Transcript Advanced Biology and Genetics DNA Outline

The History Of DNA
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.
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...
Experimental Results
– live rough --> mice okay
– live smooth --> mice pushing up daisies
– killed (boiled) rough --> mice okay
– killed (boiled) smooth --> mice okay
– live smooth + killed rough --> mice kick the
bucket
– live rough + killed smooth --> MICE CROAK!
This was a surprise!
Experimental Results
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?!
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
Avery’s 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
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
Bacteriophages and Genetics
Scientists began to wonder
what the differences were
in the life cycle of the lytic
and lysogenic phages
The Blender Experiment
Martha Chase and Albert Hershey
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.
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
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.
X-Ray Crystallography
Rosalind’s 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.
A=T
C=G
The second species-invariant
observation was that
Chargaff's first parity rule also
applies, to a close
approximation, to singlestranded 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:
Chargaff’s 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 6amino compounds equals that of the 6-oxo
derivatives.
Maurice Wilkins
The Design of the Hershey and Chase Experiment
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)
Conclusions Led to the Concept of Semi-Conservative
Replication
Protein Synthesis
Nirenberg and Khorana
a. 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 acids
The 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.
UUGUUGUUG..., the cells synthesized poly-leucine, poly-cysteine and poly-valine;
UGUGUGUGU... gave one peptide with alternating valines and cysteines;
GGUGGUGGU... gave poly-glycine, poly-valine, and poly-tryptophan.
Cell Overview for Transcription and Translation
Transcription of a Gene begins at the Promoter with RNA
Polymerase forming a transriptional complex
Transcription – The production of the messenger RNA
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' -> 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.)
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 EFG) and the energy of another molecule of GTP.
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
Protein Product – Tertiary Structure
Gene regulation - Prokaryotes