One of the most important event of discoveries in molecular biology is by James Watson and Francis Crick who published article in the journal Nature on April 25, 1953, revealed the structure of DNA. Their discovery was the conclusion of a decade of intense research following Oswald T. Avery, Colin MacLeod, and Maclyn McCarty’s demonstration that DNA, not protein, is the hereditary molecule. Today, the history of DNA is often told as though it started with these fundamental discoveries. Therefore, in this “Discoveries in Molecular Biology” we are going to discuss some of the series of previous experiment that lead to the discovery of DNA. Without the scientific foundation provided by these pioneers, Watson and Crick may never have reached their groundbreaking conclusion of 1953: that the DNA molecule exists in the form of a three-dimensional double helix.

Table of Contents

• Friedrich Miescher: 1869
• Feulgen: 1912
• Phoebus Aaron Theodor Levene: 1919
• Frederick Griffith:1928
• Oswald Avery: 1944 (DNA, and the transformation of biology)
• Beadle and Tautum: 1940 (One-gene–one-enzyme hypothesis)
• Chargaff’s Rule: 1944
• Roger Harriot: 1951
• Alfred Hershey and Martha Chase: 1952
• James Watson and Francis Crick
• Meselson and Stahl: 1958

Friedrich Miescher: 1869

In 1869 Swiss physiological chemist Friedrich Miescher identified thematerial which he called “nuclein” from the nuclei of human white blood cells.

• He collected the hospital bandages and studied the pus cells (leukocytes).
• He identified some phosphorus containing compounds that were acidic in nature.
• As they were being identified within the nucleus of the cell, these compounds were named as nuclein. (The term “nuclein” was later changed to “nucleic acid” and eventually to “deoxyribonucleic acid,” or “DNA.”).

Feulgen: 1912

Feulgen, in 1912, discovered that when DNA is subjected to hydrolysis and Schiffs reagent is added to it, then a staining reaction occurs resulting in reddish purplish colour (Feulgen reaction). This specificity of Feulgen reaction paved the way for a number of quantitative studies, especially cytophotometry, involving the measurement of amount of light transmitted through Feulgen stained preparations. Through such studies, it has been established that the amount of DNA in the nuclei of cells of an organism is always constant, except for gametes (n) and polyploid cells.

Phoebus Aaron Theodor Levene: 1919

Levene work leads to major discoveries like:-

• He was the first to discover the order of the three major components of a single nucleotide (phosphate-sugar-base).
• The first to discover the carbohydrate component of RNA (ribose).
• The first to discover the carbohydrate component of DNA (deoxyribose); and
• The first to correctly identify the way RNA and DNA molecules are put together.

Levene proposed a tetranucleotide structure, in which the nucleotides were always linked in the same order (i.e., G-C-T-A-G-C-T-A and so on). Indeed, many new facts and much new evidence soon emerged and caused alterations to Levene’s proposal but Levene’s polynucleotide structure was accurate in many regards.

Frederick Griffith:1928

In 1928 Frederick Griffith carried out the classical experiment where he infected mice with two separate strains of Pneumococcus. This is also the important discoveries in molecular biology.

• One strain that contained capsular glycoproteins, had smooth surface (type III- ‘S’ strain) and was virulent while the other strain had rough cell surface (type II ‘R’ strain) and was non-virulent.
• He showed that infection with live bacteria of ‘S’ strain resulted in disease and lead to death of mice while the mice were not killed if infected with ‘R’ strain. Further, the mice survived when infected with heat-killed bacteria of ‘S’ strain.
• However, the co-infection with bacteria of heat killed ‘S’ strain and the live ‘R’ strain together resulted in death of the animals.
• This suggested that some factor(s) present in the killed ‘S’ bacteria was capable of transforming the ‘R’ bacterium resulting in its (the bacteria of non-virulent ‘R’ strain) conversion to a virulent strain.
• Furthermore, when the DNA from heat killed ‘S’ strain was injected into the mice along with live ‘R’ strain bacteria, the combination was found to be virulent and lethal.
• It clearly demonstrated that DNA from killed ‘S’ strain bacteria was able to transform the ‘R’ strain bacteria and this was responsible for the pathogenicity of the transformed bacteria.

However, doubts were expressed about the homogeneity and purity of the DNA preparation (many of the refined techniques for purification of subcellular components were not available at that time) and a number of scientists still thought that the contaminating proteins might have been responsible for transformation of the ‘R’ bacteria resulting in the death of mice. The dilemma continued till 1943 and many workers still believed that proteins were the genetic material.

Oswald Avery: 1944 (DNA, and the transformation of biology)

Their analysis included elemental analysis as well as physical characterizations such as optical properties, ultracentrifugal behaviour, electrophoretic migration and diffusion properties. Further, they also showed that removal of even the last traces of lipids and proteins from the transforming principle has no effect on its effectiveness as transforming factor. The treatment of this factor with either proteases or RNases did not result in loss of its activity. But treatment with DNase resulted in total loss of its transforming property.

On 1 February 1944, the Journal of Experimental Medicine published one of the breakthrough discoveries of the 20th century: Oswald Avery (1877–1955), together with his colleagues Colin MacLeod (1909–1972) and Maclyn McCarty (1911–2005), reported that the transformation of pneumococcus bacteria from one type to another occured through the action of a ‘transforming principle’ that they identified as being composed of ‘sodium desoxyribonucleate’ or DNA.

Beadle and Tautum: 1940 (One-gene–one-enzyme hypothesis)

Clarification of the actual function of genes came from research in the 1940s on Neurospora by George Beadle and Edward Tatum, who later received a Nobel Prize for their work.

Experimental Procedure

1. They first of all irradiated Neurospora to cause mutations and then tested cultures from ascospores for interesting mutant phenotypes.
2. They identified numerous auxotrophs (these are strains that cannot grow on a minimal medium unless the medium is supplemented with one or more specific nutrients).
3. In each case, the mutation that generated the auxotrophic requirement was inherited as single gene mutation each gave a 1:1 ratio when crossed with a wild type.
4. One set of mutants strains required arginine to grow on a minimal medium. These strains provided the focus for much of Beadle and Tatum’s further work.

• First, they found that the mutations mapped into three different locations on separate chromosomes, even though the same supplement (arginine) satisfied the growth requirement for each mutant. Let’s call the three loci the arg-1, arg-2, and arg-3 genes.
• Beadle and Tatum discovered that the auxotrophs for each of the three loci differed in their response to the chemical compounds ornithine and citrulline, which are related to arginine.
• The arg-1 mutants grew when supplied with ornithine, citrulline, or arginine in addition to the minimal medium.
• The arg-2 mutants grew on either arginine or citrulline but not on ornithine.
• The arg-3 mutants grew only when arginine was supplied.

It was already known that cellular enzymes often interconvert related compounds such as these. On the basis of the properties of the arg mutants, Beadle and Tatum and their colleagues proposed a biochemical model for such conversions in Neurospora.

Note: For better understanding, compare the above table with the above figure

• The arg-1 mutants have a defective enzyme X, so they are unable to convert the precursor into ornithine as the first step in producing arginine. However, they have normal enzymes Y and Z, and so the arg-1 mutants are able to produce arginine if supplied with either ornithine or citrulline.
• The arg-2 mutants lack enzyme Y, but have normal enzyme Z, and so the arg-2 mutants are able to produce arginine if supplied with citrulline.
• and the arg-3 mutants lack enzyme Z. So the arg-3 mutants are able to produce arginine if supplied with only with arginine.
• Thus, a mutation at a particular gene is assumed to interfere with the production of a single enzyme.
• The defective enzyme, then, creates a block in some biosynthetic pathway. The block can be circumvented by supplying to the cells any compound that normally comes after the block in the pathway.
• Now, look at this biochemical model presentation

This model, which has become known as one-gene-one-enzyme hypothesis was the source of the first exciting insight into the functions of genes: genes somehow were responsible for the function of enzymes, and each gene apparently controlled one specific enzyme.

Let’s Summarize Beadle and Tautum Results

• Biochemical reactions in vivo (in the living cell) consist of a series of discrete, step-by-step reactions.
• Each reaction is specifically catalyzed by a single enzyme.
• Each enzyme is specified by a single gene.

Chargaff’s Rule: 1944

Chargaff, an Austrian biochemist, after studying Oswald Avery and his colleagues work at Rockefeller University, which demonstrated that hereditary units, or genes, are composed of DNA, he launch a research program that focussed around the chemistry of nucleic acids. In his research plan Chargaff set out to see whether there were any differences in DNA among different species.

After developing a new paper chromatography method for separating and identifying small amounts of organic material, Chargaff reached two major conclusions.

• First, he noted that the nucleotide composition of DNA varies among species. In other words, the same nucleotides do not repeat in the same order, as proposed by Levene.
• Second, Chargaff concluded that almost all DNA–no matter what organism or tissue type it comes from–maintains certain properties, even as its composition varies. In particular, the amount of adenine (A) is usually similar to the amount of thymine (T), and the amount of guanine (G) usually approximates the amount of cytosine (C). In other words, the total amount of purines (A + G) and the total amount of pyrimidines (C + T) are usually nearly equal. (This second major conclusion is now known as “Chargaff’s rule.”).

Roger Harriot: 1951

In 1951 Roger Harriot determined the structure of bacteriophages.

• The phages have needle like structure with a head and a sharp pointed tail.
• Nucleic acid was localized in the head region.
• During infection the tail of phage pierces the host bacteria and facilitates the transfer of phage nucleic acid into the host cell, but the outer envelope (mainly made up of the proteins) remains outside the infected host.
• The phage nucleic acid was implicated to be responsible for the host cell transformation.
• This observation gave further support to the theory that DNA was the genetic material.

Alfred Hershey and Martha Chase: 1952

Though all these studies what we have discussed so far made it clear that DNA is the genetic material and is responsible for the transfer of virulent characters in original experiment of Griffith, but some doubts still persisted. Finally in 1952 Alfred Hershey and Martha Chase with their double labeling experiment proved that DNA is the genetic material.

Experimental Procedure

1. They radiolabeled the DNA of phage T2 with ³²P and its protein with ³⁵S.
2. Bacteria were infected with the double-labeled phage and the fate of radioactivity was carefully followed.
3. It was found that while ³²P (i.e. phage DNA) was taken up by the host cells and was detected inside the infected host cells, ³⁵S (i.e. the phage proteins) did not enter the host cells and remained outside in the culture medium.

This confirmed that DNA was the genetic material that was responsible for the transformation of the host bacteria beyond any doubt. It is now well established that DNA is the genetic material for all the living cells except in certain viruses.

James Watson and Francis Crick

The actual race for the discovery of DNA structure began in 1950. The 1950’s saw 3 separate groups working intensively on the DNA structure:

• Maurice Wilkins and Rosalind Franklin at King’s College in London

• Linus Pauling, an American Chemist at the California Institute of Technology

• James Watson and Francis Crick at Cambridge

The credit for the discovery of double helix goes to Watson and Crick at Cambridge in 1953. The double helix model by these two satisfies all conditions. The double helix model also took into account the results obtained by scientists Erwin Chargaff in USA and Rosalind Franklin in UK.

The Watson and Crick structure of DNA (B-DNA) has following features:-

1. DNA consists of two antiparallel polynucleotide strands that turn around a common axis with a right handed twist to form a DNA double helix.
2. The ideal B-DNA helix has the diameter of 20 Å. The bases are 3.4 Å apart along the helix axis and the helix rotates 36° per base pair.  Therefore, the helical structure repeats after 10 residues on each chain, i.e., at intervals of 34 Å. It can also be said as that each turn of the helix contains 10 nucleotide residues.
3. The phosphate and deoxyribose are present on the outer side of the helix, whereas the nitrogenous bases (purine and pyrimidine) occur in the centre.
4. The planes of the nitrogenous bases are perpendicular to the DNA helix axis.
5. Each nitrogenous base is hydrogen bonded to a base present on the opposite strand (like; A with T and G with C). This leads to the formation of a planar base pair.
6. The planes of the sugars are almost at right angles to those of the bases.
7. The hydrogen bonding between the bases takes place either between the –NH₂ group of one base and =O of the other base or between =NH of one base and the –N of the other base. For stable bond formation, the distance between N-N is 0.30 nm and that between O-N is 0.28-0.29 nm.
8. The DNA double helix when runs antiparallel generates major and minor grooves.
9. When the ion such as Na⁺ and the relative humidity is >92%, fiibers of DNA assume the so called B- Conformation
10. B-form of DNA is the most stable structure for a random sequence of DNA.

Rosalind Franklin at King’s College, London carried X-ray diffraction analysis of DNA using technique developed by Maurice Wilkins. X-ray pattern obtained when a crystallized DNA fibre is bombarded with X-rays revealed that DNA is a helix with two regular periodicities of 3.4 Å and 34 Å along the axis of the molecule.

Taking into consideration all these aspects, Watson and Crick deduced that the only structure that fitted the facts, was “the double helix”. For their discovery of ‘the double helix’ Watson and Crick shared the 1962 Nobel Prize with Maurice Wilkins.

Meselson and Stahl: 1958

After the discovery of DNA double helix, one big question concerned was DNA replication. Meselson and Stahl reasoned that if by any method the parental DNA be labeled that it could be distinguished from the daughter DNA, the replication mechanisms could be recognised. They put the hypothesis that,

• If DNA replication is semiconservative, then after a single round of replication, all DNA molecules should be hybrids of parental and daughter DNA strands.
• If replication is conservative, then after a single round of replication, half of the DNA molecules should be composed only of parental strands and half of daughter strands.
•  IIf replication is dispersive, DNA replication results in two DNA molecules that are mixtures, or “hybrids,” of parental and daughter DNA. In this model, each individual strand is a patchwork of original and new DNA.

Meselson and Stahl used “heavy” nitrogen (¹⁵N) to differentiate parental DNA from daughter DNA,. This isotope contains an extra neutron in its nucleus, giving it a higher atomic mass than the more abundant “light” nitrogen (¹⁴N).

Experimental Procedure

1. They began by growing E. coli in medium, containing a “heavy” isotope of nitrogen, ¹⁵N for many generation. Therefore, after growing in ¹⁵N medium for several generations, the bacteria contained only ¹⁵N-labeled DNA.
2. Then Meselson and Stahl abruptly changed the medium to one containing ¹⁴N as the sole nitrogen source.
3. Therefore, after growing in ¹⁵N medium for several generations, all the DNA synthesized by the bacteria would incorporate ¹⁴N, rather than ¹⁵N, so that the daughter DNA strands would contain only ¹⁴N.
4. As the bacteria continued to grow and replicate their DNA in the ¹⁴Ncontaining medium, samples were taken periodically, and the bacterial DNA was analyzed using technique of equilibrium density-gradient centrifugation.

Equilibrium Density-Gradient Centrifugation

In this technique,

• A DNA sample is mixed with a solution of cesium chloride (CsCl₂).
• Due to high-speed centrifugation the CsCl₂ forms a gradient, and the DNA migrates to the position where the density of the DNA is equal to that of the CsCl₂.
• If the DNA sample contains molecules of different densities, they will migrate to different positions in the gradient.
• Because ¹⁵N has a greater density than ¹⁴N labeled DNA. The higher-density (¹⁵N) DNA will sediment to a different position than the lower density (¹⁴N) DNA.
• Hybrid DNA molecules, containing both ¹⁵N and ¹⁴N, will sediment at an intermediate density, depending on the ratio of heavy nitrogen to light.

Results

• Before any DNA replication had occurred in the ¹⁴N-containing medium, all DNA sedimented as a single species, corresponding to ¹⁵N-labeled DNA.
• As DNA replication proceeded, the amount of (¹⁵N)-DNA decreased, and a second DNA species, consisting of hybrid DNA molecules containing ¹⁵N- and ¹⁴N-labeled strands, appeared. DNA collected after completion of the first round of replication was found to sediment with the second species.
• When the DNA produced during a second round of replication was analyzed, two distinct species were observed. One corresponded to hybrid molecules; the other corresponded to ¹⁴N-labeled DNA.
• With each subsequent round of replication the proportion of hybrid DNA decreased as the amount of ¹⁴N-labeled DNA increased.