Molecular Genetics by HC120917224727

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									   Molecular Genetics

What are chromosomes made of?
I.   Introduction

• A. Current definition of a gene
•    1. a unit factor controlling the inheritance of
     some external trait
•    2. a spot on a chromosome which influences
     some trait
•    3. a poppit bead on a string of poppet beads
     in the laboratory
•    4. going to flesh out in more detail what a
     gene is made of and what it does in our next
     unit
• B. Two questions are going to be answered in
     this part of the course
•    1.    What is a gene made of?
•    2.    What do genes do? How do genes
     control the activity of a cell?
II.  Two traits are necessary to be a good
candidate for the stuff genes are made of
• A.   a genetic molecule must be able to replicate with very few
       mistakes


• B.   a good genetic candidate must be able to direct the activities of
       the cell’s cytoplasm from the confines of the nucleus
III. Protein vs. DNA

• A. Basic structure of the
  two
•     1.   protein
•     2.   DNA
• B. Which is likely
  candidate?
B. Series of historical experiments that determined the
chemical nature of chromosomes

1.   Friedrich Miescher (1869)
      – a. 1869-not too long ago
      – b. exposed cells to pepsin-a
        proteolytic enzyme
      – c. the proteins in the cell’s
        cytoplasm were liquefied but
        the contents of the nucleus
        were left relatively intact
      – d. it was believed at this time
        that the genes or controlling
        factors of inheritance were
        housed in the cell’s nucleus
2.        Feulgen
     • a. developed a stain specific
       for nucleic acids
     • b. what region of the cell took
       up the stain?
     • c. all the somatic cells of an
       individual stained with the
       same intensity-what does that
       tell us about developmental
       processes?
     • d. the germ cells of an
       organism stain with half the
       intensity as the somatic cells
3. Griffiths Work

a. Work involved
   two different
   strains of
   pneumococcus
b. Smooth-virulent
   strain
c. Rough-benign
   strain
d. protocol
e. results

• Dead S bacteria
  transformed the living R
  bacteria into a virulent
  strain
• Later determined that the
  fraction of the dead S
  bacteria that was the
  transforming agent was
  DNA
4. Herschey and Chase
a. structure of a bacteriophage
b. how do viruses work?
c. phage infection
d. protocol
IV. Molecular Structure of DNA


• A. Monomers of nucleic acids
  are nucleotides which consist of
  three basic parts
• 1. pentose sugar
• 2. phosphate and nitrogen
  base
• 3. notice that there are two
  classes of nitrogen containing
  bases
•     a.     purines
•     b.     pyrimidines
Four examples of DNA nucleotides
B. Polymers

• 1. Polymers of nucleotides
  are produced by dehydration
  synthesis of nucleotides in a 5’
  to 3’ direction
• 2. this represents a single
  strand of DNA
• 3. If another nucleotide were
  to join this existing strand-to
  where would it attach? Which
  end?
C. Watson and Crick built their model of DNA on
the work of others

Early sixties
Done in England
Physically built model
    seen to fit data of
    others
Pieced together
    information
Which one is which?
1. Chargaff’s rule

•   a.    a number of species were
    investigated
•   b.    the percentage of different
    bases found in the DNA of each
    species was investigated
•   c.    found that the amount of
    adenine was about the same as
    the thymine
•   d.    the amount of guanine was
    about the same as the amount of
    cytosine
•   e.    at the time, did not know
    what to make of the discovery
•   f.    Chargaff data
•   h.    analysis
2. Rosalind Franklin and X-ray diffraction study
2. Rosalind Franklin

• a. Watson came across knowledge of the work
  at a tea party
• b. had not been exposed to the information
  before
• c. like a light bulb going off in his head
• d. recurring periodicities seen
•     -.34 nm
•     -3.4 nm
•     -2.0nm
3. Work of Linus Pauling


• a. worked on helical
  nature of proteins
• b. saw parallel in nucleic
  acid structure
• c. denied travel visa to
  attend a DNA conference
  that was being held in
  England
• d. linked to the communist
  movement in USA
  because he had opposed
  nuclear bomb used against
  Japan
Diary of Pauling

• Diary shows
  complementary base
  pairing before proposed
  by Watson and Crick
I believe that Linus Pauling won
the Nobel Peace prize later in life
V. DNA double helix

• A. Complementary base
  pairing as suggested by
  work of Chargaff and
  Franklin
B. Double helix
C. Properties



•   1.   antiparallel
•   2.   complimentary base pairing
•   3.   periodicities seen
•   4.   covalent vs. hydrogen bonding
    – Importance of accessibility of information
    – Where is information stored in the molecule
• 5. The information in the DNA molecule is
  carried in the base pair sequence of the
  molecule
• 6. Notice also that because of complimentary
  base pairing, the two sides of the molecule are
  mirror images of each other
• 7. each strand contains the information needed
  to produce the second strand
• 8. Watson and Crick proposed an early model
  of replication in their first paper describing the
  structure of DNA
VI. Replication
• A. Size of the problem
• 1.   approximately 40,000 human genes in a nucleus
• 2.   the length of the DNA in one human cell is
       approximately 3 meters
• 3.   (length of 1 bp)(number of bp per cell)(number of cells in the
       body) = (0.34 x 10-9 m)(6 x 109)(1013) = 2 x 1013 meters
• 4.   That is the equivalent distance from the earth to the sun and
       back.
B. Semi-conservative model as proposed
C. There were three possible modes of replication

1. conservative mode-the parent molecule would
   remain intact while the newly synthesized
   molecule is totally brand new
2. semi-conservative model-each new molecule of
   DNA is half new and the other half comes as a
   template from the parent
3. dispersive model-the two strands of the old
   molecule would break up and be scattered in
   the composition of the two new molecules
D. Work of Meselsohn and Stahl
   1. Verified the semiconservative nature of DNA
      replication
   2. Used heavy nitrogen technology introduced by
      Kornberg along with in vitro DNA replication
3. Protocol
E. Replication-the real story

• 1. Mentioned above the sheer size of the DNA
  molecules in the nucleus
•    a. the amount of information held in the
     nucleus is enough to fill 600 volumes
     of 500 pages each
•    b. the enzymes copying DNA add 100
     nucleotides together per second in mammals
•    c. 1000 nucleotides together per second in
     bacterial cells
•    d. huge amount of information to be
     processed
2. Very accurate process-not a duplication
event


• a. one in every billion base pairs there is a
  mistake
• b. in the typing of the 600 volumes above,
  that would translate to about 4 mistakes
3. Replication origins
• replilcation can start at the beginning of
  the molecule as well as internally at
  special recognition sites
4.     Helicases unwind double helix
•     a.       double helix must be unwound in order for the
  templates to be accessed by free nucleotides
•     b.       single stranded binding proteins also keep the
  separated strands separated
 5. DNA polynucleotide strands cannot start from
 nothing
• a. the first nucleotide
  must connect to
  something else
• b. RNA primer piece
  must be laid down first
  c. notice that with
  many replication forks,
  this piece of primer
  must be laid down
  every time a new
  section of DNA is manu.
6. Nucleotides add to a growing chain in the
5’ to 3’ direction

a. new bases cannot add
   onto the 5’ end of the
   polynucleotide chain
b. DNA is an antiparallel
   molecule
c. at the replication fork
   this causes confusion
d. DNA polymerase can
   only operate in one
   direction
6. cont.

e. terms to know:
   leading strand, lagging
   strand, Okazaki
   fragments, ligase
f. the Okazaki fragments
   are 1000-2000
   fragments long in
   bacteria
g. the Okazaki fragments
   are 100-300 fragments
   long in mammals
h. the Okazaki fragments
   are joined by an
   enzyme called ligase
7. Summary Slide
8. The final problem



  •   a. Chromosomes
      shorten with repeated
      divisions
  •   b. The position where
      the primer RNA was
      found on the leading
      strand leaves a gap at
      the end with each
      replication
  •   c. The daughter
      molecules get shorter
      and shorter
  •   d. Important
      information can be
      eroded
• e. telomeres are basically like buffers
• f.   able to be eroded away without getting into the coding sequence of
  the chromosomes
• g. repeating nucleotide base sequence TTAGGG
• h. there may be over 100 to 1000 of these repeats
8. The final problem cont.

• i. telomerase not present in all cells
• j. lack of enzyme and shortening of
  chromosomes may be a built in clock for
  life of cell
• k. telomerase is present in germ lines
• l. also present in tumors which exhibit
  uncontrolled mitotic divisions
VII. What is a chromosome anyways?

• A. Recall that each nucleus possesses
  about 3 meters of DNA
• B. It appears as chromatin in a
  nondividing cell
• C. In a mitotic cell the chromatin must be
  organized to accomplish an efficient
  division process
D. Terms to remember

• 1. Histone proteins
• 2. Nucleosomes
• 3. Scaffolding proteins

								
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