The History Of DNA

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The History Of DNA
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Meischer and DNA

• The history of deoxyribonucleic acid (DNA)
  research begins with Friedrich Miescher, a Swiss
  biologist who in 1868 carried out the first
  carefully thought out chemical studies on the
  nuclei of cells. Using the nuclei of pus cells
  obtained from discarded surgical bandages,
  Miescher detected a phosphorus-containing
  substance that he named nuclein. He showed that
  nuclein consists of an acidic portion, which we
  know today as DNA, and a basic protein portion
  now recognized as histones, a class of proteins
  responsible for the packaging of DNA. Later he
  found a similar substance in the heads of salmon
  sperm cells. Although he separated the nucleic
  acid fraction and studied its properties, the
  covalent structure of DNA did not become known
  with certainty until the late 194Os.

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Griffiths Experiment

• Rough Pneumococcus are harmless. They lack a gel
  capsule that would protect them from a host
  organism's immune system attack.
• Smooth Pneumococcus are pathogenic (they cause
  disease), and when injected, give a mouse fatal
  pneumonia.
• Griffiths injected several combinations of rough
  and smooth into hapless mice, and found...

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Experimental Results

• live rough --gt mice okay
• live smooth --gt mice pushing up daisies
• killed (boiled) rough --gt mice okay
• killed (boiled) smooth --gt mice okay
• live smooth killed rough --gt mice kick the
  bucket
• live rough killed smooth --gt MICE CROAK! This
  was a surprise!

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Experimental Results
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The Puzzle

• When Griffiths autopsied the mice, he found LIVE
  SMOOTH PNEUMOCOCCUS!! He replicated this many
  times, in case there had been an accidental
  injection of some live smooth bacteria into the
  mice, but there was no mistake. Somehow, the
  harmless live, rough bacteria had been
  TRANSFORMED into deadly smooth bacteria. But
  how?!

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Conclusions

• Whatever the culprit, it had to have several
  properties in order to fit the bill
• It had to be duplicated whenever a cell divided,
  so it could be passed on unchanged.
• It had to be in the form of an informational code
  
• It had to be (mostly) stable and resistant to
  change

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Averys Experiment

• Oswald Avery (1944)
• demonstrated that the substance responsible for
  the transformation of harmless bacteria into
  disease-causing monsters was DNA
• He exposed the extract of boiled bacteria
  Griffiths had used to various substances that
  would destroy one of the compounds (gel capsule,
  proteins or nucleic acids), one at a time.
• He found that DNase (an enzyme which breaks down
  DNA) would stop the transformation process.
  Boiled DNase (destroyed by heat) did not stop
  transformation

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Luria and Delbruck at Cold Spring Harbor Worked
on the life cycle of lysogenic and lytic
bacteriophages
Bacteriophages- viruses that attack bacteria
And paved the way for
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Bacteriophages and Genetics
Scientists began to wonder what the differences
were in the life cycle of the lytic and lysogenic
phages
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The Blender ExperimentMartha Chase and Albert
Hershey
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Side by side experiments are performed with
separate bacteriophage (virus) cultures in which
either the protein capsule is labeled with
radioactive sulfur or the DNA core is labeled
with radioactive phosphorus. The radioactively
labeled phages are allowed to infect bacteria.
Agitation in a blender dislodges phage particles
from bacterial cells. Centrifugation
concentrates cells, separating them from the
phage particles left in the supernatant.
Results Radioactive sulfur is found
predominantly in the supernatant. Radioactive
phosphorus is found predominantly in the cell
fraction, from which a new generation of
infective phage can be isolated. Conclusion The
active component of the bacteriophage that
transmits the infective characteristic is the
DNA. There is a clear correlation between DNA and
genetic information.
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Joshua Lederberg and Edward L. Tatum publish on
conjugation in bacteria. The proof is based on
the generation of daughter cells able to grow in
media that cannot support growth of either of the
parent cells. Their experiments showed that this
type of gene exchange requires direct contact
between bacteria. At the time Lederberg began
studying with Tatum, scientists believed that
bacteria reproduced asexually, but from the work
of Beadle and Tatum, Lederberg knew that fungi
reproduced sexually and he suspected that
bacteria did as well.
Conjugation in Bacteria- Bacterial Sex
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Rosalind Franklin

• The elegant and comprehensive X-ray diffraction
  studies of Rosalind Franklin and Maurice Wilkins
  at King's College, (London, England) yielded a
  characteristic diffraction pattern from which it
  was deduced that DNA fibers have two
  periodicities along their long ans a major one
  of 0.34 nm and a secondary one of 3.4 nm.

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X-Ray Crystallography
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Rosalinds accomplishments

• The technique with which Rosalind Franklin set
  out to do this is called X-ray crystallography.
  With this technique, the locations of atoms in
  any crystal can be precisely mapped by looking at
  the image of the crystal under an X-ray beam. By
  the early 1950s, scientists were just learning
  how to use this technique to study biological
  molecules. Rosalind Franklin applied her
  chemist's expertise to the unwieldy DNA molecule.
  After complicated analysis, she discovered (and
  was the first to state) that the sugar-phosphate
  backbone of DNA lies on the outside of the
  molecule. She also elucidated the basic helical
  structure of the molecule.

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Erwin Chargaff
AT CG
The second species-invariant observation was that
Chargaff's first parity rule also applies, to a
close approximation, to single-stranded DNA (his
"second parity rule"). If the individual strands
of a DNA duplex are isolated and their base
compositions determined, then A T, and C
G (Rudner et al., 1968). Thus it was noted that
there is an
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Chargaffs conclusions

• 1) A G T C
• (2) A T
• (3) G C and as a logical consequence of these
  three equations
• (4) A C G T, i.e., the sum of the 6-amino
  compounds equals that of the 6-oxo derivatives.

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Watson and Crick
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Maurice Wilkins
He also studied the orientation of purines and
pyrimidines in tobacco mosaic virus and in
nucleic acids, by measuring the ultraviolet
dichroism of oriented specimens, and he studied,
with the visible-light polarizing microscope, the
arrangement of virus particles in crystals of TMV
and measured dry mass in cells with interference
microscopes. He then began X-ray diffraction
studies of DNA and sperm heads.
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The Design of the Hershey and Chase Experiment
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Results
Heavy DNA made with N15( two heavy strands) Light
DNA made with N14 ( two light strands ) H/L- One
strand heavy and one strand light( Intermediate
band)
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Conclusions Led to the Concept of
Semi-Conservative Replication
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Protein Synthesis
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Nirenberg and Khorana

• Ribo-oligonucleotides - Khorana developed methods
  to synthesize short RNA molecules of specific
  sequence UUUUUUUUUUU,AAAAAAAAAAAA
• b. Cell-free extracts - simply the stuff inside
  cells after they are broken open these extracts
  contain the protein synthesis machinery
• c. 14C-labeled amino acidsThe idea was to give
  the cell-free extracts RNAs of known sequence to
  translate. The amino acids that were incorporated
  into protein in response to a specific RNA could
  be identified by providing radiolabeled amino
  acids in each experiment then purifying and
  sequencing the radiolabeled peptides.

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UUGUUGUUG..., the cells synthesized poly-leucine,
poly-cysteine and poly-valineUGUGUGUGU... gave
one peptide with alternating valines and
cysteinesGGUGGUGGU... gave poly-glycine,
poly-valine, and poly-tryptophan.
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Cell Overview for Transcription and Translation
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Transcription of a Gene begins at the Promoter
with RNA Polymerase forming a transriptional
complex
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Transcription The production of the messenger
RNA
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Note
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Initiation
The small subunit of the ribosome binds to a site
"upstream" (on the 5' side) of the start of the
message. It proceeds downstream (5' -gt 3') until
it encounters the start codon AUG. Here it is
joined by the large subunit and a special
initiator tRNA. The initiator tRNA binds to the
P site (shown in pink) on the ribosome. In
eukaryotes, initiator tRNA carries methionine
(Met). (Bacteria use a modified methionine
designated fMet.)
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Elongation
An aminoacyl-tRNA (a tRNA covalently bound to
its amino acid) able to base pair with the next
codon on the mRNA arrives at the A site (green)
associated with an elongation factor (called
EF-Tu in bacteria) GTP (the source of the needed
energy) The preceding amino acid (Met at the
start of translation) is covalently linked to the
incoming amino acid with a peptide bond (shown in
red). The initiator tRNA is released from the P
site. The ribosome moves one codon downstream.
This shifts the more recently-arrived tRNA, with
its attached peptide, to the P site and opens the
A site for the arrival of a new aminoacyl-tRNA.
This last step is promoted by another protein
elongation factor (named EF-G) and the energy of
another molecule of GTP.
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Termination

• The end of the message is marked by one or more
  STOP codons (UAA, UAG, UGA).
• There are no tRNA molecules with anticodons for
  STOP codons.

• However, a protein release factor recognizes
  these codons when they arrive at the A site.
• Binding of this protein releases the polypeptide
  from the ribosome.
• The ribosome splits into its subunits, which can
  later be reassembled for another round of protein
  synthesis.
• Polysomes

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Protein Product Tertiary Structure
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Gene regulation - Prokaryotes