Slide 1 : PART –I
DNA AS GENETIC MATERIAL
9-8-2011 5.15 PM PART- II
DNA STRUCTURE
11-8-2011 5.30 PM THE GENETIC MATERIAL : DNA : STRUCTURE BY
Dr. Ichha Purak
http://www.dripurak.com/
http://drichhapurak.webnode.com/ 1 8/8/2011
Slide 2 : It is well known fact that transmission of traits takes place from one generation to other.The offsprings are similar to both the parents in some traits
Gregor Johann Mendel (1866 ) on the basis of his hybridization experiments on Sweet pea gave the idea that transmission of traits over generations take place through Factor or Determiner or Gene which carries information for expression of trait or phenotype. THE GENETIC MATERIAL : DNA : STRUCTURE 2 8/8/2011
Slide 3 : Genes are present on the chromosomes which are distributed equally into the two daughter cells during cell division. The biochemical studies reveal that chromosomes are composed of proteins (60% ) and DNA (40% ) 3 8/8/2011
Slide 4 : Genetic material must be capable of
Replication ( Make its copy )
Storage of information for expression of trait
Control expression of traits
Change in controlled way (undergo mutation)
Must be stable
Is the Genetic Material Protein or DNA 4 8/8/2011
Central Dogma of Molecular Genetics : Central Dogma of Molecular Genetics 5 8/8/2011
Slide 6 : Topic of Discussion till 1944 was which chromosomal component DNA or Protein carries hereditary information or is the genetic material
Untill 1940 Proteins were considered as genetic material as Proteins are polymer of 20 protein amino acids and present in larger quantity, encode more and variety of information.
DNA is polymer of only 4 different deoxyribonucleotides (ATP, CTP, GTP & TTP) and is present in smaller quantity 6 8/8/2011
Slide 7 : Most geneticists focused on “transmission genetics” and passively accepted proteins as the genetic material
But On the basis of certain experiments conducted from time to time ,it was ultimately demonstrated that DNA carries genetic information and not the proteins
There are some direct evidences and some indirect evidences which prove DNA as Genetic Material 7 8/8/2011
Slide 8 : Direct evidences come from :
Frederick Griffith’s (1928) experiment on Bacterial
Transformation
Oswald Avery, Colin Macleod and Maclyn McCarty’s
(1944 ) experiment on Transformation
Alfred D. Hershey and Martha Chase (1952 )
experiment on T- Even (2,4 ) Bacteriophage 8 8/8/2011
Slide 9 : Frederick Griffith (1928) Studied Diplococcus
pneumoniae, having Two strains
SIII strain was virulent, possessed a lipopolysaccharide capsule and could kill mice by causing disease Pneumonia and made round colonies on a culture plate
RII strain was avirulent and lacked a Lipopolysaccharide (LPS) capsule, growing in rough –shaped colonies on a culture plate 9 8/8/2011
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Slide 11 : 11 STEPS IN THE EXPERIMENT 1 LIVE
SIII LIVE
RII 3 H K S III 4 H K S III & LIVE RII 8/8/2011
Griffith’s Experiment : Griffith’s Experiment RII SIII transformation takes place in step 4 give clue for DNA as
genetic material 1 2 3 4 12 8/8/2011
Slide 13 : STEPS AND RESULTS OF GRIFFITH’S EXPERIMENT 13 8/8/2011
Slide 14 : GRIFFITH’S CONCLUSION
On the basis of Result of step IV Griffith concluded that there was transformation of Avirulent RII type to Virulent SIII type by picking up the genetic material encoding the LPS capsule from the Heat Killed S III .
This bacterial transformation clearly shows the role of DNA as Genetic Material and is further confirmed by results of step V which shows no transformation as DNase digests DNA and step VI again shows transformation as protease only digests protein 14 8/8/2011
Slide 15 : Avery, MacLeod, & McCarty, 1944 repeated Griffith’s experiment of transformation using purified cell extracts and concluded
Removal of all protein from the transforming material did not destroy its ability to transform R strain cells
DNA-digesting enzymes destroyed all transforming ability
The transforming material is DNA 15 8/8/2011
Avery,macleod And McCARTY (1944) : Avery,macleod And McCARTY (1944) 16 8/8/2011
Avery, MacLeod AND McCarty EXPERIMENT : Avery, MacLeod AND McCarty EXPERIMENT Is based on transformation
Cell free extract of SIII strain Bacterium was subjected to DNase,RNase and Protease
Each treated extract was mixed with RII and mixture injected to mouse to see transformation.
In case of Protease and RNase transformation was recorded
In case of DNase no transformation was recorded 17 8/8/2011
Slide 18 : Avery et al (1944) revealed the chemical nature of the transforming substance to be DNA. With the help of experiment they showed that DNA isolated from SIII strain Bacteria could confer the pathogenic properties to R II strain Bacteria . Two conclusion were derived
Active factor is DNA which can cause transformation
SIII strain contains the Active factor 18 8/8/2011
Hershey AND Chase Experiment : Hershey AND Chase Experiment Alfred Hershey and Martha Chase, 1952
- investigated bacteriophages: viruses that infect bacteria
- the bacteriophage was composed of only DNA and protein
- they wanted to determine which of these molecules is the genetic material that is enters into the bacteria 19 8/8/2011
Life Cycle of T-2 Phage : Life Cycle of T-2 Phage Phage is made of DNA and protein coat
Only DNA enters in the Bacterial cell and protein coat is left out side 20 8/8/2011
Slide 21 : EVENTS WHICH TAKE PLACE IN LIFE CYCLE OF BACTERIOPHAGE
Attachment of Bacteriophage to receptor site on
bacterial cell wall by its tail
Bacterial cell wall dissolves at the point of contact
Phage DNA enters into Bacterial cell, protein coat
remains outside
Phage DNA replicates forming many copies
Assembly of phage DNA into new protein shells
Lysis or breakdown of host cell and release of infective phage particle. 21 8/8/2011
Slide 22 : HERSHEY & CHASE (1952) EXPERIMENT WITH T2 BACTERIOPHAGE In culture I Bacteriophage was grown in medium containing Radioactive Phosphorus (32P))To make DNA Radioactive
In culture II Bacteriophage was grown in medium containing radioactive Sulphur (35S) To make proteins Radioactive
Both kinds of Bacteriophage particles were allowed to infect Bacteria
The infected bacteria were observed for radioactivity
Radioactive Phosphorus was found with bacterial cells
Radioactive Sulphur was not traced in bacterial cells ( Only in Ghosts)
Bacteriophage progeny carried only radioactive phosphorus and not radioactive sulphur 22 8/8/2011
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Slide 25 : HERSHAY & CHASE CONCLUSION As ghosts or coat of Bacteriophage were not labelled with 32P and only with 35S. The results of experiment clearly indicate that only DNA and not the proteins enter the bacterial cell. Protein coat is left outside. The DNA entering the host cell carries all the genetic information for synthesis of new phage particle. This certainly proves that DNA is the genetic material in Bacteriophage and not proteins 25 8/8/2011
Slide 26 : 26 Bacteriophage DNA was labeled with radioactive phosphorus (32P)
Bacteriophage protein was labeled with radioactive sulphur (35S)
Radioactive molecules were tracked
only the bacteriophage DNA (as indicated by the 32P) entered the bacteria and was used to produce more bacteriophage
Conclusion: DNA is the genetic material Summary of Hershey & Chase (1952 ) experiment 8/8/2011
Slide 27 : INDIRECT EVIDENCES FOR DNA AS GENETIC MATERIAL Presence of DNA regularly in nuclei of all types of cells
Equal amount of DNA is present in all cells of an organism
Amount of DNA is proportional to the ploidy of cell . Haploid cells have ½ the amount of DNA than diploid cell.
Nuclear Division occurs only after DNA duplication during S phase of Interphase
Vaiation in Diploid amount of DNA amongst different species 27 8/8/2011
Slide 28 : 6. DNA has same physical and chemical properties in all organisms yet allow to produce great diversity of organisms
7. Indefinite number of combinations are possible with four bases A T G C
8. Of all macromolecules DNA is metabolically most stable
9. In prokaryotes DNA is not linked with proteins still characters are inherited 28 8/8/2011
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Slide 30 : some organisms HAVE RNA as Genetic Material Some Viruses have RNA as Genetic Material
RNA Virus ( Ribo Virus ) may have ssRNA or dsRNA enveloped by Protein Coat
Notable human diseases caused by RNA viruses include SARS, Influenza ,Hepatitis C., Measels,Mumps and Rabies
Rous Sarcoma Virus (causing cancer ) and HIV (causing AIDS) both are Retrovirus using Reverse Transcription for DNA synthesis in host cell 30 8/8/2011
Slide 31 : EXAMPLES OF PLANT RNA VIRUSES
Tobbaco Mosaic Virus (TMV ) was crystallized by Stanley (1935) for the first time
TMV causes Tobacco mosaic disease
TMV can also cause disease in Tomato, Pepper, Petunia, Snapdragon, Delphinium, and Marigold.
Papaya Ring Spot virus (PRV)
Potato Leaf Roll Virus (PLRV)
Holmesrib-grass Virus (HRV) –Plantago
Alfa-alfa Mosaic Virus (AMV) 31 8/8/2011
Slide 32 : TMV HIV HEPATITES C VIRUS RNA VIRUSES 32 8/8/2011
Slide 33 : EVIDENCE IN FAVOUR OF RNA AS GENETIC MATERIAL A Gierer and G Schramm ( 1956 ) when inoculated tobacco plants with Purified RNA isolated from TMV - Leisons appeared on leaves of healthy plant
H Fraenkel Conrat and B Singer (1957) separated RNA from protein of TMV in first step .
In second step reconstituted virus with protein from mutant strain of TMV 33 8/8/2011
Slide 34 : Inoculated Hybrid TMV into healthy tobacco plant
Tobacco Mosaic disease appeared
TMV progeny isolated from diseased plant showed parental RNA only but not parental proteins
This provided evidence that RNA carries information for assembly of Virus particles and not proteins
RNA thus is the Genetic Material in TMV 34 8/8/2011
Slide 35 : SUMMARY OF THE CLASS ON GENETIC MATERIAL Gene (Mendelian Factor) carries information for expression of Phenotype
Genes are present on chromosomes
The chromosomes contain 60% protein and 40% DNA
Genetic Material must be stable, capable of replication, storing information for expression and undergo mutation 35 8/8/2011
Slide 36 : Expression is controlled through Central Dogma of Molecular Biology involving Replication,Transcription and Translation
Results of Experiments performed by Griffith’s (1928), Avery et al (1944) and Hershey and Chase (1952) provide direct evidences in favour of DNA as Genetic Material
There are some indirect evidences also in favour of DNA as Genetic Material
In some organisms RNA is the Genetic Material
Evidences are given by Experiments on TMV 36 8/8/2011
Slide 37 : DNA preRNA RNA
Protein DNA Replication
& Repair Transcription Processing Translation Folding
Assembly
Processing Central Dogma The modern framework of Molecular Biology 37 8/8/2011
Slide 38 : THANK YOU 38 8/8/2011