Introduction:

Watson and Crick:

• Published their analysis of DNA structure in 1953.
• Two long, antiparallel, polynucleotide chains coiled around a central    axis, forming a right-handed double helix.
• "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."

• met each other as graduate students at Woods Hole in 1954, the second summer after the  Watson-Crick DNA model had been announced.
• They discussed ways to test the prediction of  the semi-conservative model of DNA replication.
• Considered the possible use of density as a label for parental DNA molecules.
• Initially they tried to label bacteria with deuterium-labeled phage particles.  However due to difficulties they finally decided to use bacteria.

• They were right!!

• Previous studies have indicated that deoxyribonucleic acid can carry and transmit hereditary information and direct its own replication.  Hypotheses for the mechanism of DNA replication contrasted in their predictions of  the distribution of the of parental molecules among progeny molecules

• In order to support the semi-conservative theory of replication, alternative theories on the subject had to be considered as well.
• Conservative replication - The two newly created strands come together and the parental strands reassociate as well.
• Dispersive replication - The parental strands cleave during replication and the DNA is dispersed into two new double helices.

-Escherichia Coli was grown at 36⁰ C, in a glucose salts medium containing      ammonium chloride as the sole nitrogen source.
-Bacterial growth was measured using microscopic cell counts in conjunction with colony assays.
-Bacteria were labeled with ¹⁵N and were then abruptly changed to a medium containing ¹⁴N.
-Samples of cells were taken before the transfer and at various intervals during growth in the unlabeled medium.
-The bacterial cells were then lysed and the DNA isolated for density gradient centrifugation.
-If several different density species of DNA are present, each will form a band at the position where the density of the CsCl solution is equal to the buoyant density of that species.
-In this way, DNA labeled with heavy nitrogen may be resolved from unlabeled DNA. ( See Figure.)
-The cells' lysate was centrifuged at 140,000X g ( 44,770 rpm) for 20 hrs in CsCl solution until the DNA was isolated from other cellular components in a region of density of 1.71 g/cm³ .
-UV absorption photographs were taken throughout centrifugation with a recording microdensitometer.
-This allows the concentration and difference in buoyant density to be determined and compared.
-The buoyant density of the DNA molecules is expected to vary directly with the fraction of  ¹⁵N label it contains. The degree of labeling of a species of DNA may be determined from the position of the band  relative to other bands.
-Two experiments were performed using this method. One was a sort of "trial run," while the second experiment reinforced their findings and experimental method as well.
-The first experiment was performed by running DNA samples retrieved at increments of 0.4 of the generation time (0.81hrs).
-The following experiment wasn't as precise.  See Photographs.

grow E. coli on medium containing N¹⁵

transfer bacteria to medium containing N¹⁴

after each generation of growth on N¹⁴ isolate DNA and run on cesium chloride (CsCl) gradient in ultracentrifuge

observe location of band of DNA

Meselson and Stahl proved that each time the DNA reproduced, half of the old DNA stayed the same while the other half was newly built. See it for yourself!
 

Here are some key points...

1.    After growing the bacteria in the heavy isotope of nitrogen for a long time, both strands of DNA would be heavy. This was the heaviest layer in the centrifuge.

2.    After the first replication in light hydrogen, each heavy DNA split and built a new half using light nitrogen. All of this DNA would have a medium weight because it had one heavy strand and one light strand. This caused one medium layer in the centrifuge.

3.    After the second replication, each of these medium DNA molecules split. The heavy strand built another new, light weight half, producing a medium weight DNA. The light half made another new, light weight half, producing a DNA even lighter than any before. This gave the two different layers in the centrifuge.

4.    After the third replication, there will be only one hybrid DNA produced compared to three N¹⁴-labeled strands. As a result, the lighter band will have a
higher concentration of DNA and thus will appear more intense. The experiment continues through a fourth and final generation, and milestone generations are compared with the T=0 result.

AT=2 hydrogen bonds; GC=3 hydrogen bonds

More energy is required to disrupt the three hydrogen bonds of a GC base pair than is required to disrupt the two hydrogen bonds of an AT base pair

The denaturation of double stranded DNA is easily followed spectroscopically. The purine and pyrimidine bases in DNA absorb UV light maximally at a wavelength of approximately 260 nm.

In double-stranded DNA, however, the absorption is decreased due to base-sticking interactions. When DNA is denatured, these interactions are disrupted and an increase in absorbance is seen. This change is called the hyperchromic effect.

results in change in interactions between the electron systems of the bases, as they are no longer stacked

They wanted to see if the DNA was in fact double stranded as Watson and Crick had proposed so...

   They heat denatured both E. coli and salmon sperm DNA

   salmon sperm DNA showed density increase of 0.0014 gm. cm. ^-3

   No change in apparant molecular weight

   However, in heated bacterial DNA, molecular weight is reduced to   approximately half that of unheated material

   Heated hybrid DNA results in loss of original half and appearance of two new density species in equal amounts, each with ~ 1/2 initial m.w.

   Density difference = 0.015 gm. cm. ^-3  ~ same as N¹⁵ label

   Suggest dissociation into two distinct subunits

   Theory tested by heating N¹⁴ and N¹⁵ separately

These findings resulted in two new theories...
  1)    subunits of salmon sperm DNA are more tightly bound
    2)    bacterial DNA in a more complex molecule, challenging Watson & Crick
 

   By means of density-gradient centrifugation, the distribution of N¹⁵ was observed among molecules of bacterial DNA following the transfer of a uniformly N¹⁵-substituted exponentially growing E. coli population to N¹⁴ medium. Here is a nice summary figure...

   "We find that the nitrogen of a DNA molecule is divided equally between two physiologically continuous subunits; that, following duplication, each daughter molecule receives one of these; and that the subunits are conserved through many duplications."

Questions? Comments? E-mail Ben and Jason anytime...