DNA as Genetic Material-Experimental Proof Part III- The Blender Experiments- Dr. C R Meera

 

DNA as Genetic Material-Experimental Proof

Part III- The Blender Experiments- Dr. C R Meera

An elegant confirmation of genetic nature of DNA came from the experiments with E.coli phage T₂ by Hershey and Chase.

·        This experiment is called Blender experiment as “Kitchen Blender” was used as the major apparatus.

·        Done by Alfred Hershey and Martha Chase, who demonstrated that DNA injected by phage particle into bacterium contains all information required to synthesize progeny phage particles.

·        T₂ phage used in the experiment consisted of DNA encased in a protein shell.

·        Radioactive phosphate and sulfur were used to mark the phage.

·        DNA is the only phosphorous-containing particle in the phage. So radioactive phosphate would be incorporated in phage DNA during multiplication in a media containing radioactive phosphate .

·        Proteins of the shell which contain the amino acids methionine and cysteine, have only sulfur atoms in it. So protein shells would take up the radioactive sulfur during multiplication in a media containing radioactive sulfur.

·        In this experiment, Phage with radioactive DNA was used which was prepared by growing phages in a nutrient medium in which radioactive phosphate          (³²PO₄^3-) is the sole source of phosphorus.

·        Phage with radioactive capsid- was also used which was prepared by growing phages in a medium containing radioactive sulfur (³⁵SO₄^2-) as the sole source of sulfur.

·        These two kinds of radiolabeled phages were used in the infection of E.coli so that phage DNA and capsid can be located by the radioactivity.

·        T₂ phage has a long tail with which it attaches to the host bacterium, E.coli. Hershey and Chase showed that the attached phage can be separated from the bacteria by violent agitation using the kitchen blender.

·        They conducted two experiments known as ³⁵S and ³²P Experiments.

                                   Fig 1. ³⁵S and ³²P Experiments by Hershey and Chase                                         

  (Image Courtesy:www.mun.ca)

 

³⁵S Experiment

·         Phage with radioactive capsid was also used in this experiment which was prepared by growing phages in a medium containing radioactive sulfur.

·         S labeled phages were allowed to adsorb to bacteria for a few minutes.

·         Phage-attached bacteria were separated from unattached phages by centrifugation of the mixture and the pellet formed is the phage-bacterium complex.

·         This complex was resuspended in a liquid and blended and the suspension was again centrifuged.  

·         Now, the pellet received is bacteria and the supernatant was also collected.

·         80% of the radioactivity or  ³⁵S was in the supernatant and 20% was in the pellet. This experiment thus showed that capsid remains outside the bacteria and not acting as the genetic material.

·         Many years later, it was found that 20% radioactivity in the pellet was due to the tail segment of the phage being too tightly attached to the bacterial cell that were not removed by blending.

³²P Experiment

·         Phage with radioactive DNA was used in this experiment which was prepared by growing phages in a nutrient medium containing radioactive phosphate. The experiment was performed same as above.

·         A very different result with 70% of radioactivity or ³²P was found in the pellet (ie; associated with the bacteria) and 30% radioactivity in the supernatant.

·         30% radioactivity in the supernatant may be due to the breakage of bacteria during the blending process or due to the defective phage particles that could not inject their DNA into the bacterial host.  

·         This experiment proved that the DNA is getting into the bacterial cell and hence acting as the genetic material.

·         Confirmation Experiment: Pellet was resuspended in the growth medium and re-incubated. It was capable of phage production, indicating that the genetic message for phage progeny production had been introduced by phage DNA, not by phage protein. Thus, DNA is the genetic material.

·         Later, a series of experiments called “transfer experiments” analyzed the progeny for ³⁵S and  ³²P.

·         ³⁵S was not found in the progeny.

·         Half of  ³²P injected was found in the progeny.

·         Interpretation: Transfer of only half of ³²P in progeny is because that DNA is selected at random for packing into protein coats.