DNA As Genetic material- Key experiments

DNA As Genetic material

1. GRIFFTH EXPERIMENT

• It is also called as as transformation principle.
• It involve in bacterial transformation. 
• Frederick giffth proposed this experiment.
• The pneumonia bacteria which causes pneumonia is used for this experiment

Strains of bacteria: 

1.Rough strain 

• It has rough appearance 
• It is avirulent and non pathogenic 
• It does not have capsule 

2.Smooth strain 

• It has smooth glistering appearance 
• It has capsule made of polysaccharide 
• It is virulent pathogenic

Strains 

• S types- SI, SII and SIII 
• R types-RI,RII and RIII 
• Both S and R types differ in each other antigen 
• Smooth forms may be mutated to rough forms but the changes are not reversible

Procedure 

Step 1 

• The laboratory mice inject With live RII pneumonia 
• The mice suffered no illness 
• Because RII pneumonia is avirulent 

Step 2 

• But when mice inject with Virulent SIII pneumonia 
• The mice suffered from pneumonia and died

Step 3 

• When he inject the killed SIII bacteria in mice they does not suffer from pneumonia 

Step 4 

• But when mice inject the mixture of living avirulent RII and heat killed SIII virulent 
• The unexpected sign of pneumonia appear on high mortality occurs
• But postmorteming mice it has blood with both RII and SIII pneumonia 
• Griffth concludes that presence of heat killed SIII bacteria cause by transformation of living RIII so as to restore them to capsule formation that had earlier lost by gene mutation 
• This is called Griffth effect or bacterial transformation

2. Avery,Mcleod and McCarthy Experiment 

• Griffth could not understand the cause of bacterial transformation. 
• It is identified by the experiment.
•  It is identified by Oswald Avery,Colin Macleod and Maclyn McCarthy 

Step 1 

• They partially purify the transformation principle from cell extract.
• They prepare samples of DNA from SIII bacteria. 
• They add this DNA to live R bacterial culture. 
• After a period they place a sample of SIII containing RII bacterial culture on agar. 
• It is grown as colonies were SIII type. It is dispersed and placed on second agar. 
• The resulting colony is SIII type. 

Step 2 

• The RII colony arise from original mixture is dispersed then RII bacteria grow. 
• The RII colony retains RII Character whereas the transformed SIII colonies as SIII.

The transformation principle is found by four method: 

1. Chemical analysis showed the major components was deoxyribose contains nucleic acid. 
2. Physical measurement show that sample has high viscous substances has property of DNA. 
3. Transformation principle not lost either treat with Purified proteolytic enzyme like trypsin,chymotrypsin RNAase(an enzyme depolymerizes RNA), Lipase 
4. The treatment with material known to contains DNA ase inactivated transformation principle.

3.Blender experiment 

• A confirmation of genetic nature of DNA came from E.coli phage T2 
• It is known as blender experiment because the kitchen wearing blender is used as apparatus. 
• It was performed by Alfred harshey and Martha chase Bacteriophage 
• The DNA injected by phage particle into bacterium contains all information require synthesis of progeny phage. 
• Phage T2 single stranded DNA encased in protein shell.

Radioactive labelled –Selection 

• The DNA has only phosphate containing substances phage particle. 
• The protein shell has amino acid methionine and cysteine have Sulphur atoms. 

Step 1-Labelling

•  The phage DNA is radioactively labelled with 𝑃^32 by growing infected bacteria on medium radioactive 𝑃^32. 
• Phage protein has no P^32 and only protein labelled as sulfur radioactivity.
• The phage protein labelled as 𝑠^35. 
• Thus DNA has no sulphur. 
• The labelling is due to make differences in DNA and RNA. 

Step 2 

• They allow labelled phage on to infect E.coli. 
• The infected bacteria agitate with blender. 
• After shaking radioactive 𝑃^32 found associated with bacterial cell, where 𝑠^35 is in surrounding medium.

Step 3 

• Phage progeny studied for radioactivity only 𝑃^32 is carried the progeny was not labelled with 𝑆^35 
• It indicate that DNA and not protein inject into bacterial cells
• The empty protein coat left out. 
• DNA enters the host carry all genetic information. 
• This confirmed DNA as carrier of genetic information.

Thank you 

References

1. CELL BIOLOGY, GENETICS, MOLECULAR BIOLOGY, EVOLUTION AND ECOLOGY-Verma and Agarwal

2.Lewin's Genes