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Part Two – Lecture I

Part Two – Lecture I. Forms of DNA. A DNA. Rosalind Franklin focused on this form Prevalent under high salt concentrations More compact Modification of major and minor grooves. Z DNA discovered. 1979 – Andrew Wang – synthetic oligonucleotide 1.8 nm in diameter 12 base pairs per turn

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Part Two – Lecture I

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  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 • Density • Mass • 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

  27. Native Protein Gels

  28. 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

  29. 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.

  30. 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.

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

  32. Polar side chains

  33. Non Polar Hydrophobic side chains

  34. Electrical charged hydrophilic

  35. 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

  36. 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

  37. 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

  38. 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.

  39. The causes of Tertiary structure

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