1. Part Two – Lecture I

2. Forms of DNA

3. A DNA  Rosalind Franklin focused on this form  Prevalent under high salt concentrations  More compact  Modification of major and minor grooves

4. Z DNA discovered  1979 – Andrew Wang – synthetic oligonucleotide  1.8 nm in diameter  12 base pairs per turn  G-C base pairs

5. Ultracentrifugation and the Svedburg coefficient  DNA and RNA may be analyzed by ultracentrifugation  RNAs are differentiated according to their sedimentation behavior when centrifuged at high speeds in a concentration gradient

6. Sedimentation Behavior  Sedimentation behavior depends upon the molecule’s 1.Density 2.Mass 3.Shape

7. Sedimentation equilibrium centrifugation  A density gradient is created that overlaps the densities of the individual components of a mixture of molecules.  The gradient is usually made of a heavy metal salt such as CsCl  During centrifugation, the molecules migrate until they reach a point of neutral buoyant density

8. Sedimentation equilibrium centrifugation  Can also be used to study the GC content  The number of GC pairs in the DNA molecule is proportional to the molecule’s buoyant density

9. Denaturation and Renaturation of DNA Molecules  When denaturation of the double stranded DNA occurs, the hydrogen bonds open, the duplex unwinds, and the strand separate  No covalent bonds break so that the strands stay intact  Strand separation can be induced by heat

10. Denaturation and uv spectrophotometry  Nucleic acids absorb ultraviolet light most strongly at wavelengths of 254- 260 nm due to the interaction of the UV light and the rings of the purines and pyrimidines

11. UV spectrophotometry  The increase of UV absorption of heated DNA is referred to as the hyperchromic shift and is easiest to measure

12. Renaturation  Denaturation can be reversed – by slowly cooling the DNA  Single strands of DNA can randomly find their complementary strands and reassociate  The hydrogen bonds will form slowly and then more and more duplexes or double helixes will form

13. Molecular Hybridization  This technique is based upon the denaturation and renaturation of DNA  In this case DNA from two different sources can be mixed  DNA and RNA and be mixed together – a transcript can find its complementary sequence in DNA

14. Molecular Hybridization  Used to determine the amount of complementarity or similarity between two different species

15. Proteins are polymers  Proteins are polymers of amino acids. They are molecules with diverse structures and functions.  Polymers are made up of units called monomers  The monomers in proteins are the 20 amino acids

16. Blotting Procedures

17. Autoradiograph

18. Fluorescent in situ hybridization - FISH  In this procedure mitotic or interphase cells are fixed to slides and subjected to hybridization conditions.  Biotin is complexed with the DNA and then bound to a fluorescent molecule such as fluorescein

19. Examples of fluorescence

20. Reassociation kinetics - Britten  Used with small fragments of DNA  DNA is then denatured  Temperature is lowered and reassociation monitored  Used to compare different organisms  Originally uncovered repetitive DNA sequences due to a greater than anticipated complmentarity

21. Reassociation kinetics and repetitive DNA

22. Electrophoresis  Separates molecules ina mixture by causing them to migrate under the influence of an electric field  A sample is placed in a porous media such as agarose or polyacrylamide gel  They are then placed in a solution (buffer) which conducts an electric current

23. Separation of DNA  DNA has a strong negative charge due to the phosphate groups  When the DNA is placed in the gel, it will migrate toward the positive electrode

24. Agarose Gel Electrophoresis

25. Staining

26. SDS Polyacrylamide Gels  Vertical gel  SDS used to denature proteins  Proteins run or separate according to their molecular mass

30. Native Protein Gels

32. Native Gels  In native gels, the proteins migrate according to a mass/charge ratio  In the case of hemoglobin the variant forms are able to be separated based upon a difference of charge due to the substitution of amino acids from the Beta globin chain

33. Protein Facts  Proteins: Polymers of Amino Acids  Proteins are polymers of amino acids. They are molecules with diverse structures and functions.  Each different type of protein has a characteristic amino acid composition and order.  Proteins range in size from a few amino acids to thousands of them.  Folding is crucial to the function of a protein and is influenced largely by the sequence of amino acids.

34. Proteins: Polymers of Amino Acids  Each different type of protein has a characteristic amino acid composition and order.  Proteins range in size from a few amino acids to thousands of them.  Folding is crucial to the function of a protein and is influenced largely by the sequence of amino acids.

35. Proteins are complex molecules  They have levels of structure  Structure based upon the sequence of the amino acids

36. Polar side chains

37. Non Polar Hydrophobic side chains

38. Electrical charged hydrophilic

39. Function of Proteins - continued  Enzymes – Biological catalysts  Transport of small molecules – Albumin and haptoglobin  Transport of oxygen – hemoglobin and myoglobin  Membrane proteins – to assist in support  Channels in membranes – to allow the passage of molecules or ions  Electron carriers in electron transport in the production of ATP

40. Functions( continued)i  Clotting proteins  Immune proteins to fight infectious agents  Histones – DNA binding proteins  Toxins to repel or kill other organisms  Bacteriocins – molecules produced by bacteria against bacteria

41. Functions of proteins  Hormones – Growth hormone  Receptors – to Receive information so that cell can communicate with other cells  Neurotransmitters – messenger molecules – to send information between neurons  Cytoskeleton – actin, myosin, and collagen – the structure of connective tissue and muscles  Antibodies – Immunoglobulins to fight disease

42. Four levels of Protein Structure  There are four levels of protein structure: primary, secondary, tertiary, and quaternary.  The precise sequence of amino acids is called its primary structure.  The peptide backbone consists of repeating units of atoms: N—C—C—N—C—C.  Enormous numbers of different proteins are possible.

46. The causes of Tertiary structure