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IB 2 DNA by huangyuarong

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									LE 16-5
             Sugar–phosphate          Nitrogenous
                backbone
                   5 end
                                         bases
                                                            DNA
                                                            Building blocks?

                                      Thymine (T)




                                      Adenine (A)




                                      Cytosine (C)




          Phosphate                                  DNA nucleotide

                Sugar (deoxyribose)
                   3 end
                                      Guanine (G)
DNA strands are antiparallel
    LE 16-8

Base pairing in DNA

Purines?
Pyrimidines?


                  Sugar

                                               Sugar
                          Adenine (A)   Thymine (T)




                  Sugar


                                                 Sugar



                          Guanine (G)   Cytosine (C)
    LE 18-2



Size
comparison       Virus
of virus,
bacterium
&
eukaryotic      Bacterium
cell
                 Animal
                 cell
                                           nucleoid




                          Animal cell nucleus
              0.25 µm
LE 18-4b
             Capsomere
                                 DNA
                                       Viral structure:
                                       Nucleic acid +
                                       protein capsid


           Glycoprotein
               70–90 nm (diameter)




                               50 nm
                Adenoviruses
LE 18-4c
                  Membranous
                  envelope
                           Capsid

                                     RNA



                                           Retrovirus

           Glycoprotein
              80–200 nm (diameter)




                                 50 nm
                Influenza viruses
LE 18-4a
           Capsomere          RNA
           of capsid




                18  250 mm




                             20 nm
             Tobacco mosaic virus
16 media lytic cycle animation
LE 18-4d



            Head
                              DNA
           Tail
           sheath
            Tail
            fiber




                80  225 nm




                             50 nm
             Bacteriophage T4
       LE 18-6


  Lytic cycle
                                          Attachment



                                                              Entry of phage DNA
                                                              and degradation of
                                                              host DNA
   Phage assembly              Release




Head     Tails   Tail fibers


                               Assembly                Synthesis of viral
                                                       genomes and proteins
        LE 18-7
                                              Lysogenic cycle
                  Phage             The phage attaches to a                                          Daughter cell
                  DNA               host cell and injects its DNA.                                   with prophage
                                                                                                                         Many cell divisions
                                                                                                                         produce a large
                                                            Phage DNA                                                    population of
Phage                                                       circularizes                                                 bacteria infected with
                                                                                                                         the prophage.
                                    Bacterial
                                    chromosome                              Occasionally, a prophage
                                                                            exits the bacterial chromosome,
                                                                            initiating a lytic cycle.




                                      Lytic cycle                                  Lysogenic cycle
                                                              Certain factors                           The bacterium reproduces
                                                            determine whether                           normally, copying the prophage
The cell lyses, releasing phages.
                                                    Lytic cycle or Lysogenic cycle                      and transmitting it to daughter cells.
                                                    is induced             is entered      Prophage or Provirus




                   New phage DNA and proteins are                          Phage DNA integrates into the              Human
                   synthesized and assembled into phages.                  bacterial chromosomes, becoming a
                                                                           prophage.                                  example?
 Genetic traits are a result of proteins
• Alcaptonurea
• rare inherited
  genetic disorder
  of phenylalanine
  and tyrosine
  metabolism
• Early Hypothesis:
  chromosomes are
  master copies of
  proteins
     Griffith’s experiment (1928) with
        Streptococcus pneumoniae




Called it Transforming factor, heat
stable, genetic material may not be
proteins
Avery’s experiment (1943)

                 Purified chemicals
                 in heat killed cells:
                 determined
                 transforming factor
                 to be DNA

                 DNA 1st isolated in
                 1869… “too simple”
    Hershey-Chase Experiment (1952)
Bacteriophage:
nucleic acid +
protein coat,
one of them
must be the
genetic
information
Chargaff studied purine vs pyrimidine content in various species: A=T, C=G




    Rosalind Franklin               Franklin’s X-ray diffraction
                                    photograph of DNA
                                             Concluded it was a helix
Watson & Crick: determined double helix model w/
Chargaff & Franklin’s help
LE 16-UN298




              Purine + purine: too wide




              Pyrimidine + pyrimidine: too narrow




              Purine + pyrimidine: width
              consistent with X-ray data
  LE 16-7




                                    5 end
                                                      Hydrogen bond
                                                                      3 end

 1 nm

                         3.4 nm




                                         3 end

                          0.34 nm
                                                                         5 end

Key features of DNA structure          Partial chemical structure                 Space-filling model
                     DNA size
• DNA fiber diameter 20 Angstroms (20 x 10-10m)
   – Hair is 5000x DNA diameter
   – Thread is 50,000x DNA diameter
• Human diploid nucleus 174 cm DNA (50 miles if
  thick as thread)

• Humans have 100 trillion cells, total DNA 2.5 x
  1014 km = 100 x distance Earth to sun
• 600 g DNA/human (1% body weight)
  LE 19-2d




                                   700 nm




                                   1,400 nm



 Metaphase chromosome



http://spine.rutgers.edu/cellbio/assets/flash/
chrompack.htm
70 inches of DNA/cell packed down to
.0000012 inches
 LE 19-2c




                      Protein scaffold
                                                  Loops

                                         300 nm
                                                  Scaffold

Looped domains (300-nm fiber)
   LE 19-2a




                                              2 nm

              DNA double helix

His-            Histone
tones           tails

                                 Histone H1   10 nm



    Linker DNA             Nucleosome
    (“string”)             (“bead”)
 Nucleosomes (10-nm fiber)




Nucleosome = DNA wrapped around 9
histones (1 in middle)
Central Dogma
LE 16-9_2

                       DNA Replication




  The parent molecule has             The first step in replication is
  two complementary                   separation of the two DNA
  strands of DNA. Each base is        strands.
  paired by hydrogen bonding
  with its specific partner, A with
  T and G with C.
LE 16-9_4




The parent molecule has     The first step in replication   Each parental strand now    The nucleotides are
two complementary           is separation of the two        serves as a template that   connected to form the
strands of DNA. Each base   DNA strands.                    determines the order of     sugar-phosphate back-
is paired by hydrogen                                       nucleotides along a new,    bones of the new strands.
bonding with its specific                                   complementary strand.       Each “daughter” DNA
partner, A with T and G                                                                 molecule consists of one
with C.                                                                                 parental strand and one
                                                                                        new strand.
   LE 16-13


         New strand           Template strand
           5 end                 3 end                        5 end   3 end



    Sugar
                       Base
Phosphate




                                           DNA polymerase
              3 end


                                                                3 end
                                                Pyrophosphate


         Nucleoside
        triphosphate              5 end                                 5 end
             DNA Replication
• 50 nucleotides added/sec
• 6 billion base pairs in 46 DNA molecules
  copied w/in few hours w/ few mistakes due
  to repair enzymes. Read rest chapt 16, fill in
  Messelson & Stahl exp
LE 16-10
                                        First         Second
                          Parent cell   replication   replication

           Conservative
           model. The two
                                                                    Early hypothesis
           parental strands                                         for DNA
           reassociate after
           acting as                                                replication
           templates for
           new strands,
           thus restoring
           the parental
           double helix.



           Semiconservative
           model. The two
           strands of the
           parental
           molecule
           separate, and
           each functions as
           a template for
           synthesis of a
           new, comple-
           mentary strand.


           Dispersive model.
           Each strand of
           both daughter
           molecules
           contains
           a mixture of
           old and newly
           synthesized
           DNA.
LE 16-11
           Bacteria                             Bacteria
           cultured in                          transferred to      Messelson & Stahl
           medium                               medium              Experiment to test
           containing                           containing
           15N                                  14N                 DNA replication
                                                                    hypothesis
               DNA sample             DNA sample        Less
               centrifuged            centrifuged       dense
               after 20 min           after 40 min
               (after first           (after second
               replication)           replication)      More
                                                        dense
                          First replication    Second replication

           Conservative
           model




           Semiconservative
           model




           Dispersive
           model
  LE 16-12

Origins of replication in eukaryotes
                              Parental (template) strand
  Origin of replication                                                            0.25 µm
                                  Daughter (new) strand




        Bubble                Replication fork




             Two daughter DNA molecules


 In eukaryotes, DNA replication begins at may sites        In this micrograph, three replication
 along the giant DNA molecule of each chromosome.          bubbles are visible along the DNA
                                                           of a cultured Chinese hamster cell
                                                           (TEM).


 Origin of replication clip 16 media
     LE 16-14
                                                                3
                          Parental DNA                               5
                                                                               Leading strand

                5
                     3
                                                     Okazaki
                                                     fragments
                                                                               Lagging strand
 Synthesis of leading
 and lagging strands                                                  3
                                                                          5
 Replication fork                                 DNA pol III
 Always start @ 3’ end
 New DNA synthesized
 in 5’ to 3’ direction                          Template
                                                strand



                                                Leading strand
                                             Lagging strand




                                  Template
                                  strand               DNA ligase

HHMI movie                               Overall direction of replication

Leading strand ch 16 media
LE 16-15_1                      Primase joins RNA
               3            nucleotides into a primer.
                                                   5

                                 5             3
               Template
               strand




Lagging strand replication 16 media clip


                          Overall direction of replication
LE 16-15_2
                           Primase joins RNA
                        nucleotides into a primer.
             3                                                 5

                               5             3
             Template
             strand
                                                             DNA pol III adds
                                                           DNA nucleotides to
                                                           the primer, forming
                                                           an Okazaki fragment.
             3                                                  5
                        RNA primer                         3

                               5




                        Overall direction of replication
LE 16-15_3
                                     Primase joins RNA
                                  nucleotides into a primer.
                     3                                                         5

                                         5             3
                     Template
                     strand                                      DNA pol III adds
                                                              DNA nucleotides to
                                                              the primer, forming
                                                              an Okazaki fragment.

                     3                                                         5
                                  RNA primer                          3

                                         5




                After reaching the
             next RNA primer (not
               shown), DNA pol III                               Okazaki
                                                                fragment       3
                     3   falls off.                                            5

                                         5




                                  Overall direction of replication
LE 16-15_5
                                      Primase joins RNA
                                   nucleotides into a primer.
             3                                                                  5

                                             5          3
             Template
             strand                                               DNA pol III adds
                                                               DNA nucleotides to
                                                               the primer, forming
                                                               an Okazaki fragment.

             3                                                                  5
                                    RNA primer                         3

                                             5




                      After reaching the
                   next RNA primer (not
                     shown), DNA pol III                          Okazaki
                                falls off.                       fragment       3
             3
                                                                                 5

                                             5


                   After the second fragment is
              primed, DNA pol III adds DNA
              nucleotides until it reaches the
                5
              first primer and falls off.
             3
                                                                                3
                                                                                     5




                                           DNA pol I replaces
                                         the RNA with DNA,
                  5                     adding to the 3 end                   3
             3                          of fragment 2.                              5




                                   Overall direction of replication
LE 16-15_6
                                         Primase joins RNA
                                      nucleotides into a primer.
                3                                                                  5

                                                5          3
                Template
                strand                                               DNA pol III adds
                                                                  DNA nucleotides to
                                                                  the primer, forming
                                                                  an Okazaki fragment.

                3                                                                  5
                                       RNA primer                         3

                                                5




                         After reaching the
                      next RNA primer (not
                        shown), DNA pol III                          Okazaki
                                   falls off.                       fragment       3
                3
                                                                                    5

                                                5


                     After the second fragment is
                 primed, DNA pol III adds DNA
                 nucleotides until it reaches the
                 first primer and falls off.

                     5                                                            3
                3
                                                                                        5




                                          DNA pol I replaces
                                      the RNA with DNA,
                                      adding to the 3 end
                     5               of fragment 2.                               3
                3
                                                                                        5




               DNA ligase forms a
             bond between the newest                                    The lagging
             DNA and the adjacent DNA                                strand in the region
             of fragment 1.                                          is now complete.
                   5                                                              3
                3                                                                      5




                                      Overall direction of replication
     LE 16-16




                  Overall direction of replication              Leading                       Lagging
                                                                        Origin of replication
                                                                strand                        strand




                                                                Lagging                       Leading
                                                                strand      OVERVIEW          strand
                          DNA pol III

                                                        Leading
                                                        strand

                                                                                 DNA ligase
5                             Replication fork
 3                                                            DNA pol I
                            Primase
Parental DNA                             DNA pol III Lagging
                              Primer                 strand
                                                                                                      3
                                                                                                    5




      DNA polymerase II:
      repair enzyme edits &
      fixes mistakes
   LE 16-17
                                        A thymine dimer
                                     distorts the DNA molecule.   Nucleotide
Common w/ uv                                                      excision repair of
exposure                                                          DNA damage
                                         A nuclease enzyme cuts
                                     the damaged DNA strand
                                     at two points and the
                                     damaged section is
                                     removed.
                                      Nuclease

                                                               Common error rate 1 in
                                                               100,000
                                                               Permanent error rate 1
               DNA                       Repair synthesis by   in 10 billion,
               polymerase            a DNA polymerase II
                                     fills in the missing      most errors fixed
                                     nucleotides.




                            DNA
                            ligase
                                         DNA ligase seals the
                                     free end of the new DNA
                                     to the old DNA, making the
                                     strand complete.
      LE 16-18
                                     5
Shortening of     End of parental                               Leading strand
                  DNA strands                                   Lagging strand
the ends of                          3
linear DNA
molecules
                                             Last fragment     Previous fragment

Telomere end is                           RNA primer
usually “junk      Lagging strand 5

DNA” no genes                       3

                  Primer removed but                     Removal of primers and
                   cannot be replaced                    replacement with DNA
Exercise = more     with DNA because                     where a 3 end is available
telomerase          no 3 end available
                  for DNA polymerase
                                               5
                                    3
                                                         Second round
                                                         of replication

                                               5
                         New leading strand 3
                         New leading strand 5
                                    3
                                                         Further rounds
                                                         of replication

                                               Shorter and shorter
                                               daughter molecules
LE 16-19
           Telomeres




                       1 µm
    LE 17-3-5

                                                             Transcription & Translation
                                                    DNA
                     TRANSCRIPTION

Prokaryotes:                                        mRNA
                                                             DNA code        RNA code
No editing of         TRANSLATION
                                                  Ribosome


mRNA                                                         RNA Code        20 amino
                                          Polypeptide
                                                                             acids
                Prokaryotic cell
                                                                             proteins

                                                 Nuclear
                                                 envelope




                    TRANSCRIPTION          DNA
                                                             Eukaryotes: Nucleus
                                           Pre-mRNA
                                                             separates transcription and
                    RNA PROCESSING
                                                             translation,
                                   mRNA                      editing of pre-mRNA


                                            Ribosome          DNA & RNA code
                 TRANSLATION
                                                              comes in?
                                      Polypeptide
                                                              Called?

                Eukaryotic cell
LE 17-4

                                         Gene 2
          DNA
          molecule
                            Gene 1


                                                  Gene 3




          DNA strand   3                                  5
          (template)



          TRANSCRIPTION


                                                                Linear 500-
          mRNA         5                                  3   10,000
                                                                nucleotides
                             Codon
           TRANSLATION


          Protein


                            Amino acid
    LE 17-5
                 Second mRNA base



64 codons
3 stop codons
AUG: met/start
codon

Page 79: amino
acid property




                                    Third mRNA base (3 end)
review
  LE 17-7

            Transcription




      Promoter        Transcription unit

5                                        3
3                                        5
                             DNA
         Start point
RNA polymerase
LE 17-7
          Promoter   Transcription unit

 5                                        3
 3                        DNA             5
           Start point
  RNA polymerase
                               Initiation




  5                                       3
  3                                       5
           RNA Template strand
   Unwound tran- of DNA
   DNA     script
       LE 17-7
                      Elongation
                                                  Non-template
                                                  strand of DNA
                                                                    RNA nucleotides
  Usually                              RNA
  expose 10                            polymerase
  bases at a
  time

                 3
                                                    3 end


Sense strand =
DNA that is 5
complementary
to mRNA (light
blue). Results
in protein                             Direction of transcription
               5
Antisense                              (“downstream”)
                                                                       Template
strand (dark                                                           strand of DNA
blue)
                          Newly made
                          RNA
LE 17-7
                Promoter   Transcription unit

          5                                     3
          3                     DNA             5
                   Start point
          RNA polymerase
                                     Initiation




          5                                     3
          3                                     5
                  RNA Template strand
          Unwound tran- of DNA
          DNA     script
                                     Elongation

                  Rewound
                  DNA
          5                           3       3
          3                                     5
                  5

                  RNA
                  transcript
LE 17-7
                Promoter    Transcription unit

          5                                              3
          3                      DNA                    5
                   Start point
          RNA polymerase
                                      Initiation




          5                                             3
          3                                             5
                  RNA Template strand
          Unwound tran- of DNA
          DNA     script
                              Elongation


                  Rewound
                  DNA
          5                            3              3
          3                                             5
                   5

                  RNA
                  transcript
                                      Termination


          5                                             3
          3                                             5
                   5                              3
                         Completed RNA transcript
    LE 17-8
                                            Eukaryotic promoters
                                Promoter
                                                                        Initiation of transciption
              5                                                  3    at a eukaryotic promotor
              3                                                  5
                          TATA box      Start point   Template
                                                      DNA strand
                                            Several transcription
                                            factors

                        Transcription
                        factors

              5                                                  3
              3                                                  5

                                            Additional transcription
                                            factors




                   RNA polymerase II            Transcription factors



              5                                                  3
              3                           5                     5
                                                   RNA transcript
HHMI movie
                         Transcription initiation complex
 LE 17-9

                RNA processing: addition of the 5’ cap and poly-A tail




                               Cap & tail:
                               * enhance exit thru nuclear
                               pores
                               *Protect RNA from hydrolytic
                               enzymes which eventually
                               digest it in cytoplasm


                      Protein-coding segment       Polyadenylation signal
           5


  5 Cap   5 UTR    Start codon      Stop codon         3 UTR             Poly-A tail

Ribosome
attachment
LE 17-10

                         RNA processing: RNA splicing

                         Exon: usually code for polypeptide, exit nucleus

                         Intron: don’t exit nucleus, regulatory?, “junk”?




                5 Exon Intron             Exon         Intron           Exon     3
 Pre-mRNA   5 Cap                                                                     Poly-A tail
                     1      30        31          104            105            146

                                                   Introns cut out and
                                        Coding
                                                   exons spliced together
                                       segment


                     5 Cap                                               Poly-A tail
                                  1                                146
                         5 UTR                                        3 UTR
LE 17-11
                            RNA transcript (pre-mRNA)
           5
                   Exon 1               Intron              Exon 2


                Protein
                snRNA                              Other proteins    Role of snRNPs &
                                                                     spliceosomes in
                          snRNPs
                                                                     pre-mRNA splicing
                                     Spliceosome



                     5




                    Spliceosome
                    components
                                                              Cut-out
                                                              intron
                                       mRNA
                            5
                                   Exon 1    Exon 2
LE 17-12
                                             Gene
      DNA
                      Exon 1      Intron     Exon 2   Intron   Exon 3


                          Transcription
                                                         Alternative RNA splicing can
                                                         lead to different polypeptides
                       RNA processing                    Evolution

                               Translation               Explains why humans have
                                                                 fewer genes than
                                                                 originally thought
                                                               Domain 3




           Domain 2

                                                               Domain 1



                                       Polypeptide
    LE 17-13




Translation Requires:
Ribosomal units (rRNA)
                                                             Amino
mRNA                      Polypeptide                        acids
t RNA
Amino acids
                                                            tRNA with
HHMI movie                                                  amino acid
                                                            attached
                                                 Ribosome




                                                            tRNA

                                                            Anticodon




                     5                 Codons                  3

                          mRNA
LE 17-14a
                                 3
            Amino acid
            attachment site                            tRNA 2 & 3D structures
                                       5
                                                       75-85 nucleotides



                                            Hydrogen
                                            bonds




                             Anticodon
                    Two-dimensional structure
                                                   Amino acid
                                  5               attachment site
                                             3



                                       Hydrogen
                                       bonds




                                                  3          5
                         Anticodon                     Anticodon
             Three-dimensional structure     Symbol used in this book
LE 17-15
             Amino acid             Aminoacyl-tRNA
                                    synthetase (enzyme)

                                          Aminoacyl-tRNA synthetase joins a
                                          specific amino acid to a tRNA




           Pyrophosphate


             Phosphates
                           tRNA




                              AMP




                    Aminoacyl tRNA
                    (an “activated
                    amino acid”)
   LE 17-16b
      Anatomy of a ribosome: 1500-2900 nucleotides + proteins on exterior

      Prokaryote: 70s                          Eukaryote: 80s



P site (Peptidyl-tRNA
binding site)
                                           A site (Aminoacyl-
                                           tRNA binding site)
     E site
     (Exit site)
                         E   P   A           Large
                                             subunit
      mRNA
      binding site                           Small
                                             subunit
   Schematic model showing binding sites
LE 17-16c




            Amino end            Growing polypeptide


                                          Next amino acid
                                          to be added to
                                          polypeptide chain

                        E                 tRNA
   mRNA                                          3


       5                   Codons

Schematic model with mRNA and tRNA
LE 17-16a




                           Exit tunnel
             Growing
tRNA         polypeptide
molecules


                                         Large
                                         subunit
                  E P
                      A

                                         Small
                                         subunit


            5
                 mRNA        3
Computer model of functioning ribosome
     LE 17-17

                          Translation Initiation

                                                                                     Large
                                                                                     ribosomal
                                                                                     subunit
                                                        P site




 Initiator tRNA                        GTP   GDP
                                                                  E          A
          mRNA
     5                                            5                                       3
                                 3
                  Start codon
G cap                           Small
                                ribosomal
  mRNA binding site             subunit                    Translation initiation complex
  LE 17-18
                                   Amino end
                                   of polypeptide




                                                    E
                                                                            3
Elongation                            mRNA
                                                             P A
             Ribosome ready for                             site site
             next aminoacyl tRNA        5
                                                                        2    GTP

                                                                                 2 GDP




                     E                                                                   E


                         P   A                                                               P   A




                                    GDP
                                         GTP




                                                        E


                                                              P    A
      LE 17-19

                    Translation Termination




                        Release
                        factor                                             Free
                                                                           polypeptide


                                                                                   5
                                  3                                       3
                                                                                                                 3
5                                       5

                 Stop codon
                 (UAG, UAA, or UGA)
      When a ribosome reaches a stop            The release factor hydrolyzes the        The two ribosomal subunits
      codon on mRNA, the A site of the          bond between the tRNA in the             and the other components
      ribosome accepts a protein called         P site and the last amino acid of the    of the assembly dissociate.
      a release factor instead of tRNA.         polypeptide chain. The polypeptide
                                                is thus freed from the ribosome.
   LE 17-20
                                                                Completed
                                       Growing                  polypeptides
Polyribosomes
                                       polypeptides


                Incoming
                ribosomal
                subunits



                            Start of
                            mRNA                             End of
                            (5 end)                         mRNA
                                                             (3 end)
                 An mRNA molecule is generally translated simultaneously
                 by several ribosomes in clusters called polyribosomes.




                                                      Ribosomes

                                                         mRNA




                                                                      0.1 mm
                 This micrograph shows a large polyribosome in a prokaryotic cell (TEM).
Transcription & Translation can occur at same time in prokaryote cell
       RNA polymerase



                        DNA
                                               mRNA

               Polyribosome



                         Direction of           0.25 mm
 RNA                     transcription
 polymerase
                                                DNA




Polyribosome

Polypeptide
(amino end)

                                  Ribosome


                              mRNA (5 end)
   LE 17-21




        Ribosomes



             mRNA
           Signal
           peptide                       ER
                                         membrane
Signal-
                               Signal
recognition
                               peptide    Protein
particle SRP
                               removed
(SRP)    receptor
      protein
CYTOSOL

ER LUMEN       Translocation
               complex
       LE 17-26
                              TRANSCRIPTION                           DNA


Gene: region
                                                                 3
of DNA where
final product is   5     RNA
                          transcript
                                                                        RNA
                                                                        polymerase

either a                 RNA PROCESSING                Exon

polypeptide or                                                       RNA transcript
                                                                     (pre-mRNA)

RNA molecule                                                           Intron

                                                                                                      Aminoacyl-tRNA
                                                                                                      synthetase

                                                   NUCLEUS

                                          FORMATION OF                                        Amino
                                       INITIATION COMPLEX                                     acid                     AMINO ACID ACTIVATION
                   CYTOPLASM
                                                                                              tRNA




                                                              mRNA          Growing
                                                                            polypeptide
                                                                                                         Activated
                                                                                                         amino acid                            3
                                               A
                                           P
                                       E             Ribosomal
                                                     subunits




                    5
                                                                                                                      TRANSLATION


                                                                            E             A
                                                                                                       Anticodon




                                                                                       Codon

                                                                            Ribosome

                                                                                                                                    mRNA will be
                                                                                                                                    degraded by enzymes
     Molecular basis of sickle-cell disease: point mutation


     Wild-type hemoglobin DNA                Mutant hemoglobin DNA
3                              5     3                             5




     mRNA                                   mRNA


5                              3     5                             3



        Normal hemoglobin 450 bp             Sickle-cell hemoglobin




 DNA: 5’ ATG 3’
mRNA: 3’ UAC 5’       Read 5’ CAU 3’
                                                    Mutagens?
    LE 17-24
               Wild type

Base-pair      mRNA
                     5                                             3
substitution   Protein
                                                             Stop
               Amino end                              Carboxyl end
               Base-pair substitution

                No effect on amino acid sequence
                                                  U instead of C
                                                                         “wobble”
                                                                         3rd nucleotide
                                                             Stop        flexibility
                Missense
                                            A instead of G



                                                                         How similar is
                                                             Stop        the substituted
                Nonsense                                                 amino acid (pg
                           U instead of A
                                                                         79)


                                  Stop
LE 17-25
            Wild type
                                                                Base-pair insertion
           mRNA 5                                         3   or deletion
           Protein
                                                      Stop
            Amino end                            Carboxyl end

            Base-pair insertion or deletion
             Frameshift causing immediate nonsense
                          Extra U




                              Stop

             Frameshift causing
             extensive missense               Missing




             Insertion or deletion of 3 nucleotides:
             no frameshift but extra or missing amino acid
                                Missing




                                                Stop
   Origins of Restriction Enzymes
1) Bacteria produce restriction enzymes to
   protect against invading viral DNA/RNA.
   Origins of Restriction Enzymes
2) The enzymes cut the invading DNA/RNA,
   rendering it harmless.
    Restriction Enzyme in Action




                               Sticky Ends




1) DNA strand with EcoRI restriction site highlighted.
2) EcoRI restriction enzyme added (outline of separation about to occur).
3) Restriction fragments separate, with “sticky ends” at each edge.
                Adding DNA Ligase




                                Sticky Ends




   DNA ligase bonds sticky ends cut with the same restriction enzyme.
   Sticky ends cut with different restriction enzymes will not bond together.
      Why?
      Because the base pair sequence of the two sticky ends will be
       different and not match up.
LE 20-3
                                  Restriction site
                                                              Using restriction
                   DNA 5                              3     enzymes & DNA ligase to
                       3                              5
                                                              make recombinant DNA
          Restriction enzyme cuts
          the sugar-phosphate
          backbones at each arrow.




                                          Sticky end




          DNA fragment from another
          source is added. Base pairing        Fragment from different
          of sticky ends produces              DNA molecule cut by the
          various combinations.                same restriction enzyme




                               One possible combination

          DNA ligase
          seals the strands.



                               Recombinant DNA molecule
Restriction enzymes
From bacteria (used for protection, protect own DNA by
adding methyl (CH3) groups to A & C
Recognize specific nucleotide sequence
Leave “sticky or blunt” ends
LE 20-8
            Gel Electrophoresis

                            Mixture      Longer
                            of DNA       molecules
      Cathode               molecules
                            of differ-
                            ent sizes



                                         Shorter
     Power                               molecules
     source
                            Gel

                            Glass
                            plates


          Anode
    Activity 3
    Plasmid Restriction analysis                Gel Electrophoresis



         BamHI                                                        Marker DNA
                                  Hind III      A     B    C    D
                  2000 bp                                             25000 bp
                                                                       9400 bp
                                      4200 bp                          6500 bp
        2300 bp
                                                                       4300 bp
                            amp

                                                                       2300 bp
EcoRI                                                                  2000 bp

             3000 bp


                            EcoRI
                                  Bam +
Uncut   BamHI   EcoRI   HindIII   EcoRI   Marker 2
48514

                                           23130
        16841

                                           9416
                                            6682
    Timeline DNA – The Code of Life
•   1928 Griffith (transforming factor)
•   1944 Avery DNA is genetic material
•   1952 Hershey & Chase/Rosalind Franklin
•   1953 Watson & Crick
•   1971 restriction enzymes; birth genetic engineering
•   1985 Polymerase Chain Reaction (PCR) copies DNA
•   1988 Gene mapping
•   1990 Beginning Human Genome Project
•   2002 completed HGP
    LE 20-2
                            Bacterium                                  Cell containing gene
                                                                       of interest
                                                 Gene inserted into
                                                 plasmid

Recombinant
DNA                  Bacterial
                     chromosome
                                     Plasmid
                                                                   Gene of
                                      Recombinant                  interest
                                      DNA (plasmid)                                DNA of
                                                                                   chromosome
                                                             Plasmid put into
                                                             bacterial cell

                               Recombinant
                               bacterium
                                                                         Host cell grown in culture
                                                                         to form a clone of cells
                                                                         containing the “cloned”
                                                                         gene of interest


                               Gene of                                 Protein expressed
                               interest                                by gene of interest
                     Copies of gene                                           Protein harvested

                                                Basic research and
                Basic                           various applications                    Basic
                research                                                                research
                on gene                                                                 on protein




              Gene for pest         Gene used to alter      Protein dissolves      Human growth hor-
              resistance inserted   bacteria for cleaning   blood clots in heart   mone treats stunted
              into plants           up toxic waste          attack therapy         growth
LE 20-4_3
                                                     Bacterial cell    lacZ gene          Human
            Isolate plasmid DNA                                        (lactose           cell
            and human DNA.                                             breakdown)

                                                                       Restriction
                                                                       site
                                           ampR gene      Bacterial
                                           (ampicillin    plasmid              Gene of
                                           resistance)                         interest
                                                                         Sticky
            Cut both DNA samples with                                                        Human DNA
                                                                         ends
            the same restriction enzyme.                                                     fragments




            Mix the DNAs; they join by base pairing.
            The products are recombinant plasmids
            and many nonrecombinant plasmids.
                                                                  Recombinant DNA plasmids

            Introduce the DNA into bacterial cells
            that have a mutation in their own lacZ
            gene.

                                                             Recombinant
                                                             bacteria
            Plate the bacteria on agar
            containing ampicillin and X-gal.
            Incubate until colonies grow.



                                                       Colony carrying non-           Colony carrying
                                                       recombinant plasmid            recombinant
                                                       with intact lacZ gene          plasmid with
                                                                                      disrupted lacZ gene


                                                                                            Bacterial
                                                                                            clone
LE 20-19
              Agrobacterium tumefaciens



               Ti
               plasmid                    Producing transgenic plants
            Site where
            restriction
            enzyme cuts

                              T DNA
               DNA with
               the gene
               of interest
           Recombinant
           Ti plasmid




                                 Plant with
                                 new trait
   LE 20-6


             Genomic libraries

                                              Foreign genome
                                              cut up with
                                              restriction
                                              enzyme
                                         or


Bacterial                      Recombinant
clones                         plasmids                        Phage
                                         Recombinant
                                         phage DNA             clones




             Plasmid library                           Phage library
    LE 20-9
                                     Normal b-globin allele

RFLPs
Restriction                     175 bp      201 bp          Large fragment

Fragment Length          Ddel        Ddel            Ddel                    Ddel
Polymorpisms
                                Sickle-cell mutant b-globin allele

Diagnosing
                                     376 bp                 Large fragment
Sickle Cell
                         Ddel                        Ddel                    Ddel
                  Ddel restriction sites in normal and sickle-cell alleles of
                  b-globin gene


                                         Normal      Sickle-cell
                                         allele      allele



                         Large
                         fragment



                                                                   376 bp
                            201 bp
                            175 bp


                  Electrophoresis of restriction fragments from normal
                  and sickle-cell alleles
LE 20-10
                                                                                                                     Heavy
      DNA + restriction enzyme          Restriction                                                                  weight
                                        fragments           I   II      III              Nitrocellulose
                                                                                         paper (blot)

                                                                                         Gel


                                                                                       Sponge

I Normal    II Sickle-cell III Heterozygote                                                                              Paper
b-globin    allele                                                                       Alkaline                        towels
allele                                                                                   solution

  Preparation of restriction fragments.          Gel electrophoresis.                     Blotting.




                          DNA Profiling



                                                           Probe hydrogen-
                                                           bonds to fragments
    Radioactively                                          containing normal                          I   II   III
    labeled probe                                          or mutant b-globin
                             I     II    III
    for b-globin
    gene is added
    to solution in
    a plastic bag                                                    Fragment from
                                                                                                                      Film over
                                                                     sickle-cell
                                                                                                                      paper blot
                                                                     b-globin allele

                                                                     Fragment from
                                                                     normal b-globin
                      Paper blot                                     allele

  Hybridization with radioactive probe.                                                    Autoradiography.
    LE 20-17
                Defendant’s   Blood from defendant’s   Victim’s
                blood (D)             clothes          blood (V)
DNA
fingerprints
from a murder
case
Using Mitochondrial DNA to identify recovered MIA soldiers

 In old bones the only surviving DNA
 is mtDNA. Amplify sample genes

 Blood samples from maternal
 relatives of missing soldiers provide
 data base. MtDNA only inherited
 from mother

 Match bone mtDNA fingerprint with
 data base.
      LE 20-7
                                                                 5   3

                                                                           Target     http://learn.genetics.utah.edu
PCR                                                                        sequence
                                                                                      /content/labs/pcr/index.html
Polymerase Chain                       Genomic DNA               3   5


Reaction                              Denaturation:
                                      Heat briefly
                                                            5               3

                                      to separate DNA
                                      strands


                                                            3               5

                                      Annealing:
                                      Cool to allow
                      Cycle 1         primers to form
                      yields          hydrogen bonds
                                      with ends of               Primers
                         2
                     molecules        target sequence                                                DNA
                                                                                                     Primers
                                      Extension:
                                      DNA polymerase                                                 DNA
                                      adds nucleotides to
                                      the 3 end of each
                                      primer
                                                                  New
                                                                 nucleo-
                                                                                                     polymer
                                                                  tides                              -ase
                                                                                                     A,T,C,G

                      Cycle 2
                      yields
                         4
                     molecules




                        Cycle 3
                        yields 8
                      molecules;
                     2 molecules
                   (in white boxes)
                     match target
                       sequence
        LE 18-7
                                              Viral Lytic & Lysogenic cycles
                  Phage             The phage attaches to a                                          Daughter cell
                  DNA               host cell and injects its DNA.                                   with prophage
                                                                                                                         Many cell divisions
                                                                                                                         produce a large
                                                            Phage DNA                                                    population of
Phage                                                       circularizes                                                 bacteria infected with
                                                                                                                         the prophage.
                                    Bacterial
                                    chromosome                              Occasionally, a prophage
                                                                            exits the bacterial chromosome,
                                                                            initiating a lytic cycle.




                                      Lytic cycle                                  Lysogenic cycle
                                                              Certain factors                           The bacterium reproduces
                                                            determine whether                           normally, copying the prophage
The cell lyses, releasing phages.
                                                    Lytic cycle or Lysogenic cycle                      and transmitting it to daughter cells.
                                                    is induced             is entered      Prophage or Provirus




                   New phage DNA and proteins are                          Phage DNA integrates into the              Human
                   synthesized and assembled into phages.                  bacterial chromosomes, becoming a
                                                                           prophage.                                  example?
Class/Family   Envelop   Examples/Disease
               e                                             Classes of Animal Viruses
I. Double-stranded DNA (dsDNA)

Adenovirus     No        Respiratory diseases, animal        How does a retrovirus get it’s
                         tumors
                                                             RNA to convert to DNA and
Papovavirus    No        Papillomavirus (warts, cervical
                         cancer): polyomavirus (animal
                                                             thereby incorporate into host
                         tumors)                             cell?
Herpesvirus    Yes       Herpes simplex I and II (cold
                         sores, genital sores); varicella
                         zoster (shingles, chicken pox);    Class/Family   Envelop   Examples/Disease
                         Epstein-Barr virus                                e
                         (mononucleosis, Burkitt’s
                         lymphoma)
                                                            IV. Single-stranded RNA (ssRNA); serves as mRNA
Poxvirus       Yes       Smallpox virus, cowpox virus


                                                            Picornavirus   No        Rhinovirus (common cold);
Class/Family   Envelop   Examples/Disease                                            poliovirus, hepatitis A virus,
               e                                                                     and other enteric (intestinal)
                                                                                     viruses
II. Single-stranded DNA (ssDNA)
                                                            Coronavirus    Yes       Severe acute respiratory
                                                                                     syndrome (SARS)
Parvovirus     No        B19 parvovirus (mild rash)
                                                            Flavivirus     Yes       Yellow fever virus, West Nile
                                                                                     virus, hepatitis C virus
III. Double-stranded RNA (dsRNA)

                                                            Togavirus      Yes       Rubella virus, equine
Reovirus       No        Rotavirus (diarrhea), Colorado                              encephalitis viruses
                         tick fever virus
LE 18-9         HIV: retrovirus that causes AIDS
Glycoprotein                                       Viral envelope


                                                           Capsid




                                                   RNA
Reverse                                            (two identical
transcriptase                                      strands)
LE 18-10
                  Membrane of
           HIV    white blood cell                                  HIV Reproduction



                                                          HOST CELL

                                                               Reverse
                                     Viral RNA                 transcription     High Rate of
                                                                                 mutation, no
                                     RNA-DNA
                        0.25 µm      hybrid                                      editing/proofing
           HIV entering a cell
                                     DNA


                                                           NUCLEUS
                                                                   Provirus
                                                 Chromosomal
                                                 DNA

                                     RNA genome
                                     for the
                                     next viral         mRNA
                                     generation




           New HIV leaving a cell
                                                                               Sumanas HIV
      LE 20-16
                   Cloned gene
Gene Therapy                                   Insert RNA version of normal allele
                                               into retrovirus.
using retroviral
vector
                                             Viral RNA


                                               Let retrovirus infect bone marrow cells
                    Retrovirus                 that have been removed from the
                    capsid                     patient and cultured.




                                                     Viral DNA carrying the normal
                                                     allele inserts into chromosome.


                    Bone
                    marrow
                    cell from
                    patient




                                                                       Bone
                       Inject engineered                               marrow
                       cells into patient.
Potential Uses of Gene
Therapy

Cystic fibrosis
Familial
Hypercholesterolemia
Atherosclerosis
Muscular Dystrophy
SCIDs (severe combined
immunodeficiency disease)

Administered by:
Aerosol
Direct injection
Delivery to extracted cells
DNA guns
Making cDNA from mRNA




                        Produces DNA w/o introns
Antisense Technology
                Flavr Savr Tomato's
               Antisense technology
PG gene: produces
enzyme which breaks
down pectin. Fruit gets
soft. Antisense strand
blocks the translation
of PG gene. Pectin
doesn’t break down.
Fruit can stay on vine
longer, get sweeter
rather than picking it
green
Genome Sizes and Estimated Number of Genes
          Human Genome Project




                          Pros & Cons?
          Human Genome Project
 Benefits                       Possible Ethical Issues
• Improved diagnosis            • Who controls the genetic
• Detection of genetic            information?
   predisposition to diseases   • Will researchers be
• Drug design & gene              prevented by rival
   therapies                      commercial interests from
• Greater understanding of        developing new
   human evolution                tests/therapies?
• Developments in forensics     • Will our genetic profile be
                                  used against us by
                                  employers, insurers or
                                  governments?
                                • Is it ethical to patent genes?
      LE 20-11
                   Cytogenetic map                       Chromosome
                                                         bands


                                         Genes located
                                         by FISH


                     Genetic (linkage)
                     mapping



                                            Genetic
                                            markers


                     Physical mapping



                                                Overlapping
                                                fragments




                     DNA sequencing




Dolan learning
center animation
Manual DNA Sequencing: Sanger
          Method
LE 20-12
           DNA                  Primer      Deoxyribonucleotides   Dideoxyribonucleotides
           (template strand)        3                             (fluorescently tagged)    Dideoxy chain-
            5
                                                                                             termination
                                    5
                                                                                             method for
                               DNA
                               polymerase                                                    sequencing DNA


            3

                    DNA (template              Labeled strands
           5       strand)                                                             3




           3

                 Direction
                 of movement
                 of strands




                 Laser                                Detector
LE 20-13
           Cut the DNA from
           many copies of an
           entire chromosome
           into overlapping frag-
           ments short enough
           for sequencing




           Clone the fragments
           in plasmid or phage
           vectors




           Sequence each fragment




           Order the
           sequences into one
           overall sequence
           with computer
           software
Proteomics: What do the sequences mean?
What do the proteins look like? What do they do?
Proteomics and Drug development
                  Drug binding to active
                  sites
                  Drugs to block protein
                  activity
    LE 21-7
                                                                   Reproductive cloning of a
                Mammary                   Egg cell
Animal we       cell donor                donor                    mammal by nuclear
want to clone                                                      transplantation

                                      Egg cell
                                      from ovary         Nucleus
                                                         removed
                Cultured              Cells fused
                mammary cells
                are semistarved,
                                                     Use electricity
                arresting the cell
                cycle and causing
                dedifferentiation
                                              Nucleus from
                                              mammary cell
                   Grown in culture

                                             Early embryo

                   Implanted in uterus
                   of a third sheep


                                                Surrogate
                                                mother

                   Embryonic
                   development
                                             Lamb (“Dolly”) genetically identical
                                             to mammary cell donor
“Pharming” making transgenic
organisms w/ ability to produce
drugs in milk or fruit
        LE 21-9
                                    Embryonic stem cells               Adult stem cells
Stem cells         From early                                                             From bone marrow
                   embryo                                                                 or blood




                                            Totipotent                    Pluripotent
                                              cells                          cells

                      Cultured
                      stem cells




                      Different
                      culture
To initiate           conditions

transcription/translation of
specific proteins
                      Different      Liver cells         Nerve cells        Blood cells
                      types of
                      differentiated
                      cells
DNA Hybridization
       LE 20-14

DNA                 Isolate mRNA.
                                                                     Tissue sample
microarray
assay


                                                         mRNA molecules
                    Make cDNA by reverse
                    transcription, using
                    fluorescently labeled
                    nucleotides.


                    Apply the cDNA mixture to a
                                                          Labeled cDNA molecules
                    microarray, a microscope slide
                                                          (single strands)
                    on which copies of single-
                    stranded DNA fragments from
                    the organism’s genes are fixed,
                    a different gene in each spot.
                    The cDNA hybridizes with any
                    complementary DNA on the
                                                      DNA
                    microarray.
                                                      microarray

                    Rinse off excess cDNA; scan
                    microarray for fluorescent.
                    Each fluorescent spot
                    (yellow) represents a gene
                    expressed in the tissue                        Size of an actual
                    sample.                                        DNA microarray
                                                                   with all the genes
http//learn.genetics.utah.edu/units/biotech                        of yeast (6,400 spots)
      LE 18-15
Can a bacterial cell acquire genes   Mixture
from another bacterial cell?



    Mutant                                                              Mutant
    strain                                                              strain
   arg+ trp–                                                           arg– trp+




                                      Mixture

    Mutant                                                                 Mutant
    strain                                                                 strain
   arg+ trp–                                                              arg– trp+
                    No                                        No
                 colonies            Colonies              colonies
                 (control)            grew                 (control)

   Video clip: transformation, transduction, conjugation, transposition
      LE 18-14

                                                     Replication fork
 Asexual
 reproduction
 called:                                              Replication of
                      Origin of
                      replication                     bacterial chromosome
                                                      Mesosome
                                                      4.6 million base pairs
                                                      – 4300 genes




E. Coli replace the 2 x 1010
                                    Termination
cells lost each day in feces.       of replication
Spontaneous mutation rate of
1 x 10-7/cell
(2 x 1010)(1 x 10-7) = 2000
E. coli mutants/day
     LE 18-19b
      Transposable genetic elements in bacteria. DNA moving from one
      location in genome to another. Provides greater possibility of
      genetic variation


                                     Transposon

                   Insertion       Antibiotic            Insertion
                   sequence        resistance gene       sequence


5                                                                      3
3                                                                      5


                 Inverted repeat                     Transposase gene
Barbara McClintock Jumping genes (transposons) in Indian corn
       LE 18-16
                                                  Generalized transduction
                                 Phage DNA

                                 A+ B+            Phage infects a bacterial cell that has
                                                  alleles A+ & B+


                              A+ B+               Host DNA (brown) is fragmented and phage
                                                  DNA & proteins are made
Mice: 1% total DNA
                                      Donor
is of viral origin,                   cell
10% of mRNA may
be retroviral                                     A bacterial fragment w/ A+ allele may be
                                                  packaged in phage capsid
Virus’s & Cancer                         A+

Hepatitis B > liver
                      Crossing
cancer                over                        Phage w/ A+ allele from donor cell infects a
Papilloma >                      A+
                                                  recipient A-B- cell & crossing over between
cervical cancer                  A– B–
                                                  donor DNA (brown) & recipient DNA (green)
HIV > Kaposi                          Recipient   occurs at 2 places (dotted lines)
                                      cell
sarcoma
                                                  Genotype of resulting recombinant cell
                                                  (A+B-) differs from geneotypes of both the
                                 A+ B–            donor (A+B+) & recipient (A-B-)
                        Recombinant cell
LE 18-17




           Sex pilus   5 µm
 LE 18-18_4                                    Conjugation: Fertility Plasmid
       F plasmid        Bacterial chromosome

    F+ cell                                                        F+ cell
Mating
bridge
    F– cell                                                        F+ cell
                         Bacterial
                         chromosome
       Conjunction and transfer of an F plasmid from and F+ donor to an F– recipient



              F+ cell                                                    Hfr cell

                                                      F factor


  Hfr cell


   F– cell

                    Temporary
                    partial                                      Recombinant F–
                    diploid                                      bacterium
      Conjugation and transfer of part of the bacterial chromosome from an
      Hfr donor to an F– recipient, resulting in recombiination
Incorporation of drug resistant plasmids (R)




                                               Transformation: free
                                               plasmid incorporated
                                               usually by heat shock &
                                               salt or electroporation

                                               Dolan DNA Learning
                                               center: Transformation 2


                                                Amgen pARA/pKAN-R ppt
 Transformation Lab: Transforming E. coli w/ pARA-R plasmid




               _                   _
 +                     +
                                             Pick up E. coli w/
pARA         H2O      pARA        H2O        inoculating loop
                                             or sterile
                                             toothpick,

Control protocol:     Varied protocol:       thoroughly
250ul CaCl + E.       250ul CaCl + E.        mix w/ pipette
coli                  coli
*Ice tubes for 15 minutes
*Heat shock 45 sec in 42C hot
water bath
*ice 2 minutes
*Add 150 ul Lurea broth
*Incubate tubes for 15 minutes
in incubator at 37C
*Add 100 ul w/ micropipette,
*Spread w/ sterile spreader




     LB        LB/amp+        LB/amp/ara+
     +



     LB            LB/amp -
     -

   Control protocol plates
   Need 5 more for changed protocol
   LE 18-20
                 Regulation of enzyme                 Regulation of enzyme
Negative                                              production
                 activity
Feedback                          Precursor
              Feedback
              inhibition
                                        Enzyme 1   Gene 1




                                        Enzyme 2   Gene 2
                                                                      Regulation
                                                                      of gene
                                                                      expression
                                        Enzyme 3   Gene 3




                                        Enzyme 4   Gene 4




                                        Enzyme 5   Gene 5




                           Tryptophan
      LE 18-22a
                   The lac operon: regulated synthesis of inducible enzymes
Operon:
DNA               Regulatory                               Promoter
                  gene
required for
protein                                                               Operator
                                                                                 switch
production
   DNA                     lacl                                                           lacZ

                                                                                      No
                                                                       off            RNA
                                                                                      made


                                     3

   mRNA                                       RNA
                   5                         polymerase


                                                                        Prokaryotes only
  Continually
                                                                        Inducible operons
  transcribed &
                                              Active
                                                                        usually function in
  translated
   Protein                                    repressor                 catabolic
                                                                        pathways;
      Lactose absent, repressor active, operon off
                                                                        breakdown of
                                                                        nutrients
   LE 18-22b

               Lac Operon



                                                 lac operon


DNA            lacl                              lacZ           lacY          lacA

                           RNA
                      3   polymerase
mRNA                             mRNA 5
          5



Protein                                    b-Galactosidase    Permease   Transacetylase

                              Inactive      Enzymes needed to break down lactose
  Allolactose
                              repressor
  (inducer)

   Lactose present, repressor inactive, operon on
   LE 18-21a

   Trp operon: regulated synthesis of repressible enzymes (usually
   function in anabolic pathways)


                                                       trp operon

   Promoter                        Promoter
                                                       Genes of operon
DNA            trpR                             trpE      trpD      trpC   trpB   trpA
                                      Operator/switch
Regulatory                 RNA         Start codon Stop codon
gene                  3   polymerase
                                    mRNA 5
mRNA
          5
                                                 E         D         C      B      A

Protein                Inactive                     Polypeptides that make up
                       repressor                 enzymes for tryptophan synthesis

  Tryptophan absent, repressor inactive, operon on


Regulatory gene can be located anywhere
Inactive repressor continually transcribed/translated
LE 18-21b_2




 DNA

                                                          No RNA made



                                                        Switch is off,
                                                        can’
 mRNA




 Protein                                           Active
                                                   repressor



                Tryptophan
                (corepressor)

Tryptophan present, repressor active, operon off
LE 19-14
                             Exons (regions of genes coding
                            for protein, rRNA, or tRNA) (1.5%)




                          Repetitive
                          DNA that                     Introns and
                          includes                     regulatory
                          transposable                 sequences
                          elements                     (24%)
                          and related
                          sequences                 Gene families
                          (44%)
                DNA                                 Ribosomes/histones
                plasticity/
                Ab                                      Unique
                                                        noncoding
                production           Repetitive         DNA (15%)
                                     DNA
                                     unrelated to
                                     transposable
                                     elements
                                     (about 15%)
           Alu elements                                              Pages 376-381
           (10%)
                                                                     gene changes
                                                                     lead to evolution
                   Simple sequence       Large-segment               (details not
                   DNA (3%)              duplications (5–6%)
     Structural:                                                     important)
     centromere/telomere
       LE 19-3
                      Signal

                                                                      Stages in gene expression that
                                                     NUCLEUS
                                             Chromatin
                                                                      can be regulated in eukayotic
Potential                                                             cells
places for
controlling         DNA
                                                  Gene available
                                                                      20% genes expressed in any
                                                  for transcription
gene                           Gene                                   given moment
expression                               Transcription
                      RNA               Exon
                                               Primary transcript
                                  Intro
Most                                    RNA processing

commonly                                 Tail
                                          mRNA in nucleus
                  Cap
done at?                              Transport to cytoplasm


                                                         CYTOPLASM
                                          mRNA in cytoplasm

                 Degradation
                  of mRNA

                                           Translation


                                           Polypeptide
                                        Cleavage
                                  Chemical modification
                                   Transport to cellular
                                       destination


                                       Active protein


                                  Degradation of protein

                                       Degraded protein
  LE 19-2d




                               700 nm




                               1,400 nm



 Metaphase chromosome




Heterochromatin: DNA highly coiled around + charged histones, not
transcribed
Euchromatin: DNA loosely coiled, DNA more accessible for transcription
Gene location
           DNA methylation (CH3)
• Prokaryotes: add methyl groups to DNA to protect it from
  restriction enzymes
• Eukaryotes: methylate DNA, 1* cytosine, inactivates DNA
   – Barr Body
   – Twin studies: identical methylation patterns at birth &
     diverge w/ different environments
   – Smoking: hypermethylation around tumor suppressor
     genes. Potential warning signal (genetic test for lung
     cancer)
   – Human Epigenome Project: map where methyl markers
     can attach change the expression of a given gene
LE 19-4
                                  Histone acetylation (COCH2)




                                                    Histone
                                                    tails




            DNA
            double helix                            Amino acids
                                                    available
                                                    for chemical
                                                    modification
           Histone tails protrude outward from a nucleosome




          Unacetylated histones        Acetylated histones

           Acetylation of histone tails promotes loose chromatin
           structure that permits transcription
LE 19-6
          Distal control      Enhancers and transcription activators
          element                Activators                             Promoter
                                                                                         Gene
                DNA

                           Enhancer                                TATA
                                                                   box


                                                                                       General
                                                                                       transcription
                                                                                       factors
                                                   DNA-bending
                                                   protein

                                                                                    Group of
                                                                                    mediator proteins




                                                                                   RNA
                                                                                   polymerase II




                                                                                      RNA
                                                                                      polymerase II



                                                   Transcription
                                                                                   RNA synthesis
                                                   Initiation complex
    LE 19-7

Cell specific transcription                             Liver cell               Lens cell
Each gene has its own                                   nucleus                  nucleus

promoter & control elements
(no operons)

                                                Available                Available
                                                activators               activators

              Enhancer   Promoter



   Control                   Albumin
   elements                  gene

                                                                             Albumin
                              Crystallin                                     gene not
                                                   Albumin
                              gene                                           expressed
                                                   gene
                                                   expressed




                                                 Crystallin gene
                                                 not expressed            Crystallin gene
                                                                          expressed

                                           Liver cell                Lens cell
LE 19-8
               Alternative RNA splicing

               Post transcriptional regulation



                             Exons




 DNA




 Primary
 RNA
 transcript
                                           Producing more than 1
              RNA splicing       or        type of polypeptide from
                                           same gene

 mRNA
       LE 19-9
Regulation of gene expression by microRNAs (miRNAs) & small interfering RNAs
(siRNAs)                    RNA interference




                             Protein
                             complex                          Degradation of mRNA


              Dicer
                                                                    OR

                                       miRNA
                                               Target mRNA



   Hydrogen
   bond
                                                              Blockage of translation

                      Degradation of mRNA w/in minutes in
                      prokaryotes, hours/days in eukaryotes
   LE 19-10

  Degradation of a protein by a proteasome




                                                                      Proteasome
              Ubiquitin                                               and ubiquitin
                                                                      to be recycled
                                    Proteasome




Protein to                Ubiquitinated                                            Protein
be degraded               protein                                                  fragments
                                                 Protein entering a                (peptides)
                                                 proteasome
    LE 19-11              Cancer: diseases of uncontrolled cell division

Genetic changes that can turn proto-oncogenes into oncogenes
                                               proteins for normal cell growth              too many growth
                                                                                            proteins or change in
                                                                                             the protein
                                                    Proto-oncogene

                                            DNA



                                                                         Point mutation
Translocation or transposition:                                                                    Point mutation
                                                                         within a control
gene moved to new locus,             Gene amplification:                                           within the gene
                                                                         element
under new controls                   multiple copies of the gene


New
promoter                                                                    Oncogene                Oncogene



Normal growth-stimulating         Normal growth-stimulating
protein in excess                 protein in excess
                                                                     Normal growth-stimulating     Hyperactive or
                                                                     protein in excess             degradation-
                                                                                                   resistant protein
  LE 19-12_3
                                               Growth                                  MUTATION
                                               factor                                      Hyperactive
                                                                                           Ras protein
                                                                   G protein               (product of
                                                                                           oncogene)
               Cell cycle-stimulating                                                      issues signals
               pathway                                                                     on its own



Proto-oncogene >                            Receptor        Protein kinases                                                   Ras gene
                                                            (phosphorylation
oncogene                                                    cascade)
                                                                                                        NUCLEUS               mutated in
                                                                                Transcription
                                                                                factor (activator)                            30%
                                                                                  DNA

                                                                                 Gene expression
                                                                                                                              cancers
                                                                                       Protein that
                                                                                       stimulates
                                                                                       the cell cycle



                   Cell cycle-inhibiting
                   pathway                                      Protein kinases
                                                                                                         MUTATION

Tumor suppressor                                                                                            Defective or
                                                                                                            missing
                                                                                                            transcription
genes mutated – 50%                          UV
                                                                            Active
                                                                                                            factor, such as
                                                                                                            p53, cannot
                                                                                                            activate
                                                                            form
of cancers                                   light
                                                        DNA damage
                                                                     of        p53
                                                                                                            transcription


                                                        in genome  DNA



                                                                      Protein that
                                                                      inhibits
                                                                      the cell cycle


                   Effects of                                   EFFECTS OF MUTATIONS
                   mutations
                                           Protein overexpressed                                Protein absent



                                           Cell cycle overstimulate      Increased cell         Cell cycle not
                                                                         division               inhibited
    p53 Tumor Suppressor Gene
• Implicated in 52 human cancers
• p53 protein acts as a transcription factor that
  promotes synthesis of growth inhibiting
  proteins as well as DNA repair enzymes
• Activates suicide genes: cell death - apoptosis
   LE 19-13

Multistep model for the development of colorectal cancer

Usually several mutations needed
Cancer is a disease of old age
15% cancers due to viral infections/mutations (HIV – Kaposi sarcoma
                                               HPV – cervical cancer)
                 Colon




                         Loss of                         Activation of                         Loss of
                      tumor-                          ras oncogene                          tumor-
                      suppressor                                                            suppressor
           Colon wall gene APC (or                                                          gene p53
                      other)

                                                         Loss of                              Additional
                         15%                          tumor-                                mutations
                         inherit                      suppressor
  Normal colon                       Small benign     gene DCC           Larger benign                     Malignant tumor
  epithelial cells       defective   growth (polyp)                      growth (adenoma)                  (carcinoma)
                         gene
             BRCA 1 & BRCA 2
•   Tumor suppressor genes
•   5-10% breast cancers
•   Test positive 65% get cancer
•   Grama or aunt: 14% chance
•   Mom or sister: 10-20% chance
•   Mom & sister: 11-48% chance

								
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