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Dr. Kornberg’s key contributions to nucleic acid research:
Demonstration of the antiparallel orientation of complementary strands
10/16/2006
Lecture 9: 1
Geiger Counter
An ion or electron penetrating the tube (or an electron knocked out of the wall
by X-rays or gamma rays) tears electrons off atoms in the gas, and because of
the high positive voltage of the central wire, those electrons are then attracted
to it. In doing so they gain energy, collide with atoms and release more
electrons, until the process snowballs into an "avalanche" which produces an
easily detectable pulse of current. With a suitable filling gas, the flow of
electricity stops by itself, or else the electrical circuitry can help stop it.
The instrument was called a "counter" because every particle passing it
produced an identical pulse, allowing particles to be counted (usually
electronically) but not telling anything about their identity or energy (except that
they must have sufficient energy to penetrate the walls of the counter).
Dr. Kornberg’s key contributions to nucleic acid research:
Demonstration of the antiparallel orientation of complementary strands
Detection by autoradiography
Silver bromide (AgBr), a soft, paleyellow, insoluble salt well known
(along with other silver halides) for
its unusual sensitivity to light. This
property has allowed silver halides to
become the basis of modern
photographic materials.
The photographic emulsion is usually
10 to 30 µm thick, and is composed of
silver halide grains dispersed within
gelatin. The grains are 1 µm or greater
in diameter; large grains facilitate
greater sensitivity, small grains enable
finer resolution. The grains consist of
silver, bromine, and iodine ions
arranged in a crystal lattice (see Fig. 2).
Sulfur-containing compounds are often
added in order to form specks silver
sulfide, which increase photosensitivity.
10/16/2006
Lecture 9: 1
Dr. Kornberg’s key contributions to nucleic acid research:
Demonstration of the antiparallel orientation of complementary strands
Detection by autoradiography
Chemical properties of photographic film
The film base is usually plastic such as tri-acetate or polyester which is
coated with a light sensitive emulsion.
The emulsion consists of gelatin containing light sensitive silver halide
crystals such as silver bromide and silver chloride. In practice the film will
consist of many other layers. Photographic emulsion is not a true emulsion,
it is a dispersion of small solid particles in a liquid medium which is then
allowed to cool and set.
The light sensitive crystals are prepared by the combination of silver-Agand a halogen. Due to the very low solubility of silver halides mixing
aqueous solutions of silver ions and halide ions will result in the
precipitation of silver halide crystals. e.g.
silver nitrate (AgNO3) + potassium bromide -----> silver bromide (AgBr) +
potassium nitrate (KNO3)
Or Ag+ (silver ion in solution) + Br- (bromide ion in solution) --------> Ag+Br(silver bromide crystal)
Silver bromide is a lattice crystal containing millions of pairs of ions.
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Dr. Kornberg’s key contributions to nucleic acid research:
Demonstration of the antiparallel orientation of complementary strands
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Lecture 9: 4
Detection by autoradiography
Formation of the Latent Image
Step 1: Light Activation
It is not fully understood exactly what happens during exposure but the energy
released when a photon of light strikes a silver halide crystal frees an electron
from the bromide ion. The former bromide ion is released from the crystal as
bromine and is absorbed by the gelatin.
Step 2: Movement of electrons
The free electron moves through the crystal to a 'sensitivity speck' caused by
imperfections in the crystal structure or created during the sensitizing process
during manufacture.
Step 3: Deposition of Silver Ions
This now negatively charged speck attracts positive silver ions which are
neutralized to form silver atoms. If enough silver atoms form at a single point
then a latent image is created. The latent image is not visible, even under a
microscope so the only way to tell if it is present is to chemically develop the
film to reveal the image.
Dr. Kornberg’s key contributions to nucleic acid research:
Demonstration of the antiparallel orientation of complementary strands
10/16/2006
Lecture 9: 5
Detection by autoradiography
Development of the latent image
Development:
During development the developing agent supplies electrons to the latent image thus attracting
and neutralizing silver ions to produce metallic silver which will eventually form a visible image.
The latent image acts as a catalyst encouraging development to take place faster in exposed
areas. Development takes place in both exposed and unexposed areas of the film just at
different rates. Developing agents: Metol, Phenidone, Hydroquinone.
Stop:
When the predetermined development time has been reached the film is moved from the
developer to a 'stop bath' which neutralizes the developer and prevents any further
development of the image from taking place. Developers work most effectively in an alkaline
environment which is why an acid stop bath is used. Stop bath: 1% solution acetic acid.
Fixing:
After development the emulsion still contains unexposed and undeveloped silver halides. The
film will look cloudy or milky and given exposure to light the remaining silver halides will be
reduced to silver. The fixer, commonly sodium thiosulphate, converts the unexposed silver
halide to soluble salts which can be washed out of the emulsion.
Washing:
The processed film is washed thoroughly to remove any chemical residue before being dried.
Dr. Kornberg’s key contributions to nucleic acid research:
Discovery of poly(P)
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Lecture 9: 6
We (Dr. Arthur Kornberg and his associates) have switched the focus of our research from DNA replication
to an entirely new subject: inorganic polyphosphate (poly P). Poly P is a linear polymer of many tens or
hundreds of orthophosphate (Pi) residues linked by high-energy, phosphoanhydride bonds. Likely a
prominent precursor in prebiotic evolution, poly P is now found in volcanic condensates, deep-oceanic
steam vents and in every living thing-bacteria, fungi, protozoa, plants and mammals. Ignored in textbooks
and dismissed as a "molecular fossil," our mission is to bring this molecule back to life and demonstrate
that poly P is truly a "molecule for many reasons."
Our approach is to discover the enzymes for the synthesis and utilization of poly P in bacteria, yeast and
animal cells. These enzymes will reveal novel mechanisms and insights and when purified will open the
route of reverse genetics: the peptide sequence leads to the gene and thereby the means to knock it out and
overexpress it. By manipulating expression of the gene and the cellular levels of its product, phenotypes are
created which provide clues to metabolic functions. Most immediate and decisive, the enzymes provide
unique and invaluable reagents for analytic and preparative work.
Among the several current directions are:
DNA entry into cells: The mechanism whereby the inclusion of poly P in a membrane complex enables a cell
to become competent to take up DNA and then genetically transformed.
Survival in the stationary phase: The basis for poly P regulation of cellular responses to stresses and
adjustments for survival in the stationary phase of culture growth and development. In view of the
universality and complexity of basic biochemical mechanisms, it would be surprising if some of the variety
of poly P functions already observed in microorganisms did not apply to aspects of human growth and
development, to aging and to the aberrations of disease.
Regulation of development: Developmental changes in microorganisms-fruiting body and spore formation in
Myxobacteria (e.g., M. xanthus), sporulation in bacteria (e.g., Bacillus) and fungi, and heterocyst formation in
cyanobacteria (e.g., Anabaena)-occur in response to starvation of one or another nutrient. In view of the
involvement of poly P in the stationary stage of E. coli, poly P may well participate in other instances of
cellular adjustments to deprivation.
Dr. Kornberg’s key contributions to nucleic acid research:
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He (Dr. Authur Kornberg) was married in 1943 to Sylvy Ruth Levy,
who died in 1986. He has three sons and eight grandchildren.
Roger is a Professor of Structural Biology at Stanford; Thomas is
a Professor of Biochemistry and Biophysics at the University of
California in San Francisco; Kenneth is an architect and founder
of Kornberg Associates in Menlo Park and Delmar, California,
specializing in laboratory design.
http://kornberg.stanford.edu/
Dr. Kornberg’s key contributions to nucleic acid research:
XI. Good offspring
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Lecture 9: 8
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Lecture 9: 10
Fermentation
1. Hexokinase catalyzes: glucose + ATP
2. Phosphoglucose Isomerase catalyzes:
glucose-6-phosphate (aldose)
glucose-6-phosphate + ADP
fructose-6-phosphate (ketose)
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Fermentation
3. Phosphofructokinase catalyzes:
fructose-6-phosphate + ATP
fructose-1,6-bisphosphate + ADP
4. Aldolase catalyzes:
fructose-1,6-bisphosphate
dihydroxyacetone phosphate + glyceraldehyde-3-phosphate.
Fermentation
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5. Triose Phosphate Isomerase (TIM) catalyzes::
dihydroxyacetone phosphate (ketose)
glyceraldehyde-3-phosphate (aldose)
6. Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) catalyzes:
glyceraldehyde-3-phosphate + NAD+ + Pi
1,3,bisphosphoglycerate + NADH + H+
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Fermentation
7. Phosphoglycerate Kinase catalyzes:
1,3-bisphosphoglycerate + ADP
3-phosphoglycerate + ATP
8. Phosphoglycerate Mutase catalyzes: 3-phosphoglycerate
2-phosphoglycerate
Fermentation
9. Enolase catalyzes: 2-phosphoglycerate
10. Pyruvate Kinase catalyzes: phosphoenolpyruvate + ADP
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phosphoenolpyruvate + H2O
pyruvate + ATP
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Fermentation
Balance sheet for high energy bonds of ATP:
2 ATP consumed
4 ATP produced (2 from each of two 3C fragments from glucose)
Net production of 2 ~P bonds of ATP per glucose.
Glycolysis Pathway (omitting H+):
glucose + 2 NAD+ + 2 ADP + 2 Pi à 2 pyruvate + 2 NADH + 2 ATP
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Fermentation
Because glycolysis produces two
pyruvate molecules from one glucose,
each glucose is processes through the
kreb cycle twice. For each molecule of
glucose, six NADH2+, two FADH2, and
two ATP.
Acetyl-CoA
+ 3 NAD+ + FAD
+ GDP + Pi + 3 H2O
3 NADH + FADH
+ CoA-SH + GTP
+ 3 CO2
C6H12O6 + 6O2
6CO2 + 6H2O + energy
(ATP)
Fermentation
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Fermentation
Anaerobic organisms lack a respiratory chain. They must reoxidize NADH produced in
Glycolysis through some other reaction, because NAD+ is needed for the Glyceraldehyde3-phosphate Dehydrogenase reaction (see above). Usually NADH is reoxidized as pyruvate
is converted to a more reduced compound. The complete pathway, including Glycolysis and
the re-oxidation of NADH, is called fermentation.
Skeletal muscles ferment glucose to lactate during exercise. Lactate
released to the blood may be taken up by other tissues, or by skeletal
muscle after exercise, and converted via Lactate Dehydrogenase back to
pyruvate, which may be oxidized in Krebs Cycle or (in liver) converted to
back to glucose via gluconeogenesis.
Lactate serves as a fuel source for cardiac muscle as well as brain
neurons. Astrocytes, which surround and protect neurons in the brain,
ferment glucose to lactate and release it. Lactate taken up by adjacent
neurons is converted to pyruvate that is oxidized via Krebs Cycle.
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Fermentation
Glycogen is a polymer of glucose residues linked mainly by α(1
4)
glycosidic linkages. There are α(1
6) linkages at branch points. The
chains and branches are longer than shown. Glucose is stored as glycogen
predominantly in liver and muscle cells.
1) Glycogen phosphorylase
2) glucan transferase
Fermentation
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Pathways involved in the regulation of glycogen phosphorylase. See the text for details of the regulatory
mechanisms. PKA is cAMP-dependent protein kinase. PPI-1 is phosphoprotein phosphatase-1 inhibitor.
Whether a factor has positive (+ve) or negative (-ve) effects on any enzyme is indicated. Briefly,
phosphorylase b is phosphorylated, and rendered highly active, by phosphorylase kinase. Phosphorylase
kinase is itself phosphorylated, leading to increased activity, by PKA (itself activated through receptormediated mechanisms). PKA also phosphorylates PPI-1 leading to an inhibition of phosphate removal
allowing the activated enzymes to remain so longer. Calcium ions can activate phosphorylase kinase even
in the absence of the enzyme being phosphorylated. This allows neuromuscular stimulation by
acetylcholine to lead to increased glycogenolysis in the absence of receptor stimulation.
Fermentation
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invertase
Fermentation
Evolution of GAPDH
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