I.  DNA as the Genetic Material

• Mendel (1866)
  
• Deoxyribonucleic acid (DNA)
  
• Sutton (1902)
  

     A.  Fred Griffith (1928)

            transformation with Streptococcus pneumoniae
 

      B. Levene (1929)

            elucidation of the chemical composition of DNA

      C.  Oswald Avery et al. (1944)

            selectively destroyed cellular constituents of S. pneumoniae and repeated Griffith's experiment
 

       D.  Alfred Hershey and Martha Chase (1952)

            labeled T2 bacteriophage DNA with 32P and viral protein with 35S
 

        E.  Franklin and Wilson (1952)

            diffraction photos of DNA
 

        F. Watson and Crick (1953)

• structure of DNA
  
  
• Chargoff's rules
  

II.  Nucleic Acids

    Ribonucleic acid (RNA)

    A.  DNA Structure

        usually single stranded, can form hairpin-loop

        purines   pyrimidines
        adenine    uradine
        guanine    cytosine
 

    C. Organization of DNA in Cells

        procaryotes: usually circular

        eucaryotes: usually linear

        supercoiled, in eucaryotes associated with proteins called histones
 
 

  III. DNA Replication

        A. Introduction

            semi-conserved replication

            1.   bidirectional: two replication forks
 
 

            2.  rolling circle
 
 

            3.  eucaryote: linear DNA with many replication forks
 

        B. Mechanism of Replication

            1.  Unwinding

• helicases unwind DNA
  
  
• single-stranded DNA binding proteins (SSBs)
  
  
• topoisomerases (DNA gyrase)
  
  
• antimicrobial agents that inhibit DNA gyrase
   

            2.  DNA synthesis

• three DNA polymerases identified in E. coli
  
  
• nucleotides added to the 3'-OH end, synthesis occurs in 5' to 3' direction
  
  
• DNA synthesis is semi-discontinuous.
   

                 b) DNA replication complementary to template

• DNA polymerase III
  
  
• Okazaki fragments
   

        3.  RNA primer removed

• DNA polymerase I or RNase H
   

        4.  Okazaki fragments joined together

• ligase
   

        5.  summary

•  complex process
  
  
• very accurate: proof-reading by DNA polymerases
  
  
• very rapid: 750-1,000 bp/sec
   

IV. The Genetic Code

      A.  Codons

      B.  Gene structure

• Gene: linear sequence of nucleotides with fixed start and end points
  
  
• DNA segment that codes for a polypeptide, rRNA, or tRNA
  
  
• Shine-Dalgarno: common sequence found in leader sequence of procaryotes
  
  
• RNA translation initiation codon (AUG)
  
  
• Open reading frames (ORFs)
  

 

C.  General Principles of Genes

   1.  Definitions

• genome: all the genes present in a cell or virus
  
  
• genotype: specific set of genes an organism possess
  
  
• phenotype: collection of characteristics that are observed
  
  
• haploid: single set of chromosomes
  
  
• diploid: two sets of chromosomes
  
  
• clone: population of cells asexually derived from a paraental cell and are genetically identical
  
  

          2.  Central Dogma

• Crick and Gamov (1957)

 

          3.  Not all genes expressed

          4. Operons

•  sequence of bases coding for one or more polypeptide under the control of a single promoter
  
  
• advantages
  
  
• lac operon

 

          5.  Genomics

• the study of the molecular organization of genomes, their information content, and the gene products they encode

     D.  Proteomics

• Study of proteomes

V. Mutations

     A. Introduction

• first defined as altered phenotypes
  
  
• permanent, inheritable change in DNA
  
  
• mutagenesis
  
  
  1. spontaneous mutations

  
           a)  constitutive mutations
  
  

         b)  hypermutations (adaptive mutations)



  2.  induced mutations
 

      B. Types of mutations

•  wildtype

            1) forward mutation
 

            2) reversion mutation: a second mutation occurs resulting in a wildtype phenotype

                a) back mutation
 

                b) suppressor mutation
 
 

            3) point mutation

                a) silent mutation
 

                b) missense mutation

• neutral mutation
  

 

                c) nonsense mutation
 

                d) frame shift mutation
 
 
 

    C. Detection and Isolation of Mutants

        1. Mutant detection

•  must know the wildtype phenotype in order to recognize the mutation
  

            a) pigment production
 
 

            b) enzyme activity
 
 

            c) auxotrophic mutants

                replica plating
 
 

        2. Mutant selection

            a) reversion mutation
 

            b) resistance selection
 

            c) substrate selection
 

        3. Carcinogenicity (mutagenicity) testing

•  Ames test
  
  
• Salmonella Typhimurium his-
   

    VI. DNA Repair

•  Many changes to DNA are lethal
  
  
•  Reactive oxygen species and anti-oxidants

         A. Excision repair

• SOS regulon induced

 

          B. Photoreactivation
 

          C. Mismatch repair system
 

          D. Recombination repair

• RecA protein