Molecular Biology: The discovery of DNA, evidences indication DNA as the genetic material, DNA and its types ( A,B and Z DNA), closed super coiled DNA, denaturing and renaturing of DNA, hybridization

Discovery:- The Swiss scientist Friedrich Miescher discovered nucleic acid and fisrt naming it nuclein in 1868. Later in 1889 Altmann introduced the term nucleic acid. 

Nature of Genetic Material:-

•  A genetic material must:-

-   Be able to generate its replica (Replication).

-   Chemically and structurally be stable.

-   Provide the mutations that are required for evolution.

-   Be able to express itself as ‘Mendelian Characters’.

•   DNA versus RNA:-

-   DNA is stable while RNA is unstable. Due to unstable nature of RNA, RNA viruses mutate and evolve faster.

-   For the storage of genetic information DNA is better due to its stability. But for the transmission of genetic information, RNA is better.

-   RNA can directly code for the protein synthesis, hence can easily express the characters. DNA is dependent on RNA for protein synthesis.

-   The 2 DNA strands are complementary. On heating, they separate. When appropriate conditions are provided they come together.

•   RNA World:-

- RNA was the first genetic material.

- It acts as genetic material and catalyst.

- Essential life processes (metabolism, translation, splicing etc) evolved around RNA.

- DNA evolved from RNA for stability.

•   Central Dogma of Molecular Biology:-

- It is proposed by Francis Crick. 

- It states that the genetic information flows from DNA → RNA → Protein.

 

Evidences indicating DNA as Genetic Material:-

1. Griffith’s experiment (Transforming principle):-

•  Griffith used mice & Streptococcus pneumoniae. Streptococcus pneumoniae has 2 strains:-

-  Smooth (S) strain (Virulent): Has polysaccharide mucus coat. Cause pneumonia.

-  Rough (R) strain (Non-virulent): No mucous coat. Does not cause Pneumonia.

•  Experiment:-

-   S-strain → Inject into mice → Mice die

-   R-strain → Inject into mice → Mice live

-   S-strain (Heat killed) → Inject into mice → Mice live

-   S-strain (Heat killed) + R-strain (live) → Inject into mice → Mice die

•  Transforming principle:- He concluded that some, transferred from heat-killed S-strain to R-strain. It enabled R-strain to synthesize smooth polysaccharide coat and become virulent. This must be due to the transfer of genetic material.

 2. Biochemical characterization of transforming principle:-

•  Oswald Avery, Colin MacLeod & Maclyn McCarty worked to determine the biochemical nature of ‘transforming principle’ in Griffith’s experiment.

•  They purified biochemicals (proteins, DNA, RNA etc.) from heat killed S cells using suitable enzymes.

•  They discovered that:-

-   Digestion of protein and RNA (using Proteases and RNases) did not affect transformation. So the transforming substance was not a protein or RNA.

-   Digestion of DNA with DNase inhibited transformation. It means that DNA caused transformation of R cells to S cells, i.e. DNA was the transforming substance.

3. Hershey-Chase Experiment (Blender Experiment):-

•   Hershey & Chase made 2 preparations of bacteriophage - In one, proteins were labeled with S35 by putting in medium containing radioactive sulphur (S-35). In the second, DNA was labeled with P32 by putting in a medium containing radioactive Phosphorous (P-32).

•  These preparations were used separately to infect E. coli.

•  After infection, the E. coli cells were gently agitated in a blender to separate the phage particles from the bacteria.

•  Then the culture was centrifuged. Heavier bacterial cells are formed as a pellet at the bottom. Lighter viral components outside the bacterial cells remained in the supernatant.

•  They found that:-

-   Supernatant contains viral protein labeled with S-35, i.e. the viral protein had not entered the bacterial cells.

-   The bacterial pellet contains radioactive P. This shows that viral DNA labeled with P32 had entered the bacterial cells. This proves that DNA is the genetic material.

Types of Genetic Material:- There are 2 types: -

a. DNA (Deoxyribose Nucleic Acid):-

i. A – DNA

ii. B – DNA

iii. Z – DNA

b. RNA (Ribose Nucleic Acid):-

i. mRNA (mssenger RNA)

ii. tRNA (transfer RNA)

iii. rRNA (ribosomal RNA)

DNA is the genetic material in all cellular organisms. DNA viruses have DNA as genetic material and RNA viruses have RNA as genetic material. All plant viruses are RNA viruses, such as TMV. Whereas animal viruses are DNA or RNA viruses.

DNA and its types ( A,B and Z DNA):- 

Structure of Genetic Material:- A nucleotide is made up of 3 parts: -

a. Nitrogenous Base

b. Pentose Sugar

c.  Phosphate Group

Nitrogenous bases and pentose sugars are linked together by N-glycosidic bonds. Pentose sugars and phosphate groups are interconnected by ester bonds.

a. Nitrogenous Base:-   These are of two types: -

i.  Purines 

ii. Pyrimidines 

i. Purines:- Its structure consists of 2 hetero cyclic rings interconnected. One 6-membered ring is connected to another 5-membered ring.

Examples - A (Adenine), G (Guanine)   

ii. Pyrimidines:- It has only one hetero cyclic ring in its structure. It is a 6-membered ring.

Examples - C (Cytosine), U (Uracil), T (Thymine)

b. Pentose Sugar:- There are 2 types: -

i.  Ribose Sugar

ii. Deoxyribose Sugar

i. Ribose Sugar:- It is a sugar containing 5 carbons. It forms a 5-membered hetero cyclic furanose ring.

Formula - C5H10O5

ii. Deoxyribose Sugar:- It is also a sugar containing 5 carbons. It also forms a 5-membered hetero cyclic furanose ring. The only difference is that it has H at the number 2 carbon instead of OH.

Formula - C5H10O4 

c. Phosphate Group:- It is only phosphoric acid that gives negative charge to the nucleotide.

Ribonucleotides:- Ribose sugars containing nucleotides are called ribonucleotides. It is found in RNA. There are 5 types - AMP, GMP, CMP, UMP, TMP

B – Ribose – P 

Deoxyribonucleotides:- Nucleotides containing deoxyribose sugars are called deoxyribonucleotides. It is found in DNA. There are 5 types - dAMP, dGMP, dCMP, dUMP, dTMP

B – Deoxyribose – P 

Nucleoside:- If the phosphate group is removed from the nucleotide, the remaining part is called nucleoside.

Nomenclature:-

A, B and Z – DNA:- 

i. Helical Sense:- 

RH = Right Handed

LH = Left Handed

ii. Pitch:- The number of base pairs per turn is called Pitch. 1 turn = 360 ° rotation

iii. Helix diameter:-

iv. Distance between base pairs:-

v. Twist in DNA:- Rotation of one base pair with respect to a neighbouring base pair.

vi. Inclination:-

vii. Glycosidic bond configuration:- The relative position of nitrogenous base with respect to sugars.

Structure of B - DNA:-

•  The model of secondary structure of DNA was given by Watson and Crick.

•  DNA is found in the form of double-helical structure with 2 polynucleotide chains coiled around each other.

•  Each polynucleotide chain is a hetero polymer of nucleotides.

•  Both polynucleotide chains are arranged anti-parallel to each other.

•  Both polynucleotide chains are complementary to each other in base sequence.

•  The 1 full turn of spiral chain has 360 ° with 10 base pairs.

•  The length of a full turn is 34A °.

•  There is a distance of 3.4A ° between any 2 neighbouring base pairs.

•  The chain takes a turn of 36 ° at each base pair.

•  The backbone of DNA is a chain made up of sugars - phosphates in which nitrogenous bases form steps.

•  There are 4 types of nitrogenous bases found in DNA -

i. A (Adenine)

ii. G (Guanine)

iii. C (Cytosine)

iv. T (Thymine)

•  There are 2 hydrogen bonds between A and T and 3 hydrogen bonds between G and C.

Chargaff's rule:- The rule states that in dsDNA there is always equality in quantity between the bases A and T and between the bases G and C.

•  Deoxyribose sugars are found in DNA.

•  A molecule of phosphate binds 3 'carbon of one sugar to 5' carbon of another.

•  A phosphodiester bond is found between any 2 neighbouring sugar molecules.

Closed Supercoiled DNA:-

DNA supercoiling:- It refers to the over- or under-winding of a DNA strand. Supercoiling is important in a number of biological processes, such as compacting DNA. Additionally, certain enzymes such as topoisomerases are able to change DNA topology to facilitate functions such as DNA replication or transcription. 

Mathematical expressions:- They are used to describe supercoiling by comparing different coiled states to relaxed B-form DNA.

i. Linking Number (L):- Total number of crosses of two strands of ds DNA. 

ii. Twisting Number (T):-  Total number of helical turns in dsDNA. OR Total number of turns of duplex axis around the helical axis.

iii. Writhing Number (W):- Total number of superhelical turns in dsDNA. OR Total number of turns of duplex axis around superhelical axis.

Corelation:-

L = T+W

Change in L, T & W:-

(ΔL = ΔT + ΔW)

- If there is no supercoiling (i.e. relaxed DNA), then W = 0 and T = L. 

- The example below shows how twist and writhe can vary while linking number remains constant.

Figure: Supercoiled Structure of Circular DNA: This is a 

supercoiled structure of circular DNA molecules with low 

writhe. Note that the helical nature of the DNA duplex 

is omitted for clarity.

Key Points:-

- As a general rule, the DNA of most organisms is negatively supercoiled.

- DNA supercoiling is important for DNA packaging within all cells.

- The coiling of the DNA helix upon itself; can cause disruption to transcription and lead to cell death.

Denaturing and Renaturing of DNA:-

Denaturation:- The hydrogen bonds between two strands are broken giving rise to two single strands. The covalent bonds of DNA remain unaffected.

Denaturation can be brought by various methods:-

i. Thermal denaturation:- Denaturation can be done by heating (>80-90℃). The temperature at which DNA is half denatured is called critical temperature or melting temperature, Tm. Tm is dependent on the length and composition of the DNA bases and other factors such as pH and denaturing agents.

ii. Extreme pH:- At high pH (>11.3), hydrogen bonds between base pairs of two strands of DNA dissociate due to presence of abundant OH– ion. It results in denaturation of DNA.

iii. Other denaturing Agents:- Low salt concentrations destabilise hydrogen bonds. Formaldehyde and urea have a tendency to form hydrogen bonds with nitrogen bases and aldehydes also prevent hydrogen bonding between base pairs by modifying electronegative centres of nitrogenous bases.

Effect of denaturation of DNA:-

i. Increased absorption of UV light at 260nm wavelengths. The rate of absorption is directly proportional to the rate of denaturation

ii. Viscosity decreases, which reflects the physical change occurred in the DNA structure

Renaturation:- It is also known as annealing. When the temperature and pH return to optimum biological level, the unwound strand of DNA rewind and give back the dsDNA.

- If the DNA is not completely denatured, the renaturation process is fast and a one-step process, but if the DNAs are completely denatured then the renaturation process occurs in a two-step process. First complementary strands come together by random collision and then rewinding takes place forming a double helix.

- Renaturation occurs when the denatured DNAs are cooled in suitable conditions. Renaturation also depends on temperature, pH, length and constituents of the DNA structure. The renaturation rate is directly proportional to the number of complementary sequences present.

- With renaturation, absorption of UV (260nm) decreases and viscosity increases again.

Hybridization:- DNA is usually found as a double-stranded molecule. The two strands bind to one another in a complementary fashion by a process called hybridization. Naturally, when DNA is replicated, the new strand hybridizes to the old strand. In the laboratory, we can make small pieces of DNA, designed to screen for the presence or absence of certain DNA or RNA molecules in the cell. It also plays an important role in a procedure called polymerase chain reaction, known as PCR, where we amplify specific regions of the gene, and this is used in clinical testing.