3 The Search for Genetic Material

3 THE SEARCH FOR GENETIC MATERIAL  The experiments given below prove that DNA is the genetic material. (I) Evidence from bacterial transformation.  The transformation experiments, conducted by Frederick Griffith in 1928, are of great importance in establishing the nature of genetic material.  He used two strains of bacterium Diplococcus or Streptococcus pneumoniae or Pneumococcus i.e., S-III and R-II. (1) Smooth (S) or capsulated type which have a mucous coat and produce shiny colonies. These bacteria are virulent and cause pneumonia. (2) Rough (R) or non-capsulated type in which mucous coat is absent and produce rough colonies. These bacteria are nonvirulent and do not cause pneumonia. The experiment can be described in following four steps : (a) Smooth type bacteria were injected into mice. The mice died as a result of pneumonia caused by bacteria. S strain  Injected into mice  Mice died (b) Rough type bacteria were injected into mice. The mice lived and pneumonia did not occur. R strain  Injected into mice  Mice lived (c) Smooth type bacteria which normally cause disease were heat killed and then injected into the mice. The mice lived and pneumonia was not caused. S strain (heat killed)  Injected into mice  Mice lived (d) Rough type bacteria (living) and smooth type heat-killed bacteria (both known not to cause disease) were injected together into mice. The mice died due to pneumonia and virulent smooth type living bacteria could also be recovered from their dead bodies. S strain (heat killed) + R strain (living)  Injected into mice  Mice died He concluded from fourth step of the experiment that some rough bacteria (nonvirulent) were transformed into smooth type of bacteria (virulent). This occurred perhaps due to absorption of some transforming substance by rough type bacteria from heat killed smooth type bacteria. This transforming substance from smooth type bacteria caused the synthesis of capsule which resulted in production of pneumonia and death of mice. Therefore, transforming principle appears to control genetic characters (for example, capsule as in this case). However, the biochemical nature of genetic material was not defined from his experiments. Fig: In his famous series of experiments, Griffith used two strains of S. pneumoniae. The S strain is pathogenic and causes death. Mice injected with the nonpathogenic R strain or the heat-killed S strain survive. However, a combination of the heat-killed S strain and the live R strain causes the mice to die. The S strain recovered from the dead mouse showed that something had passed from the heat-killed S strain to the R strain, transforming the R strain into an S strain in the process.  Later, Avery, Macleod and McCarty (1944) repeated the experiment in-vitro to identify the biochemical nature of transforming substance. They proved that this substance is DNA.  Pneumococcus bacteria cause disease when capsule is present. Capsule production is under genetic control.  In the experiments, rough type bacteria (non-capsulated and non-virulent) were grown in a culture medium to which DNA extract from smooth type bacteria (capsulated and virulent) was added.  Later, the culture showed the presence of smooth type bacteria also in addition to rough type.  This is possible only if DNA of smooth type was absorbed by rough type bacteria which developed capsule and became virulent.  This process of transfer of characters of one bacterium to another by taking up DNA from solution is called transformation. When DNA extract was treated with DNase (an enzyme which destroys DNA), transformation did not occur. The transformation occurs when proteases and RNases were used. This clearly shows   that DNA is the genetic material. Fig: Oswald Avery, Colin MacLeod, and Maclyn McCarty followed up on Griffith’s experiment and experimentally determined that the transforming principle was DNA. Fig: In vitro experiment of Avery and others demonstrating that DNA is genetic material (II) Evidence from experiments with bacteriophage.  T2 bacteriophage is a virus that infects bacterium Escherichia coli and multiplies inside it. Fig: Martha Chase and Alfred Hershey conducted an experiment separately labelling the DNA and proteins of the T2 bacteriophage to determine which component was the genetic material responsible for the production of new phage particles.  T2 phage is made up of DNA and protein coat.  Thus, it is the most suitable material to determine whether DNA or protein contains information for the production of new virus (phage) particles.  Hershey and Chase (1952) demonstrated that only DNA of the phage enters the bacterial cell and, therefore, contains necessary genetic information for the assembly of  new phage particle. The functions of DNA and proteins could be found out by labelling them with  radioactive tracers. DNA contains phosphorus but not sulphur.  Therefore, phage DNA was labelled with P32 by growing bacteria infected with phages in culture medium containing 32P.   Similarly, protein of phage contains sulphur but no phosphorus. Thus, the phage protein coat was labelled with S35 by growing bacteria infected with  phages in another culture medium containing 35S. After the formation of labelled phages. Three steps were followed, i.e., infection, blending, centrifugation. 1. Infection: Both types of labelled phages were allowed to infect normally cultured bacteria in separate experiments. 2. Blending: These bacterial cells were agitated in a blender to break the contact between virus and bacteria. 3. Centrifugation: The virus particles were separated from the bacteria by spinning them in a centrifuge.  After the centrifugation the bacterial cells showed the presence of radioactive DNA labelled with P32 while radioactive protein labelled with S35 appeared on the outside of bacteria cells (i.e., in the medium).   Labelled DNA was also found in the next generation of phage. This clearly showed that only DNA enters the bacterial host and not the protein.  DNA, therefore, is the infective part of virus and also carries all the genetic information.  This provided the unequivocal proof that DNA is the genetic material.  Hershey and Chase's experiments with bacteriophages showing that DNA is genetic material or DNA is infective part of virus. Properties of Genetic Material :  (1) (2) Following are the properties and functions which should be fulfilled by a substance if it is to qualify as genetic material. It should chemically and structurally be stable. The genetic material should be able to transmit faithfully to the next generation, as (3) Mendelian characters. The genetic material should also be capable of undergoing mutations. (4)  The genetic material should be able to generate its own kind (replication). This can be concluded after examining the above written qualities, that DNA being more stable is preferred as genetic material, as (a) Free 2OH of RNA makes it more labile and easily degradable. Therefore, DNA in comparison is more stable. (b) (c) Presence of thymine at the place of uracil also confers additional stability to DNA. RNA being unstable, mutates at a faster rate.