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Chapter 14: DNA: The Genetic Material

Chapter Outline

Chapter 14: DNA: The Genetic Material

 

14.0 Introduction
  1. Connection Between Hereditary Traits and Chromosomes

    1. Chain of Experiments Led to Understanding Molecular Mechanisms of Heredity fig 14.1

    2. Journey Often Erratic, Path Not Always Direct

14.1 What is the genetic material?

  1. The Hammerling Experiment: Cells Store Hereditary Information in the Nucleus

    1. Hammerling's Experiment with Acetabularia fig 14.2

      1. Large, unicellular alga used as model organism

      2. Preliminary experiment

        1. Large green alga cell with distinct foot, stalk and cap

        2. Cap lacking nucleus amputated: Cap regenerates

        3. Foot with nucleus amputated: No foot regenerated

        4. Hypothesized hereditary information stored in foot

    2. Surgery on Single Cells

      1. To test hypothesis used two species that looked different fig 14.3

        1. A. crenulata: Disk-shaped cap, A. mediterranea: Flower-shaped cap

        2. A. crenulata stalk onto A. mediterranea foot

        3. Regenerated cap looked similar to A. crenulata

        4. Amputated regenerated cap, next cap looked like A. mediterranea

        5. Further supported that hereditary information in foot

      2. Initial flower-shaped cap somewhat intermediate in shape

        1. First cap used information that was already present

        2. Subsequent caps used information provided by new foot

        3. Now know that instructions for first cap based on remaining mRNA

  2. Transplantation Experiments: Each Cell Contains a Full Set of Genetic Instructions

    1. Briggs and King: Frog Nucleus Transplant Experiments

      1. Removed nucleus from frog egg: No development

      2. Added nucleus from another egg: Development occurred

      3. Concluded nucleus directed development

    2. Successfully Transplanting Nuclei

      1. Inclusive whether nucleus could direct development of entire adult

      2. Eggs with transplanted nuclei often developed abnormally

      3. Gurdon transplanted nucleus of another species from tadpoles into eggs

        1. Eggs usually developed normally

        2. Nucleus at later stage retained information to direct development

    3. Totipotency in Plants

      1. Stewart fragmented mature carrot tissue

      2. Individual cells developed roots, became adult plants when placed on solid medium

      3. Concluded each cell has full set of genetic material, can generate entire adult

  3. The Griffith Experiment: Heredity Information Can Pass Between Organisms

    1. Genes Hold Hereditary Information

    2. Discovery of Transformation

      1. Griffith injected mice with various strains of one bacteria fig 14.4

        1. Virulent, coated bacteria (S form) lethal to mice

        2. Nonvirulent, coatless strain (R form) not lethal

        3. Coat necessary for infection

      2. Questioned toxic effect of coat itself

        1. Injected mice with dead coated bacteria

        2. Mice remained healthy

      3. Dead S form and live R form bacteria mixed and injected

        1. Mice died, had live S form bacteria in blood

        2. Factor passed from one strain to other transforming it to virulent strain

  4. The Avery Experiments: The Transforming Principle Is DNA

    1. Utilized Same Bacterium

      1. Removed 99.98% of protein from dead S/live R mixture

      2. Transformation activity unaltered fig 14.5

    2. Properties of Transforming Principle Resembled Those of DNA

      1. Purified principle analyzed to resemble elements of DNA

      2. Principle acted like DNA with ultracentrifugation, electrophoresis

      3. Extracting lipid and protein did not alter activity

      4. Protein- or RNA-digesting enzymes did not affect activity

      5. DNA-digesting enzymes destroyed activity

      6. Concluded principle was indeed DNA

  5. The Hershey Chase Experiment: Some Viruses Direct Their Heredity with DNA

    1. Examined Bacteriophage Viruses that Attack Bacteria

      1. Bacteriophages possess either DNA or RNA, surrounded by protein coat

      2. Lytic virus injects viral genetic material into bacteria

      3. Causes production and release of more viruses when cell lyses

    2. Experiments to Determine If the Genetic Material Was DNA or Protein fig 14.6

      1. Used DNA bacteriophage called T2

        1. Labeled bacteriophage DNA with 32P and protein coat with 35S

        2. Viruses infect bacteria, attached viruses shaken off

        3. Agitation removed 35S from bacterial preparation

        4. Found 32P injected into bacterial cells

      2. Concluded genetic material in bacteriophages was DNA

      3. 40 years of evidence supports DNA as hereditary material in eukaryotes also

  6. The Fraenkel-Conrat Experiment: Other Viruses Direct Their Heredity with RNA

    1. Experiments to Determine Hereditary Material in Non-DNA Viruses

      1. Some viruses possess RNA, not DNA

        1. Tobacco mosaic virus (TMV)

        2. Holmes ribgrass virus (HRV)

      2. Is their genetic material RNA or protein?

        1. Separated protein coat from RNA

        2. Isolated RNA infective, isolated protein not infective

      3. Further experimented to verify hypothesis

        1. Tobacco infected with hybrid with TMV protein coat and HRV RNA fig 14.7

        2. Observed lesions characteristic of HRV

        3. Concluded hereditary material was RNA

    2. Retroviruses

      1. Other viruses also contain RNA, not DNA

        1. DNA viruses copy own DNA and insert into cell's DNA

        2. Retroviruses make intermediate double-stranded DNA from own RNA

        3. Examples: HIV virus, tumor causing viruses

          1. Transcription of virus RNA only occurs after DNA copy inserted into host DNA

          2. Integration is an obligatory step

14.2 What is the structure of DNA?

  1. The Chemical Nature of Nucleic Acids

    1. Miescher Isolated Material from Cell Nuclei

      1. White substance isolated from human cells, fish sperm

      2. Had unique proportions of nitrogen and phosphorus

      3. Named substance "nuclein"

    2. Levene's Analysis: DNA Is a Polymer

      1. Nuclein found to be acidic, renamed nucleic acid

      2. Levene determined primary structure fig 14.8

        1. Phosphate group PO4

        2. Five carbon sugars

        3. Nitrogen containing base: Purine or pyrimidine

          1. Purines = adenine (A), guanine (G)

          2. Pyrimidines = thymine (T), cytosine (C), RNA contains uracil (U) not T

      3. DNA and RNA composed of repeating units

        1. Called nucleotides

        2. Nitrogen base distinguishes nucleotide identity

      4. Numbering scheme for sugar structure fig 14.9

        1. A prime ( ˘ ) indicates that the carbon is located on the sugar molecule

        2. Phosphate attaches to 5' carbon

        3. Base attaches to 1' carbon

        4. Free hydroxyl, (–OH) attaches to 3' carbon

      5. Phosphate at 5˘ C, hydroxyl at 3˘ C enables chains to form

      6. Sugars linked by phosphodiester bond fig 14.10

      7. Nucleotide chain possesses definite direction

        1. One end of chain with free 5˘ phosphate group

        2. Other end of chain with free 3˘ hydroxyl group

        3. Sequences conventionally written in 5˘ to 3˘ direction

      8. Levene's early analysis found all four nucleotides present in equal amounts

        1. Assumed DNA a polymer of four repeating units

        2. DNA had structural role and protein had hereditary role

        3. Found to be wrong

    3. Chargaff's Analysis: DNA Is Not a Simple Repeating Polymer

      1. Found base amounts differed, depended on source tbl 14.1

      2. Composition of nucleotides varied in complex ways

        1. Suggested that DNA not a simple repeating polymer

        2. Found proportions of certain nucleotides equal to others

      3. Chargaff's rules

        1. Proportion of adenine (A) equal to thymine (T)

        2. Proportion of guanine (G) equal to cytosine (C)

        3. Proportion of purine (A + G) equal to pyrimidine (C + T)

  2. The Three-Dimensional Structure of DNA

    1. Franklin: X-Ray Diffraction Patterns of DNA fig 14.11

      1. In X-ray crystallography molecule bombarded with X-rays

        1. Resulting pattern of diffractions caused by DNA fibers

        2. Not precise since DNA sample was in fibers not true crystals

      2. Initial analysis of DNA fig 14.12

        1. Spring-like spiral with helical diameter of 2 nanometers

        2. Complete turn made every 3.4 nanometers

    2. Watson & Crick: A Model of the Double Helix fig 14.13

      1. Constructed models to determine shape

      2. Model of double helix fit all known data fig 14.14

        1. Bases pointed inward toward one another

        2. Large purine always paired with small pyrimidine

        3. Hydrogen bonds between bases stabilize antiparallel strands fig 14.15

          1. One strand ran 5' to 3'

          2. Other strand ran 3' to 5'

        4. Model explained Chargaff's results fig 14.15

          1. Adenine, thymine form two bonds

          2. Guanine, cytosine form three bonds

14.3 How does DNA replicate?

  1. The Meselson Stahl Experiment: DNA Replication Is Semiconservative

    1. Model Dependent on Complementarity of Strands

      1. Sequence of one chain determines sequence of its partner

      2. Each chain is complementary mirror image of other

      3. Unzipping molecule allows each strand to form daughter strands with same sequence

      4. Replication called semiconservative

        1. Sequence of strand conserved

        2. Duplex itself not conserved

        3. One strand of original goes into each of daughter strands

    2. Using Heavy Isotopes to Density-Label DNA Strands

      1. Labeled generations of bacteria with heavy nitrogen 15N

      2. DNA of new bacteria denser than other bacteria grown on 14N medium

      3. Transferred 15N bacteria onto 14N medium, collected DNA at intervals

    3. Separating DNA Strands by Density

      1. Experimental procedure

        1. Separated DNA strands in cesium chloride

        2. Ultracentrifuge used to spin solution

        3. Cesium ions form density gradient

        4. DNA strands migrates to position that matches density of cesium ions

      2. Experimental results

        1. 15N strands are denser than 14N strands

        2. 15N strands migrate further down tube

    4. The Key Result: Replication Alters DNA Density fig 14.16

      1. Initial bacteria all dense

      2. After one round of replication density intermediate between 15N and 14N

      3. After another round grouped into intermediate and light classes

        1. Intermediate group same as after first round

        2. Light group was equal to all 14N-DNA

    5. Interpreting the Results

      1. After first round of replication DNA had one heavy, one light strand

      2. When hybrid replicated to hybrids formed

        1. One had one light and one heavy strand

        2. Other had two light strands fig 14.17

      3. Confirmed Watson-Crick model of semiconservative replication

  2. The Replication Complex

    1. DNA Replication Must Be Fast and Accurate

      1. Replication begins at one or more origins of replication

      2. DNA replicating enzymes include DNA polymerase III

      3. Catalyzes reaction to add nucleotides to complementary strands tbl 14.2

    2. DNA Polymerase III

      1. DNA polymerase I is small, enzymes that plays supporting role

      2. DNA polymerase III is key enzyme, larger, more complex fig 14.18

        1. Contains 10 different polypeptide chains

        2. Is a dimer with two similar multisubunit complexes

      3. Variety of proteins have unique duties

        1. Large a subunit catalyses 5' to 3' addition of nucleotides

        2. Smaller e subunit proofreads 3' to 5' strand for mistakes

        3. Ring-shaped b2 dimer subunit clamps polymerase III complex around DNA helix

        4. Moves at rate of 1000 nucleotides per second

    3. The Need for a Primer

      1. DNA polymerase III cannot link first nucleotide in a newly synthesized strand

        1. RNA polymerase, primase constructs RNA primer

        2. Ten nucleotides complementary to DNA parent template

      2. DNA polymerase III recognizes primer, adds new nucleotides

      3. RNA nucleotides in primer replaced by DNA nucleotides

    4. The Two Strands of DNA Are Assembled in Different Ways

      1. DNA polymerase III can only add on to 3' end

      2. Replication occurs only in 5 ® 3 direction

      3. New strands oriented in opposite directions, replicated different ways

      4. Replication of leading strand, 5' to 3' strand

        1. Elongates towards replication fork

        2. New strand grows from 3' end

      5. Lagging strand, 3 to 5 strand replication

        1. Elongates away from replication fork

        2. Synthesized discontinuously in small batches

        3. Segments called Okazaki fragments

        4. 5' ® 3' synthesis catalyzed by DNA polymerase III

        5. DNA ligase attaches fragment to lagging strand

      6. Overall replication process is termed semidiscontinuous

  3. The replication Process

    1. Complex Process Deciphered After Decades of Research

    2. Occurs in Five Steps

      1. Opening up the DNA double helix

        1. Step One: Initiating replication

          1. Binding of initiator proteins to replication origin

          2. Starts process that opens helix

        2. Step two: Unwinding the duplex

          1. Untwisted by helicase enzyme

          2. Bind to one strand, push aside other strand

        3. Step three: Stabilizing the single strands

          1. Single-stranded binding proteins protect strands from cleavage

          2. Prevent rewinding

        4. Step four: Relieving the torque generated by unwinding

          1. If replication proceeds at 1000 nucleotides/second helix rotates 100 times/second

          2. Resulting twisting, torque, relieved by gyrases (topisomerases)

          3. Cleave strand of helix, swivels around intact strand, reseals strand

      2. Building a primer

        1. DNA polymerases require 3' primers to initiate replication

        2. Short series of RNA added by RNA polymerase called primase

        3. Multisubunit complex called a primasome

        4. Starting chains on exposed templates induces errors

        5. RNA marks initial stretch as temporary, later removes

      3. Assembling the complementary strand fig 14.20

        1. DNA polymerase III binds to replication fork

        2. Leading strand complexes with one half of the dimer

        3. Lagging strand loops around, complexes with other half of dimer

        4. Formation of complementary sequences on both strands at same time

      4. Removing the primer

        1. DNA polymerase I removes RNA primer

        2. Fills in gap and gaps between Okazaki fragments

      5. Joining the Okazaki fragments

        1. Gaps between Okazaki fragments filled in

        2. DNA ligase joins fragments to lagging strand

  4. Eukaryotic DNA Replication

    1. DNA Packaged into Nucleosomes within Chromosomes fig 14.22

      1. Individual zone of replication called replication unit or replicon

      2. Has own replication unit

      3. Multiple units may be replicating DNA at once fig 14.23

      4. Multiple origins increases speed of replication

    2. Replication Regulation Ensures Only One Copy Made

14.4 What is a gene?

  1. The One gene-One Polypeptide Hypothesis

    1. Garrod: Genetic Disorders Can Involve Specific Enzymes

      1. Examined several diseases

        1. Behaved like products of simple recessive alleles

        2. Concluded they were Mendelian traits

        3. Originated as change in heredity in ancestor to family

      2. Example: Alkaptonuria

        1. Urine contains homogensic acid that oxidizes and blackens on exposure to air

        2. Acid in normal urine broken down by enzymes

        3. Postulated that affected patients lack enzymes

      3. Concluded that information in DNA coded for enzymes

    2. Beadle and Tatum: Genes Specify Enzymes

      1. Set out to create and examine mutations in chromosomes

      2. A defined system

        1. Chose proper organism, a bread mold grown readily on a defined medium

        2. Defined medium contains known ingredients

        3. Used X-rays to induce mutations in mold spores

        4. Expected spores be damaged in areas associated with normal growth fig 14.24

          1. Lose ability to synthesize one or more compounds

          2. Affect ability to grow on normal medium

        5. Allowed progeny to grow on complete medium

          1. Contains all possible nutrients

          2. Strains unable to produce own nutrients still grew

      3. Isolating growth-deficient mutant

        1. Grow progeny on minimal medium to test for deficiencies

        2. Cells unable to make metabolite would not grow

        3. Identified numerous growth-deficient mutants

      4. Identifying the deficiencies

        1. Individually replace chemicals to determine deficiency

        2. Determine enzymes involved in deficiencies

          1. Each site coded for different enzyme in pathway

          2. Arginine mutants clustered in three areas fig 14.25

    3. One Gene-One Polypeptide

      1. Isolated a mutant strain for each enzyme in arginine pathway

        1. Mutation always located at one of a few chromosomal sites

        2. Each enzyme mutation occurred at different site

      2. Concluded genes produce effects by encoding enzyme structure

        1. Original hypothesis: One gene-one enzyme

        2. Current relationship: One gene-one polypeptide

      3. Enzymes responsible for catalyzing synthesis of all parts of an organism

        1. Mediate assembly of all biomolecules

        2. DNA therefore, specifies structure of organism itself

  2. How DNA Encodes Protein Structure

    1. Sanger: Proteins Consist of Defined Sequences of Amino Acids

      1. Identified amino acid sequence of insulin

      2. First demonstration of protein structure

      3. Information for enzymes is ordered list of amino acids

    2. Ingram: Single Amino Acid Changes in a Protein Can Have Profound Effects

      1. Analyzed normal and sickle-cell hemoglobin fig 14.26

      2. Single amino acid substitution between hemoglobins

      3. Change from glutamic acid to valine

    3. Modern Understanding of Heredity

      1. Gene: A sequence of nucleotides that usually encodes a particular protein

      2. Some genes encode produce special forms of RNA, involved in protein synthesis

      3. Central dogma of molecular genetics: DNA ® RNA ® protein

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