strand 3’ 5’ 5’ 3’ Okazaki fragment RNA Primers DNA Polymerase Helicase - Melts DNA Leading Strand 5’

Extension - Okazaki Fragments DNA Pol. 3’ 5’ RNA Primer Okazaki Fragment DNA Polymerase has 5’ to 3’ exonuclease activity. When it sees an RNA/DNA hybrid, it chops out the RNA and some DNA in the 5’ to 3’ direction. DNA Pol. 3’ 5’ RNA Primer RNA and DNA Fragments DNA Polymerase falls off leaving a nick. 3’ 5’ RNA Primer Ligase Nick The nick is removed when DNA ligase joins (ligates) the DNA fragments.

The End