1. ©2000 Timothy G. Standish Isaiah 33:22 22For the Lord is our judge, the Lord is our lawgiver, the Lord is our king; he will save us.

2. ©2000 Timothy G. Standish Molecular Basis Of Inheritance Timothy G. Standish, Ph. D.

3. ©2000 Timothy G. Standish Outline 1 How we know DNA is the genetic material 2 When DNA is replicated 3 How DNA is replicated 4 How DNA is maintained

4. ©2000 Timothy G. Standish Transformation Of Bacteria Two Strains Of Streptococcus Capsules Smooth Strain (Virulent) Rough Strain (Harmless)

5. ©2000 Timothy G. Standish Experimental Transformation Of Bacteria The Griffith Experiment - Control + Control - Control OUCH!

6. ©2000 Timothy G. Standish Avery, MacLeod and McCarty 1944 Avery, MacLeod and McCarty repeated Griffith’s 1928 experiment with modifications designed to discover the “transforming factor” Extracts from heat-killed cells were digested with hydrolytic enzymes specific for different classes of macromolecules: NoNuclease YesProtease YesLipase Transformation?Enzyme YesSaccharase

7. ©2000 Timothy G. Standish The Hershey-Chase Experiement 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 classes of macromolecules: Protein and DNA H OH P O HO O NH 2 Nucleotides contain phosphorous, thus DNA contains phosphorous, but not sulfur. H OH O H2NH2NCC CH 2 SHSH H OH O H2NH2NC CH 3 C CH 2 S Some amino acids contain sulfur, thus proteins contain sulfur, but not phosphorous. CysteineMethionine

8. Using S 35 Bacteria grown in normal non- radioactive media T2 grown in S 35 containing media incorporate S 35 into their proteins Blending causes phage protein coat to fall off T2 attach to bacteria and inject genetic material Is protein the genetic material? When centrifuged, phage protein coats remain in the supernatant while bacteria form a pellet The supernatant is radioactive, but the pellet is not. Did protein enter the bacteria?

9. Using P 32 Bacteria grown in normal non- radioactive media T2 grown in P 32 containing media incorporate P 32 into their DNA Blending causes phage protein coat to fall off T2 attach to bacteria and inject genetic material Is DNA the genetic material? When centrifuged, phage protein coats remain in the supernatant while bacteria form a pellet The pellet is radioactive, but the supernatant is not. Did DNA enter the bacteria?

10. ©2000 Timothy G. Standish When DNA Replication Occurs Typically DNA replication only occurs when cells are preparing to divide (there are some exceptions) The cell lifecycle is well defined and can be divided into four stages: –Gap 1 (G1) - The growth phase in which most cells are found most of the time –Synthesis (S) - During which new DNA is synthesized –Gap 2 (G2) - The period during which no transcription or translation occurs and final preparations for division are made –Mitosis - Cell division

11. ©2000 Timothy G. Standish G1 M M G2 S S The Cell Lifecycle Gap 1 - Doubling of cell size. Regular cellular activities. Transcription and translation etc. Synthesis of DNA - Regular cell activities cease and a copy of all nuclear DNA is made Gap 2 - Final preparation for division Mitosis - Cell division

12. OH O CH 2 Sugar H H H A Nucleotide Adenosine Mono Phosphate (AMP) OH NH 2 N N N N Base P O OH HO O Phosphate 2’3’ 4’ 5’ 1’ Nucleotide Nucleoside H+H+ -

13. Pyrimidines NH 2 O N N NH N Guanine N N Adenine N N NH 2 N O N O N Cytosine Uracil (RNA) CH 3 N O N O NH N O N O Thymine (DNA) Purines

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

15. CH 3 N O N O N H + - Thymine N N N N H N H - + Adenine Base Pairing Adenine And Thymine

16. Base Pairing Adenine And Cytosine N O H N O N N H Cytosine - + - N N N N H N H - + Adenine

17. Base Pairing Guanine And Thymine CH 3 N O N O N H + - Thymine H O N N N N N H H Guanine + + -

18. SUGAR-PHOSPHATE BACKBONE H P O HO O O CH 2 HOH P O O HO O O CH 2 H P O OH HO O O CH 2 NH 2 N N N N O O N NH N N N O NH 2 N B A S E S DNADNADNADNA O H P O HO O O CH 2 HO O H2NH2N N HN N N H H P HO O O CH 2 O O N O H2NH2N N H H2OH2O HOH P O HO O O CH 2 CH 3 O O HN N H2OH2O

19. ©2000 Timothy G. Standish The Watson - Crick Model Of DNA 3.4 nm 1 nm 0.34 nm Major groove Minor groove A T T A G C C G G C T A A T G C T A A T C G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

20. ©2000 Timothy G. Standish DNA Replication: How We Know There are three ways in which DNA could be replicated: + New Old + New Old New + Old + New Old + New Old Conservative - O ld double-stranded DNA serves as a template for two new strands which then join together, giving two old strands together and two new strands together Old Semiconservative - Old strands serve as templates for new strands resulting in double-stranded DNA made of both old and new strands Old Dispersive - In which sections of the old strands are dispersed in the new strands

21. ©2000 Timothy G. Standish The Meselson-Stahl Experiment The Meselson-Stahl experiment demonstrated that replication is semiconservative This experiment took advantage of the fact that nucleotide bases contain nitrogen Thus DNA contains nitrogen OH H P O HO O NH2NH2 N N N N The most common form of Nitrogen is N 14 with 7 protons and 7 neutrons N 15 is called “heavy nitrogen” as it has 8 neutrons thus increasing its mass by 1 atomic mass unit

22. ©2000 Timothy G. Standish After 20 min. (1 replication) transfer DNA to centrifuge tube and centrifuge Dispersive model prediction Conservative model prediction Semiconservative model prediction The Meselson-Stahl Experiment Prediction after 2 or more replications Bacteria grown in N 15 media for several replications Transfer to normal N 14 media X X X The conservative and dispersive models make predictions that do not come true thus, by deduction, the semi- conservative model must be true.

23. ©2000 Timothy G. Standish Stages of Replication Replication can be divided into three stages: 1 Initiation - When DNA is initially split into two strands and polymerization of new DNA is started 2 Elongation - When DNA is polymerized 3 Termination - When the new strands of DNA are completed and some finishing touches may be put on the DNA Both elongation and termination may involve proofreading of the DNA ensuring that mutations are not incorporated into newly formed DNA strands

24. ©2000 Timothy G. Standish Tools of Replication Enzymes are the tools of replication: DNA Polymerase - Matches the correct nucleotides then joins adjacent nucleotides to each other Primase - Provides an RNA primer to start polymerization Ligase - Joins adjacent DNA strands together (fixes “nicks”)

25. ©2000 Timothy G. Standish More Tools of Replication Helicase - Unwinds the DNA and melts it Single-Strand Binding Proteins - Keep the DNA single stranded after it has been melted by helicase Topisomerase - Relieves torsional strain in the DNA molecule Telomerase - Finishes off the ends of DNA strands

26. ©2000 Timothy G. StandishInitiation Initiation starts at specific DNA sequences called origins (Ori C = origin in E. coli chromosomes) Long linear chromosomes have many origins First the origin melts (splits into two single strands of DNA) Next primers are added Finally DNA polymerase recognizes the primers and starts to polymerize DNA 5’ to 3’ away from the primers

27. ©2000 Timothy G. Standish Initiation - Forming the Replication Eye 3’5’ 3’5’ 3’ Origin of Replication 5’ 3’ 5’ 3’ 5’ 3’

28. ©2000 Timothy G. Standish Large Linear Chromosomes Have Many Origins Of Replication 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Origins of Replication

29. ©2000 Timothy G. Standish Leading Strand Lagging Strand 3’ 5’ 3’ 5’ Extension - The Replication Fork 5’ 3’ 5’ 3’ 5’ Single-strand binding proteins - Prevent DNA from re- annealing DNA Polymerase Okazaki fragment RNA Primers Primase - Makes RNA primers 5’ 3’ 5’ Helicase - Melts DNA

30. ©2000 Timothy G. Standish Extension - Okazaki Fragments The nick is removed when DNA ligase joins (ligates) the DNA fragments. 3’5’ 3’ RNA PrimerOkazaki Fragment 3’5’ 3’ RNA Primer RNA and DNA Fragments Nick 3’5’ 3’ 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. DNA Polymerase falls off leaving a nick. DNA Pol. DNA Pol. Ligase

31. ©2000 Timothy G. Standish Mutation When Mistakes Are Made 5’3’ 5’ DNA Pol. 5’ 3’ 5’3’ 5’ DNA Pol. DNA Pol. Mismatch 3’ to 5’ Exonuclease activity

32. ©2000 Timothy G. Standish Thimine Dimer Mutation Excision Repair 3’ 5’3’ 5’ 3’ 5’ Endo- Nuclease

33. ©2000 Timothy G. Standish 5’3’ 5’ 3’ 5’ Mutation Excision Repair 3’ 5’3’ 5’ Endo- Nuclease Nicks DNA Pol.

34. ©2000 Timothy G. Standish 5’3’ 5’ Mutation Excision Repair 3’ 5’3’ 5’ 3’ 5’ DNA Pol. Endo- Nuclease

35. ©2000 Timothy G. Standish 5’3’ 5’ 3’ 5’ Mutation Excision Repair 3’ 5’3’ 5’ DNA Pol. Ligase Endo- Nuclease Nicks Nick Ligase

36. ©2000 Timothy G. Standish

37. Problem 1 Question: –If an organism’s DNA is 32 % adenine, what percent guanine, thymine, and cytosine are found in the DNA? Answer: –As adenine always pairs with thymine, there must be 32 % thymine –% GC = 100 % - (T% + A%) = 100 % - (32 % + 32 %) = 36 % –The proportion of guanine to cytosine has to be equal as they pair with one another thus G and C % = 36 % / 2 = 18 % –G = 18 %, T = 32 % and C = 18 %

38. ©2000 Timothy G. Standish Problem 2 Question: –Given the following sequence of one strand of DNA, write out the complementary strand. –5’AATACGCGATGCTGGTATC3’ Answer: –5’AATACGCGATGCTGGTATC3’ –3’TTATGCGCTACGACCATAG5’