Nucleic acids (DNA and RNA) act as genetic material in all organisms and viruses: 1. Evidences from Bacteria 2. Evidence from Bacteriophages 3. Evidence from Bacterial Conjugation 4. Evidence from RNA Viruses.
1. Evidences from Bacteria:
For the first time, an English Health officer, Frederick Griffith (1928) gave an experimental evidence that the DNA was the genetic material. He took two types of a bacterial strain, pneumococci (Streptococcus pneumonia) that causes pneumonia in humans and other animals.
There were two types of pneumococci, type II and type III. Each type exists in two forms RII, SII, and RIII, SIII forms where ‘R’ represents the rough, non-capsulated and non- virulent form and ‘S’ represent the smooth, encapsulated and virulent form.
After injection of smooth (virulent) strain, mice were killed (Fig. 5.24). When boiled smooth strain was injected, the mice were not affected and no pneumococci could be recovered from the mice. Upon injection of a mixture of heat killed smooth strain and live rough (non-virulent) strain, the mice were killed and live virulent bacteria could be recovered from blood of dead mice.
Griffith called this change of non-virulent strain into virulent strain as transformation, because the virulent strain transformed the non-virulent strain into the virulent strain. This is called Griffith’s transformation experiment (Fig. 5.24). The phenomenon of bacterial transformation is called “Griffith effect”.
Identification of transforming substance:
Griffith could not identify the nature of transforming substance. Oswald T. Avery, C.M. Mac Leod and M.J. Mc Carty (1944), set out experiments to identify the transforming principle. They destroyed cell constituents in extract of virulent pneumococci SIII using enzyme that hydrolyzed DNA, RNA, proteins and polysaccharides.
They treated the non-virulent strain separately with bacterial extracts as below:
RII Cells + Purified SIII cell polysaccharide → R colonies
RII Cells + Purified SIII cell protein → R colonies
RII Cells -H Purified SIII cell RNA → R colonies
RII Cells + Purified SIII cell DNA → SIII COLONIES
RII Cells + Purified SIII cell extract + protease → SIII colonies
RII Cells 4- Purified SIII cell extract h- RNase → SIII colonies
They concluded that a cell free and highly purified RNA extract of SIII strain could bring about transformation of RII strain into SIII strain. However, this effect was lost when the extract was treated with deoxy-ribonuclease.
Therefore, DNA is the genetic material and carries sufficient information for tons-formation. Thereafter, the transforming material (DNA) was also confirmed in several bacteria such as Bacillus subtilis, Haemophilus influenzae, Shigella para-dysenteriae, etc.
2. Evidence from Bacteriophages:
Alfred D. Hershey and Martha Chase (1952) carried out several experiments in bacteriophage T2 that proved the DNA to be the genetic material of T2. They prepared a culture medium for the growth of E. coli that contained phosphorus and sulphur.
To this culture medium known amount of isotopic phosphorus (32P) and sulphur (35S) was amended. Cell suspension of E. coli was poured onto the growth medium. After bacterial growth 32P and 35S were incorporated into the bacterial cell.
These cells were allowed to get infected by T2 bacteriophage. T2 was multiplied in E. coli cells. The 32Pwas incorporated into DNA molecule and 35S into capsid protein of T2 phage. Since DNA lacks sulphur, therefore, 35S was not incorporated into DNA. By using this method they labelled differentially the phage DNA and coat protein with the isotopes. These radiolabelled phage particles were recovered from E. coli by centrifugation method.
In another experiment, non-radiolabelled E. coli cells were allowed to be infected by radiolabelled bacteriophages. After absorption the coat protein remained outside and only radiolabelled DNA was injected. From the infected E. coli cells, the viral particles were separated from the host through agitation.
The amount of 32P and 35S in bacteriophage, particles, E. coli cells and medium was estimated. The 32p was estimated with the bacterial cells and 35S with protein coat left outside the bacterial wall i.e. the growth medium. This experiment shows that the genetic material of T2 phage resides in DNA but not in their protein.
For this work A.D. Hershey shared Nobel Prize in medicine for 1969 with M. Delbruck and S.E. Luria. Furthermore, this experiment was repeated by several other workers by taking different bacteriophages. In all the cases, DNA was found to give evidence of genetic material.
3. Evidence from Bacterial Conjugation:
Conjugation is one of the methods of transfer of DNA from donor bacterium to the recipient bacterium. Lederberg and Tatum (1946) demonstrated when F+ (bacterium containing fertility factor i.e. male) strain of E. coli cells is mixed with F– (cells devoid of fertility factor i.e. female); the F– cells were converted to F+ cells.
F+ cells + F– cells → F+ cells
The F+ factor is a segment of plasmid DNA. It is evident from this experiment that DNA is the genetic material.
4. Evidence from RNA Viruses:
In 1956, A. Gierer and S. Schramm put example that when healthy tobacco leaves are inoculated by RNA purified from tobacco mosaic virus, lesions developed on healthy leaves which have like those obtained from tobacco mosaic plants.
Like Hershey and Chase, another experiment was done by H.F. Conract and B. Singler in 1957. They separated RNA from protein coat of TMV and reconstituted them. One group contained parental RNA and protein from mutant TMV, and the other had RNA from the mutant TMV and protein coat from the parental TMV.
The reconstituted viruses were allowed to infect healthy tobacco leaves. After infection from the lesions the TMV particles were recovered. In all the cases the progeny TMV particles contained parental RNA type, but not parental protein type.