Transcript Molecular Genetics

Molecular Genetics
What are chromosomes made of?
I.
Introduction
• A. Current definition of a gene
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1. a unit factor controlling the inheritance of
some external trait
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2. a spot on a chromosome which influences
some trait
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3. a poppit bead on a string of poppet beads
in the laboratory
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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
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1.
What is a gene made of?
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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
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1.
protein
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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
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a.
purines
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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
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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
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-3.4 nm
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-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
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1.
2.
3.
4.
antiparallel
complimentary base pairing
periodicities seen
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.
• 2.
• 3.
• 4.
approximately 40,000 human genes in a nucleus
the length of the DNA in one human cell is
approximately 3 meters
(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
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
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a. the amount of information held in the
nucleus is enough to fill 600 volumes
of 500 pages each
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b. the enzymes copying DNA add 100
nucleotides together per second in mammals
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c. 1000 nucleotides together per second in
bacterial cells
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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.
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Helicases unwind double helix
a.
double helix must be unwound in order for the
templates to be accessed by free nucleotides
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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
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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