4.1 The Discovery of DNA:

Modern understanding of DNA has evolved from the discovery of nucleic acid to the development of the double-helix model.

In 1869, Friedrich Miescher began working with white blood cells which are the major component of pus from infections. He collected a lot of pus from bandages at the local hospital. He used a salt solution to wash salt solution to wash off the pus off the bandages.

When he added a weak alkaline solution to the cells, the cells lysed and nuclei precipitated out of the solution.

From the cell nuclei, he isolated a unique chemical substance to which he called nuclein. Chemically, nuclein has high phosphorus content. Moreover it showed acidic properties. Hence it was named as nucleic acid.

By the early 1900s, we knew that Miescher's nuclein was a mix (mixture) of proteins and nucleic acids. There are two kinds of nucleic acids. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid).

> At the time of Mendel, the nature of those 'factors' regulating the pattern of inheritance was not clear. Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are the two types of nucleic acids (polymers of nucleotides) found in living systems. DNA acts as the genetic material in most of the organisms.

> RNA acts as a genetic material in some viruses, mostly functions as a messenger. RNA also functions as adapter, structural and in some cases as a catalytic molecule.

The Structure of DNA

DNA, or deoxyribonucleic acid, serves as the fundamental genetic material in living organisms. It consists of a long polymer of deoxyribonucleotides, forming a double helix structure.

Composition and Structure

DNA comprises two complementary strands of deoxyribonucleotides.

These strands run antiparallel to each other, with their nitrogenous bases held together by hydrogen bonds.

The four types of nucleotides that make up DNA are adenine (A), thymine (T), cytosine (C), and guanine (G).

Length and Characteristics

The length of DNA is typically measured by the number of nucleotides or base pairs it contains.

Each nucleotide pair, consisting of a complementary A-T or C-G base pair, is referred to as a base pair (bp).

The length of DNA varies among different organisms:

Bacteriophage Φ174: 5386 nucleotides

Bacteriophage lambda: 48502 base pairs (bp)

Escherichia coli (E. coli): 4.6 x 10⁶ bp

Haploid content of human DNA: 3.3 x 10⁹ bp

Structure of Polynucleotide Chain

A polynucleotide chain is composed of nucleotides, each consisting of three main components: a nitrogenous base, a pentose sugar, and a phosphate group.

Components of a Nucleotide:

• Nitrogenous Base:

There are two types of nitrogenous bases - purines (adenine and guanine) and pyrimidines (cytosine, uracil, and thymine). Cytosine is common to both DNA and RNA. Thymine is found in DNA, while uracil replaces thymine in RNA.

• Pentose Sugar:

The pentose sugar in RNA is ribose, while in DNA, it is deoxyribose.

• Phosphate Group:

A phosphate group is attached to the 5'-OH of the pentose sugar through a phosphodiester linkage.

Formation of Nucleotides:

• A nitrogenous base is connected to the 1'C pentose sugar via an N-glycosidic linkage, forming a nucleoside.

• When a phosphate group is linked to the 5'-OH of a nucleoside through a phosphodiester linkage, a nucleotide is formed.

• Two nucleotides are connected through 3'-5' phosphodiester linkages, forming a dinucleotide. Additional nucleotides can join to form a polynucleotide chain.

RNA Specifics:

• In RNA, each nucleotide residue has an additional -OH group at the 2' position of the ribose sugar.

• Uracil replaces thymine in RNA, binding to adenine through complementary base pairing.

Structure of DNA

In 1953, James Watson and Francis Crick proposed the double helix model for the structure of DNA, based on X-ray diffraction data produced by Maurice Wilkins and Rosalind Franklin. Their proposition was also influenced by Erwin Chargaff's observations, summarized as follows:

• Purines and pyrimidines are always present in equal amounts: A+G=T+C.

• The amount of adenine equals that of thymine, and the amount of guanine equals that of cytosine: A=T and G=C. However, the total amount of A+T may not equal G+C.

• The base ratio A+T/G+C may vary between species but remains constant within a given species.

Key Features of DNA's Double-Helix Structure:

1. Polynucleotide Chains:

DNA consists of two polynucleotide chains, with the sugar-phosphate backbone forming the exterior and the bases projecting inward.

2. Anti-parallel Polarity:

The two chains have anti-parallel polarity, meaning if one chain has the polarity 5′→3′, the other has 3′→5'.

3. Base Pairing:

Bases in the two strands are paired via hydrogen bonds (H-bonds) to form base pairs (bp).

Adenine pairs with thymine via two H-bonds, and guanine pairs with cytosine via three H-bonds.

This results in purines always pairing with pyrimidines, maintaining a uniform distance between the two strands.

4. Right-Handed Coiling:

The two chains coil in a right-handed fashion, with a helical pitch of 3.4 nm and approximately 10¹⁰ base pairs per turn. This results in approximately 0.34 nm between each base pair.

5. Stacking of Base Pairs:

The plane of one base pair stacks over the other in the double helix, contributing to the stability of the helical structure along with H-bonds.

4.2 The Genetic Material is a DNA:

By the early 1900s, geneticist knew that genes control the inheritance of traits, that genes are located on chromosome and that chemically chromosomes are mainly composed of DNA and proteins. Initially, most geneticists thought that protein are large, complex molecules and store information needed to govern cell metabolism. Hence it was assumed that proteins caused the variations observed within species.

On the other hand DNA thought to be small, simple molecule whose composition varied little among species. Over the time, these ideas about DNA were shown to be wrong. In fact DNA molecules are large and vary tremendously within and among species.

Variations in the DNA molecules are different than the variation in shape, electrical charge and function shown by proteins so it is not surprising that most researchers initially favored proteins as the genetic material.

Over a period of roughly 25 years (1928-1952), geneticists became convinced that DNA and not protein, was the genetic material. Let us study three important contributions that helped cause this shift of opinion.

Griffith's experiments :

In 1928, a British medical officer Frederick Griffith performed an experiment on bacterium Streptococcus pneumoniae that causes pneumonia in humans and other mammals.

Griffith used two strains or two genetic varieties of Streptococcus to find a cure for pneumonia, which was a common cause of death at that time. The two strains used were :

i. Virulent, smooth, pathogenic and encapsulated S type.

ii. Non-virulent, rough, non-pathogenic and non-capsulated R type.

Griffith conducted four experiments on these bacteria.

1. Mice injected with strain R bacteria survived.

2. Mice injected with strain S bacteria developed pneumonia and died.

3. Mice injected with heat-killed strain S bacteria survived.

4. In fourth experiment, he mixed heat-killed S bacteria with live bacteria of strain R and injected to mice. The mice died and Griffith recovered large numbers of live strain S bacteria from the blood of the dead mice.

In these four experiments, something had caused harmless strain R bacterium to change into deadly S strain bacterium.

Griffith showed that the change was genetic. He suggested that genetic material from heat-killed strain S bacterium had somehow changed the living strain R bacterium into strain S bacterium.

Griffith concluded that the R-strain bacterium must have taken up, to what he called a "transforming principle" from the heat-killed S bacterium, which allowed R strain to get transformed into smooth-coated bacterium and become virulent. 

Avery, McCarty and MacLeod's experiment:

In 1944, after some 10 years of research and experimentation, U. S. microbiologists Oswald T. Avery, Colin M. MacLeod and Maclyn McCarty (all at Rockefeller University in New York) first evidence to prove the DNA is a genetic material (transforming principle), through the experiments.

- DNA, RNA, proteins, and other substances were extracted from S cells.

- These substances were mixed with heat-killed S cells and live R cells.

- The experiment identified which substance could transform harmless R cells into deadly S cells.

Only DNA was able to transform harmless strain R into deadly strain S.

Finally, Alfred Hershey and Martha Chase (1952) proved that DNA is the genetic material and not proteins, by using bacteriophages.

Hershey - Chase Experiment:

1. Bacteriophage Selection:

• Hershey and Chase chose bacteriophages, viruses that infect bacteria.

• These viruses are composed of primarily two components: DNA and protein.

2. Radioactive Labeling:

• Experiment 1:

o Bacteriophages were grown in a medium containing radioactive phosphorus-32 (³²P).

o Phosphorus is a component of DNA, so the phage's DNA became radioactively labeled.

• Experiment 2:

o Bacteriophages were grown in a medium containing radioactive sulfur-35 (³⁵S).

o Sulfur is a component of proteins, so the phage's protein coat became radioactively labeled.

3. Infection of E. coli Bacteria:

• The radioactively labeled phages were allowed to infect E. coli bacteria.

4. Blending and Centrifugation:

• After infection, the mixture of bacteria and phages was blended. This process detached the phage coats from the bacterial cells.

• The mixture was then centrifuged to separate the heavier bacterial cells from the lighter phage parts.

5. Analysis of Radioactivity:

• Experiment 1:

o The bacterial pellet, which contained the infected bacteria, was found to be radioactive.

o This indicated that the radioactive DNA had entered the bacterial cells.

• Experiment 2:

o The bacterial pellet was found to contain very little radioactivity.

o This suggested that the radioactive protein coats had remained outside the bacterial cells.

6. Conclusion:

• The results of the experiments demonstrated that DNA, not protein, is the genetic material.

• The DNA from the phage entered the bacterial cell and directed the production of new phages.

In other words, sometime after infection, radioactivity for 'P' and 'S' was tested. Only radioactive 'P' was found inside the bacterial cell, indicating that DNA is the genetic material.