Bio3400 Chapter 10 DNA Structure and Analysis
  1. The model for genetic expression is summarized in the central        of molecular genetics: genetic information flows from      to      to           .

      Central Dogma of Molecular Genetics. Transcription of DNA results in the synthesis of messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). Translation of mRNA occurs on rRNA-containing ribosomes complexed with tRNA to make proteins.

      Central dogma of genetics. Gene expression involves transcription of DNA into mRNA, followed by translation of mRNA on a ribosome into a protein, which is made of amino acid subunits. The translation requires a dictionary, or genetic code.
    • RNA is similar to DNA except:
    • it has Uracil (U) in place of Thymine (T). + the sugar in its nucleotides is ribose instead of deoxyribose.
  2. The building blocks of nucleic acids are              , which consist of a              base, a          sugar, and a            group.

        Nucleic acids (DNA and RNA) are composed of repeating units of nucleotides. Each nucleotide is composed of a phosphate group, a sugar, and a nitrogenous base. The 4 types of nitrogenous base in DNA are adenine (A), thymine (T), guanine (G), and cytosine (C).
    • The              bases can be          or              .

        Nitrogenous bases can be either purines ot pyrimidines; each atom in the ring is assigned a number. The double-ring purines are adenine (A) and guanine (G). The single-ring pyrimidines are cytosine (C), thymine (T), and uracil (U).
        Both DNA and RNA contain A, C, and G; only DNA contains T, while only RNA contains U.
    • A nitrogenous base and a          sugar make up a             .

        Each carbon atom in the pentose sugar is assigned a number with a prime sign ('). Ribonucleic acids (RNA) contain ribose, a 5-carbon sugar. Deoxyribonucleic acids (DNA) contain deoxyribose, which has a hydrogen atom at the 2' carbon rather than a hydroxyl group. A ribose or deoxyribose sugar with a purine or pyrimidine base attached to the 1' carbon is called a nucleoside.
    • A nucleoside and a phosphate group make up a             .

      A nucleoside with a phosphate group at the 5' carbon is called a nucleotide. Nucleosides and nucleotides are named according to the specific nitrogenous base (A, T, G, C, and U) that is part of the building block.
  3. A nucleotide is also called a nucleoside                . Adding one or two phosphate groups results in nucleoside               and                , respectively.

      A nucleotide is a nucleoside monophosphate (NMP), with one phosphate group attached to the 5' carbon. Two phosphate groups yield a diphosphate (NDP), while three phosphate groups yield a triphosphate (NTP). The molecule at right is adenosine triphosphate (ATP), an important energy molecule.
  4. Nucleotides are linked by a                 bond between the phosphate group at the     position and the OH group on the     position.

      Linkage of two nucleotides by the formation of a 3'-5' phosphodiester bond, producing a dinucleotide. Multiple phosphodiester bonds form a polynucleotide chain.
    • Q: Is this DNA or RNA?+ A: This is DNA: it contains a deoxyribose sugar.
  5. Two strands of DNA polynucleotides form a         helix, which are               to each other.

      Watson and Crick (Nobel 1962) Watson and Crick proposed the double-helix structure of DNA in 1953. Each turn of the helix contains 10 nucleotides^*, so the 3.4 angstrom (0.34 nm) internucleotide distance add up to 34 angstroms per turn, twisting around major and minor grooves.

    • The distance between nucleotides is 0.34 nm, or .34 x 10^-9 m = 3.4 x 10^-10 m.
    • You have about 6 billion base pairs per cell in 46 chromosomes (p. 264).
    • (6 x 10^9) x (3.4 x 10^-10 m.) = 2.04 m. of DNA per cell.
    • You have about 200 trillion (2 x 10^14) somatic cells.
    • If all the DNA in your cells were uncoiled and lined up end-to-end, their total length is (2 x 10^14) x 2 m. = 400 trillion m. = 400 billion km.
    • The distance from Earth to the Sun is about 150 million km, or 300M km round trip.
    • You have enough DNA to take over 1,000 round trips to the Sun. Updated Sep 12, 2007 by Peter Chen

      The 2 strands have antiparallel sugar-phosphate backbones, connected by rungs of nitrogenous base pairs, of which there are 10 per turn. These phosphodiester bonds are covalent bonds that are stronger than the hydrogen bonds between the bases. On the inside, the nitrogenous bases are joined by hydrogen bonds: 2 hydrogen bonds between A-T pairs, and 3 hydrogen bonds between G-C pairs.

      G-C base pairs form 3 hydrogen bonds.

      The 2 DNA strands run in opposite (antiparallel) directions; one in the 3' - 5' direction, the other 5' - 3'. The A-T and G-C base pairing provides complementarity of the two strands. Thus, DNA follows these base-pairing rules: A always pairs with T, and G always pairs with C.
  6. Most RNA is         stranded and synthesized from a complementary strand of      .

      In RNA, the pentose sugar in the nucleotide is ribose, and uracil (U) instead of thymine (T) base-pairs with adenine (A). The 3 classes of RNA are messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA).
  7. Nucleic acids can be separated and analyzed by sedimentation              centrifugation and sedimentation           centrifugation.

      A mixture of molecules is loaded on top of a solution, forming a concentration gradient. The tubes are spun in an ultracentrifuge for a given amount of time and stopped.

      Insedimentation equilibrium centrifugation (or density gradient centrifugation), different molecules in the mixture will settle in bands of different buoyant densities where the centrifugal force is equal and opposite to the upward diffusion force. The gradient is eluted from the tube in fractions, which can then be measured for UV absorption at 260 nm. This technique can be used to analyze base composition of double-stranded DNA.

      In sedimentation velocity centrifugation, an analytical centrifuge is used to measure the speed at which molecules migrate down the tube by monitoring with UV absorption optics while spinning. This velocity is measured in Svedberg coefficients.

      Ribosomes in mitochondria from different species exhibit various sedimentation coefficients, different from the cytoplasmic ribosome coefficient of 80S of all eukaryotes. The 80S coefficient for Tetrahymena is probably coincidental. This supports the endosymbiotic hypothesis of the origin of mitochondria.
  8. DNA base composition can also be estimated by measuring               shift during DNA               .

      Double-stranded DNA can be unwound (denatured) by applying heat to break the hydrogen bonds holding the bases together. The denatured DNA exhibits stronger UV absorption, showing a hyperchromic shift during "melting".
    • Q: Which sample of DNA in the diagram has a greater percent of GC pairs? + A: The green sample with the higher melting temperature (Tm). The Tm increases as GC content increases, since GC pairs have 3 hydrogen bonds while AT pairs have 2 hydrogen bonds.

      A-T base pairs form 2 hydrogen bonds.
  9. Denatured and other single-stranded nucleic acids can be            by cooling. This allows for molecular                of nucleic acids from different sources.

      Double-stranded DNA is transcribed to form a single-stranded RNA transcript complementary to one of the 2 DNA strands. The DNA is heated to denature it, then slowly cooled together with the RNA.

      Some of the RNA will find its single-stranded DNA complement and renature in a process called molecular hybridization. The result is a DNA:RNA hybrid duplex.
  10. Fluorescent in situ                ( FISH ) can be used to identify the chromosomal location of a DNA segment.

      Fluorescent in situ hybridization (FISH). Single-stranded DNA or RNA complementary to specific segments is added to metaphase chromosomes fixed to slides and allowed to hybridize. The hybridized segment is then localized with a fluorescent molecule.
  11.                kinetics show the size and complexity of genomic DNA.

      Denatured, single-stranded DNA fragments can reassociate into complementary double strands. The single-stranded DNA concentration (C) can be plotted against a logarithmic scale of the product of C[0]t (initial concentration of DNA single strands), and t (time). The half-reaction time C[0]t[1/2] increases as the fragment size increases, so this can be used to estimate genome sizes. Repetitive DNA sequences in some eukaryotic DNA allow them to have much shorter C[0]t[1/2].
  12. Nucleic acid fragments can be separated by gel                  .

      Gel electrophoresis. A sample is placed on a semisolid gel immersed in a solution that conducts electricity. When current is applied, the negatively charged DNA fragments migrate toward the positive electrode (anode). The fragments move at rates based mostly on fragment size: smaller molecules migrate at a faster rate through the pores of the medium than larger ones. The fragments can be visualized as bands by applying dyes or by autoradiography (exposing a radioactive molecule to photographic film).