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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 G-C base pairs 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 Sedimentation Behavior Sedimentation behavior depends upon the molecule’s 1. Density 2. Mass 3. Shape 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 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 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 Denaturation and uv spectrophotometry Nucleic acids absorb ultraviolet light most strongly at wavelengths of 254260 nm due to the interaction of the UV light and the rings of the purines and pyrimidines UV spectrophotometry The increase of UV absorption of heated DNA is referred to as the hyperchromic shift and is easiest to measure 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 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 Molecular Hybridization Used to determine the amount of complementarity or similarity between two different species 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 Blotting Procedures Autoradiograph 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 Examples of fluorescence 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 Reassociation kinetics and repetitive DNA 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 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 Agarose Gel Electrophoresis Staining SDS Polyacrylamide Gels Vertical gel SDS used to denature proteins Proteins run or separate according to their molecular mass Native Protein Gels 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 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. 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. Proteins are complex molecules They have levels of structure Structure based upon the sequence of the amino acids Polar side chains Non Polar Hydrophobic side chains Electrical charged hydrophilic 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 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 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 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. The causes of Tertiary structure