PRESENTED BY YA DVEER SINGH M PHA RM 1ST YEA R DEPT.

OF PHARMA COLOGY DHA RWAD

INTRODUCTION:

DNA is the functional unit of life That controls the cellular activity and growth Made up of many genes As long as molecular structure of gene was unknown there was no way to think about geneprotein relation DNA (genes) consist of Deoxyribonucleic acid

Some discovery in nucleic acids

Investigator
* Freidrich Miescher(1868) * Discovery : Isolated substance from pus celland called it nuclein. * Altman (1889) : Named the substance nucleic acid. * E. Chargaff (1950) :Discovered the equivalence in base ratios in DNA. * R. Franklin and M.F.H. Wilkins (1952) : Studied x-ray diffraction and revealed that DNA has a helical structure. * J.D.Watson, F.H.C. Crick (1953) : Proposed the double helical model for DNA.

DNA
It is defined as, long chain of poly nucleotide sequence running anti parallel to each other, two strands held by weak hydrogen bonds, which has capacity to self replication and expression, which is responsible for the trait and hereditary characters. DNA is a macromolecule of a high molecular weight of the order of millions of daltons. It is the mastermolecule of a biological system and forms the genetic material.

Location Of DNA
DNA is sited on chromosomes (genes sitting side by

side) Improvement in microscope made possible to see genes Isolation of pure chromosomes is much more informative about DNA DNA occurs in the central region of the spherical or polyhydral head of the virus In prokaryotes ,it occurs in the nucleoid as their chromosome . In eukaryotic plant and animal cells the DNA occurs in the chromosomes found in the nuclei. DNA is normally found inside the nuclei of the cells.

Two Main Chromosomal Components Were Almost Invariably Found

1. A Deoxyribonucleic acid (DNA)
major constituent of nucleus so called nucleic acid (1869 by Fredric Miescher) 2. A small class of +vely charged proteins called histones Basic in nature, neutralizes the acidity of nucleic acid In 1920 DNA specific purple dye is developed by Robert Feulgen ( sited on chromosome)

Cells Contains Both RNA As Well As DNA

Cells have a second kind of nucleic acid

called RNA RNA located primarily in the nucleus concentrated as a dense granules (Nucleoli) Found abundance in cytoplasm also Both DNA and RNA resemble proteins in that they are constructed from many smaller building blocks linked end to end

 Chemistry of DNA:- cemical analysis of DNA has

revealed the presence of three different chemical substances namely pentose sugars, phosphate groups and nitrogeneous bases. Adenine pairs with Thymine with two hydrogen bonds (A=T) and Guanine pairs with cytosine with three hydrogen bonds(G=C).This specific base pairing is called complementary base pairing.

 Each nucleotide contains a phosphate

group, sugar and purine or pyrimidine base When nucleotide linked together in large number then called as polynucleotide The sugar for RNA was known to be different from that of DNA

 Two purines and two pyrimidines are

found in both DNA & RNA

Diameter of DNA
DNA has asymmetrical from its behavior in
solution Direct measurement of DNA is possible only with electronic microscope Shows many thousand in length & 20 thickness All DNA molecules are unbranched Each nucleotide base was just over 3

 The helix makes one complete turn at every 34A

along its length containing 10 nucleotides. The distance between two nucleotides in a helix is 3.4A. The length of the DNA molecule varies from chromosome to chromosome and species to species . The amount of DNA per nucleus of a species is measured in terms of picograms ex: in human beings DNA per nucleus is 5.6 pg and total length is 174cm(1 pg=31cm) Nucleosides are the combination of pentose sugar (deoxyribose) and base Nucleotides are the combination of a pentose sugar, a nitrogen base and a phosphate group.

Characteristic diameter (D) bp/turn

ADNA

BDNA

Z-DNA

2.6 nm 2.0 nm 1.8 nm 11 10.4 12

degrees rotation/bp +32.7 +34.6 -30.0 axial distance/turn 2.8 nm 3.4 nm 4.5 nm axial distance between 0.25 0.33 0.38

DNA & RNA linked by 5-3 phoshodiester bonds

DNA Has Highly Regular Shape

DNA chains are very regular Contains repeating nucleotide
sequences 4 different bases can arrange any order along the back bone individuality Dry DNA appear irregular white fluffs of cotton With water becomes tachy and fibers x-ray studies of Para-crystalline pattern chows helical configuration

The Double Helix Is Held By Hbonding

DNA Self Replication

makes an exact copy of itself. The copy is called the
replica, and the process is called replication . Whenever DNA replicates, it produces two daughter molecules which are identical to each other and also to the parent Occur prior to the cell division (S- phase) The parent strands forms template for the new strand Replication is semi-conservative During cell division the DNA molecule molecule. During replication, the two strands of DNA helix unwind dueto the dissolution of hydrogen bonds at specific sites called ORI (Origin of Replication) sites by the action of certain enzymes. The region at which the DNA opens is called the eye or replication fork.

Replication Of DNA
A topoisomerase, which introduces negative

supercoils A helicase, which unwinds and splits the DNA ahead of the fork. Thereafter, single-strand binding proteins (SSB) swiftly bind to the separated DNA, A primase (in prokaryotes, RNA polymerase II), which generates an RNA primer to be used in DNA replication. A DNA holoenzyme, which in reality is a complex of enzymes that together perform the "actual" replication, i.e., the polymerization of nucleotides.
DNA polymerase I -- DNA polymerase III

Replication Of DNA

Super Coiled DNA

DNA AS PRIMARY GENETIC MATERIAL

Germ plasm theory proposed by august weisman in 1904 states that the reproductive cell or gametes are the germ cell. These germ cell are carried to the descendents generation after generation. Later it was belived that chromosomes present in the gametes carry and transmit the hereditary characters. Chomosomes cocatain nucleic acid and proteins. Molecular biologists have proved that nucleic acids located in the chromosomes are the hereditary materials and chromosomes are the vehicles of heredity now Now it has been accepted that deoxyribe nucleeic acid [DNA]is the geneticmaterial and the DNA forms genes which control all the activities of micro organisms as well ashigher organisms.

CONT
The DNA carries the genetic information
form gereration to generation. This concept may be proved by a number of experiments which will form 1.Direct avidences and 2.Indirect evidences.

1.DIRECT EVIDENCES
Experiments on microorganism give
concrete direct evidence show that DNA is the genetic material they are the following 1. Griffiths bacterial transformation experiment 2.Bacteriophase infection 3. Bacterial conjugation 4. Transduction.

1.GRIFFITHS BACTERIAL TRANSFORMATION EXPERIMENT.

Fredrick Griffith [1928] has reported a phenomenon in a bacterium Pneumococcus which is now known as genetic bacterial transformation. It is a direct evidence to show that DNA is the genetic material. There are two strains of pneumococcus bacteria.they are [i] virulent and [ii] avirulent. Virulent strains [pathogenic] have smooth [s] glistening polysaccharide capsules and cause pneumonea diseases . The avirulent [nonpathogenic] stains of pneumococcus have duli, rough [R] appearance without capsule and are unable to cause pneumonia disease . Smooth and rough stains are genetically determined. Conti

CONT.
When the virulent strains are injected into the host. Mice
they kill then by causing pncumonia. When avirulent strains are injected into mice, there is no effect and they do not cause the discase Heat killed virulent stain of pneumococcus is injected with a mixture of effect and it does not cause the disease. When mice are injeted with a mixture of avirulent and heat killed virulent pneumococcus bacteria, the mice die due to pneumonia. BY conducting post mortom of dead mice it was found out that, they ware killed because of the virulent bacteria from this result it is evedent that heat killed virulent bacteria are responrible for the transformation of avirulent once into virulent once with smooth capsule. Thenomenon of transformation is no as Griffith,s effect or bacterial transformation.

Fig. 2.2: Frederick Griffiths Transformation Experiment - 1928

Griffith effect

transforming principle demonstrated with Streptococcus pneumoniae

Griffith hypothesized that the transforming agent was a IIIS protein.

Cont
O.T. Avery, C.M.MC LEOD and M.MC Carthy
[1944] identified that this permanent change in viruence is due to the transformation of a chemical substance, cell free extract of heat killed virulent strains also produces the same effect. When the extract treated with DNA ase, the transformation property was lost. This is because, DNA ase enzyme destroys the DNA.The same extract treated with protease enzyme to remove the protein, has the transformation ability . Thus it is proved directly that DNA is the genetic material in pneumococci.

2. BACTERIOPHASE INFECTION
A. Hurshey and M.Chase 1952 by using
radio active tracars provided a direct proof that DNA is the genetic material in a bacteriophase bacteriophase is a virus infecting bacteria.T2 bacteriophase attacks the bacterium E scherichia coil. Conti

Bacteriaophage Infection

CONT
This expriment based on the fact, expressed by Harriou [1951], that
virus as such never entry the bacteria; only the tail contacts the outer memorane of the bacteria.with the help of enzyme, the virus forms a hole through which viral DNA flows into the host bacterial cells. A groups of T2 phase was labelled with radioactive isotope sulphur, is a component of protein only; Another group of same T2 phase was labelled with P32 by growing them on e. coli already labelled with P32 phosphorusis one of the components of DNA and not of protein.

835 by growing them on E coli already labelled with 835 sulphur

CONT.
These two groups of labelled phages are allowed
to infect separate cultures of E coli. After obtaining the first progeny in about 20 minutes, the daughter phages are tested for radioactivity. Phages labelled with p32 showed radio active p32 in their daughter phages also. But phages labelled with s35 showed no radioactivity in their daughter phages. This result indicates that DNA is the genetic material, which has been transmittedfrom the parent to the daughter generation. But the labelled protein is not passed on to the daughter phages, since protein coat has never entered the bacterium.

3. BACTERIAL CONJUGATION
Bacterial conjugation gives direct evidence that
the DNA is the genetic material. Conjugation is a process of temporary union of two bacteriafor the trasfer of genetic material. Laderberg and Tatum [1946] found out that a f+[male] E. coli bacterium conjugate with a F-[female] E.coli. F+ cell has a double stranded circular DNA in the cytoplasm called the plasmid. F- Cell has no such plasmid. After conjugation,through the newly formed conjugation tube one strandof the plasmid [DNA] is trasmitted to the F- cell. This DNA converts the F- cell in to F+ cell. Thus a female bacterium is converted in to a male bacterium. This proves that DNA is the genetic material.

BACTERIAL CONJUGATION

4. TRANSDUCTION

Cont
Transduction is a process by which transfer of genetic material takes place from one bacterium to the other. Some bacteria are resistant to antibiotic Streptomycin and other bacteria are sensitive to this antibiotic. The resistace is due to the presence of dominant gene in the chromosomal DNA of the bacteria. The sensitive bacteria do not posses this dominant gege.

Cont
When the lysogenic bacteriophages grow on
streptomycin resistantbacteria, the viral DNA gets a fragments of a bacterchromosomal DNA containing the streptomycin resistant gege. When these phages with a bit of bacterial chromosomes are injected in to sensitive bacterial cells, the sensitive bacteria receive the chromosomal DNA containing the resistant gege from the phages. Due to this trasfer of gege, the sensitive bacteria become resistant to streptomycin. This transmission of resistant is correlated with the transfer of chromosomalDNA. Hence, DNA is consideredas the genetic material.

2. Indirect Eviderccs
Bacteria, viruses and other microorganisms
show direct evidences to prove that DNA is the genetic material. But higher organisms show only indirect evidences to support this fact DNA is the genetic. The indirect evidences to 1.Localization 4.Effect OF Mutagenes 2.Amount of DNA 5.Composition 3.Stability 6.Integrity

1.LOCALZATION
ALL Cells of the living organisms have
DNA paticularly located in the chromosomos feulgen reaction is an evidence to show that major component of chromosome is DNA which performs the duty of the genetic material.

2.Amount of DNA
THE quantity of DNA depnds on the number of
chromosome and the sets of chromosomes. Sex cells have haploid number of chromosomes.MALE And female sex cells fuse together and become zygote with diploid number of chromosomes. In polyploid cells the amount of DNA increases with the increase in the number of sets of chromosomes The effect of extra chromosomes such as Klinefelters syndromes and 21 trisomy also prove that DNA is the genetic material.

3.Stability
Studies on chromosomes reveal that
genes are arranged in a linear manner in all the chromosomer. Genes are nothing but the series of polynucleotides. Genes are stable compounds. Genes are stable compounds. Genes DNArepicate without committing any mistake in each and every cell division. Even then stable rarely it changes causing mutations.

4.Effect of Mutagens
Mutation may occur spontancously or
through the effect of mutagens such as radiation, chemicals. Etc. mutations alter the chemical structur of DNA mutations are inherited by the offspring, which show phenotypic variations. These effects of mutagenes also give evidence to the fact DNA is the genetic material.

5.composition
Structure of DNA is the same in all
microorganisms and other higher organisms. composition of DNA is similar in organisms of the same species and they interbreed. It is different in organisms belonging to different species and they do not interbreed.

6.Integrity
DNA maintains integrity during cell metabolism
and also during embryonic development of the organisms but due to crossing over variations have been produced within the species. Mutation distrupts the proper functioning of the DNA. The direct evidences of microorganisms and indirect evidences of other higher organisms prove that DNA as the stable genetic material which will survive in this biosphere with all sorts of environmental hazards.

FUNCTIONS OF DNA
1:DNA controls all the metabolic activities of the

cell directly or indirectly DNA synthesizes RNA and directs the synthesis of protein in the cytoplasm. DNA can undergo mutation and recombination which are changes at the genetic level. The synthesis of RNA by DNA is called as transcription. This is called as the heterocatalytic function .