1. ©1999 Timothy G. Standish Molecular Basis Of Inheritance Timothy G. Standish, Ph. D.

2. ©1999 Timothy G. Standish 1999 Ig Nobel Prizes Science Education. The Kansas Board of Education and the Colorado State Board of Education, for mandating that children should not believe in Darwin's theory of evolution. Medicine. Arvid Vatle of Norway, for carefully collecting, classifying, and contemplating which kinds of containers his patients chose when submitting urine samples. Chemistry. Takeshi Makino, of the Safety Detective Agency in Japan, for his involvement with an infidelity detection spray that wives can apply to their husbands' underwear. Biology. Paul Bosland, of the New Mexico State University's Chile Pepper Institute, for breeding a spiceless jalapeno chile pepper. Managed Health Care. The late George and Charlotte Blonsky of New York City and San Jose, California, for inventing a childbirth device in which the woman is strapped to a circular table that is then rotated at high speed.

3. ©1999 Timothy G. Standish Transformation Of Bacteria Two Strains Of Streptococcus Capsules Smooth Strain (Virulent) Rough Strain (Harmless)

4. ©1999 Timothy G. Standish Experimental Transformation Of Bacteria The Griffith Experiment - Control + Control - Control OUCH!

5. ©1999 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 things: 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

6. 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?

7. 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?

8. ©1999 Timothy G. Standish When DNA Replication Occurs Typically DNA replication only occurs when cells are preparing to divide (there are some exceptions) The cell life cycle 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

9. ©1999 Timothy G. Standish G1 M M G2 S S The Cell Life Cycle 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

10. ©1999 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 - T he old double-stranded DNA serves as a template for two new strands that are then joined together, thus giving two old strands together and two new strands together Old Semi-conservative - In which old strands serve as templates for new strands resulting in double-stranded DNA made up of an old and new strand Old Dispersive - In which sections of the old strands are dispersed in the new strands

11. ©1999 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 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 OH H P O HO O NH2NH2 N N N N

12. ©1999 Timothy G. Standish After 20 min. (1 replication) transfer DNA to centrifuge tube and centrifuge Dispersive model prediction Conservative model prediction Semi-conservative 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.

13. ©1999 Timothy G. Standish Stages of Replicaton Replication can be divided into three stages: Initiation - When DNA is initially split into two strands and polymerization of new DNA is started Elongation - When DNA is polymerized 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 some proofreading of the DNA to ensure that mutations are not incorporated into the newly formed strands of DNA

14. ©1999 Timothy G. Standish Tools of Replication There are three major enzymes involved in 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”)

15. ©1999 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

16. ©1999 Timothy G. StandishInitiation Initiation starts at specific DNA sequences called origins (Ori C = origin in E. coli chromosomes) Large linear chromosomes may 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

17. ©1999 Timothy G. Standish Initiation - Forming the Replication Eye 3’5’ 3’5’ 3’ Origin of Replication 5’ 3’ 5’ 3’ 5’ 3’

18. ©1999 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

19. ©1999 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

20. ©1999 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

21. ©1999 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

22. ©1999 Timothy G. Standish Thimine Dimer 5’3’ 5’ Mutation Excision Repair 3’ 5’3’ 5’ 3’ 5’ DNA Pol. DNA Pol. Ligase Endo- Nuclease Ligase Nicks

23. ©1999 Timothy G. Standish

24. Problem 1 Question: –If an organisms 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 %

25. ©1999 Timothy G. Standish Problem 2 Question: –Given the following sequence of one strand of DNA, write out the complimentary strand. –5’AATACGCGATGCTGGTATC3’ Answer: –5’AATACGCGATGCTGGTATC3’ –3’TTATGCGCTACGACCATAG5’