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DNA: Prince of the Chemicals Timothy G. Standish, Ph. D. ©1999 Timothy G. Standish Transformation Of Bacteria Two Strains Of Streptococcus Rough Strain (Harmless) Capsules Smooth Strain (Virulent) ©1999 Timothy G. Standish Transformation Of Bacteria The Griffith Experiment OUCH! + Control - Control - Control Experimental ©1999 Timothy G. Standish 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 heatkilled cells and digesting specific classes of molecules with enzymes Enzyme Transformation? Lippase Yes Protease Yes Saccharase Yes Nuclease No ©1999 Timothy G. Standish 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 OH H H2N C C CH2 CH2 S CH3 H O H2N C C OH Methionine CH2 SH O HO P NH2 O O OH Cysteine Some amino acids contain sulfur, thus proteins contain sulfur, but not phosphorous. OH H Nucleotides contain phosphorous, thus DNA contains phosphorous, but not sulfur. ©1999 Timothy G. Standish S35 T2 grown in containing media incorporate S35 into their proteins Using S35 Bacteria grown in T2 attach to bacteria and inject genetic material normal nonradioactive media When centrifuged, phage protein coats remain in the supernatant while bacteria form a pellet 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? P32 T2 grown in containing media incorporate P32 into their DNA Using P32 Bacteria grown in T2 attach to bacteria and inject genetic material normal nonradioactive media When centrifuged, phage protein coats remain in the supernatant while bacteria form a pellet 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) Phosphate HO H+ Nucleotide OH P O Base N H O 5’CH2 4’ NH2 H N O 1’ Sugar 3’ OH 2’ H OH N N Nucleoside Purines NH2 Adenine N N N O CH3 (DNA) N Guanine NH N Thymine O NH2 Uracil (RNA) NH N O N N Pyrimidines NH O N O NH2 Cytosine N N O Base Pairing Guanine And Cytosine - + + + - Base Pairing Adenine And Thymine + - Adenine - + Thymine Base Pairing Adenine And Cytosine + - - Base Pairing Guanine And Thymine + + P HO NH2 O N O CH2 OH N N O H N O CH2 O HO P O O N O CH2 OH H H2O NH N O HO P O H O NH2 N O CH2 O H O H H2O N O CH2 N O O O CH2 O P HO H O OH HO P NH2 HO P O H O HO O D N A OH - - - - - - G - 3.4 nm 1 nm - - Minor groove C G C T A A T - The Watson - Crick Model Of DNA G C T A C G A T Major groove A T C G G C 0.34 nm T A - - - - - - - - - - - ©1999 Timothy G. Standish - Large Linear Chromosomes Have Many Origins Of Replication Origins of Replication 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ ©1999 Timothy G. Standish Extension - The Replication Fork 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ Primase - Makes RNA primers Lagging Strand Okazaki fragment 5’ RNA Primers 3’ 5’ Single-strand binding proteins Prevent DNA from reannealing DNA Polymerase 5’ 3’ Helicase Melts DNA Leading Strand 5’ 3’ ©1999 Timothy G. Standish Extension - Okazaki Fragments 5’ 3’ Okazaki Fragment DNA Pol. 3’ 5’ RNA Primer 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. 5’ 3’ DNA Pol. RNA and DNA Fragments 3’ 5’ RNA Primer DNA Polymerase falls off leaving a nick. 5’ 3’ Ligase 3’ 5’ RNA Primer Nick The nick is removed when DNA ligase joins (ligates) the DNA fragments. ©1999 Timothy G. Standish ©1999 Timothy G. Standish