Paper : 03 Structure and Function of Biomolecules II Module: 01 Introduction to Nucleic acids-Historical Perspective

Principal Investigator Prof. Sunil Kumar Khare, Professor, Department of Chemistry, IIT-Delhi

Paper Coordinators Prof. Sunil Kumar Khare, Department of Chemistry, IIT-Delhi & Prof. M.N. Gupta, Department of Biochemical Engineering and Biotechnology, IIT-Delhi

Content Writer Prof. Sunil Kumar Khare, Department of Chemistry, IIT-Delhi

Content Reviewer Dr. Arun Goyal Professor Department of Biotechnology Indian Institute of Technology Guwahati

North Guwahati, 781 039 Assam, India

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STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry INTRODUCTION TO NUCLEIC ACIDS-HISTORICAL PERSPECTIVE

DESCRIPTION OF MODULE

Subject Name Biochemistry

Paper Name STRUCTURE AND FUNCTION OF BIOMOLECULES II

Module Introduction to Nucleic acids-Historical Perspective Name/Title

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STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry INTRODUCTION TO NUCLEIC ACIDS-HISTORICAL PERSPECTIVE

1. Objectives

• History of genetic material

• Historical experiments-DNA as a hereditary material

• Understanding the timeline of DNA discovery

2. Concept Map

DNA discovery-historical perspectives

Evidences towards the Initial search for DNA Timeline of DNA discovery DNA as hereditary material

Historical Experiments towards the discovery of DNA

Avery, MacLeod and Hershey-Chase Griffith's experiment McCarty Experiment Experiment

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STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry INTRODUCTION TO NUCLEIC ACIDS-HISTORICAL PERSPECTIVE

3. History of Genetic Material

3.1. Search of DNA

The history of deoxyribonucleic acid (DNA) or genetic material originated with some basic and fundamental discoveries. Depiction of DNA dates back to 1869, with its initial finding by Friedrich Miescher. On February 26, 1869, he shared the isolation of this mysterious substance (now known as DNA), with his uncle Wilhelm His (who was a renowned physician and professor of anatomy and at University of Basel; he had discovered neuroblasts and coined the term “dendrite) in a letter. In this letter he wrote, “In my experiments with low alkaline liquids, precipitates formed in the solution after neutralization that could not be dissolved in water, acetic acid, highly diluted hydrochloric acid or in a salt medium and therefore do not belong to any known type of protein”.

Fig. 1. (A) Friedrich Miescher (1844–1895) and (B) Wilhelm His (1831–1904)

He reported the isolation of a material in the nuclei of human white blood cells, which was weakly acidic in and whose function was not yet known. He named this material "nuclein". In another few years, with more research Miescher was able to separate nuclein into protein and nucleic acid components. Further research in the last half of the 20th century, the implications of nuclein as genetic material and function as the bearer of hereditary characteristics was discovered.

3.2. Evidences towards the DNA as hereditary material-historical experiments

Till many years after Miescher’s death, nuclein had received very little attention around the world. Majority of researchers were convinced that protein is the genetic material because enormous hereditary information can only be stored in complex substances. While DNA was made up of only four different molecules, proteins were composed of 20 different amino acids. Scientist thus remained confident about proteins having more complex structures and storing genetic information.

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STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry INTRODUCTION TO NUCLEIC ACIDS-HISTORICAL PERSPECTIVE

3.2.1 Griffith’s Experiment

Fig 2: Frederick Griffith

In the year 1928, Frederick Griffith, who was a Medical Officer in British ministry of Health, performed several experiments with different strains of Diplococcus pneumoniae (now named as ). These strains were of two types i.e. Smooth (S) and Rough (R).

i. Smooth (S): Smooth cells were found virulent (infectious) as it caused in vertebrates (especially human and mice). There was mainly due to the polysaccharide capsules, which also led to formation of smooth colonies on agar plate.

ii. Rough (R): In this type, cells lacked polysaccharide capsules and were non-virulent (non- infectious). Due to absence of polysaccharide capsule, they showed dull rough colonies on agar plates.

Figure 3. Pictorial representations of Rough and Smooth cells This specific feature allows the microbiologist to easily differentiate the virulent and non-virulent strains using simple microbiological culture techniques. Each strains of Diplococcus having several serotypes like S-I, S-II, S-III, R-I, R-II, R-III etc. The specificity of the serotype again depends upon the

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STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry INTRODUCTION TO NUCLEIC ACIDS-HISTORICAL PERSPECTIVE

detailed chemical structure of the polysaccharide capsule, which can be identified through different immunological techniques.

Griffith selected serotypes S-III and R-II for his work, which led to the generation of the new concept of genetic material. Griffith already knew the fact that pneumonia is only caused by living smooth cell. If the heat killed virulent bacteria will be injected in mice there should ideally be no sign of infection as similar as the non-virulent live cells. Strategically Griffith designed his experiment, which is provided in Table 1 below.

Table 1. Experimental sets in Griffith’s experiment

Set Experimental set up

Set-1 Live cells of virulent S-III

Set-2 Live cells of non-virulent R-II

Set-3 Heat killed cells of virulent S-III

Set-4 A mixture of both heat killed cells of virulent S-III and live cells of non-virulent R-II

Griffith performed his experiments by using above sets of bacteria and injecting them into mice and found the below results (Fig 4):

a) When he injected with live cells of virulent S-III strain into the mice; the mice developed pneumonia and finally died.

b) When live cells of non-virulent R-II bacteria were injected into the mice, the mice did not develop any sign of illness and survived, thus confirming the nature of non-virulent strain.

c) Heat killed cells of the virulent S-III bacteria were also not able to develop pneumonia into mice and thus mice survived. He concluded that after heat treatment bacteria must be dead due to which no infection occurs.

d) When Griffith injected a mixture of both heat killed cells of virulent S-III and live cells of non- virulent R-II into the mice; the mice suffered from pneumonia and died. After dissection of mice, it was observed that mice blood cells were having both R-II and S-III strains of bacteria. He concluded that some factor must have been passed from heat killed virulent S-III strain to the live non-virulent R-II strains, which empowered them with the ability to produce 6

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry INTRODUCTION TO NUCLEIC ACIDS-HISTORICAL PERSPECTIVE

polysaccharide capsule and make them virulent type. Griffith hypothesized that the transforming factor was an S-III protein and called the phenomena as Transforming Principle.

Fig.4. Griffith’s transformation experiment

3.2.2 Avery, MacLeod and McCarty Experiment

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STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry INTRODUCTION TO NUCLEIC ACIDS-HISTORICAL PERSPECTIVE

Fig. 5. Avery, McCarty and MacLeod.

Several other physician and bacteriologists like Henry Dawson in 1931, Lionel J. Alloway in 1933 further carried on work in the support of Griffith’s work.

After 16 years of Griffith’s work, in 1944 Ostwald T. Avery, Colin MacLeod and Maclyn McCarty reported that DNA of the virulent S-III strain served as genetic material and was responsible for the Griffith’s result.

They started their work with large culture volume of virulent S-III strain. The cells were pelleted down by centrifugation and killed by heating at 65 oC. Further pellets were homogenized with the supernatant and extracted with the detergent deoxycholate, after which they obtained a soluble filtrate having transformation capability. To remove the protein from the soluble active filtrate, several rounds of chloroform extraction was done and subsequently to remove the polysaccharides , enzymatic digestion was also carried out. At last by ethanol precipitation, a fibrous mass was collected which had the ability to induce the transformation in non-virulent R-II strain.

To solidify their finding, they planned to eliminate all the contaminants like protein, RNA etc which is present in the soluble active fraction.

In their first experiments, soluble extract were directly tested for transformation with live non- virulent R-II type strain and injected in mice. It was observed that mice developed the infection and died. Both S-III and R-II type strains were found in the blood of mice.

In the next experiment, to remove the proteins, soluble extract were treated with proteases and further transformation assay was carried out along with non-virulent R-II type strain. As a result it was observed that mice died due to pneumonia and both strains were present. Further to remove the RNA, Avery, MacLeod and McCarty treated the soluble extract with RNase. The mixture of RNase treated extract and non-virulent R-II strain was injected in mice and it was found that mice developed the infection.

The final inference came with the experiment where DNA digesting enzyme DNase was used to treat the soluble extract. The mixture of DNase treated extract and R-II strain was inject and it was found that no transformation occurs in non-virulent R-II strain. Further, they emphasized that once transformation occurs, the polysaccharide capsules will be formed in the successive generation. Therefore, transformation is heritable. This confirmed the finding that DNA is the transforming factor, which acts as a hereditary genetic material.

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STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry INTRODUCTION TO NUCLEIC ACIDS-HISTORICAL PERSPECTIVE

Fig. 6. Schematic diagram of Avery, MacLeod and McCarty’s experiment

3.2.3 Hershey-Chase Bacteriophage Experiment

(A) (B)

Fig 7: (A) Alfred Hershey in Physiology or Medicine in 1969, (B) 9

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry INTRODUCTION TO NUCLEIC ACIDS-HISTORICAL PERSPECTIVE

In support of DNA as the genetic material, another good piece of evidence was provided during the study of bacteriophage T2 and its target bacterium Escherichia coli by Alfred Hershey and Martha Chase in 1952. The T2 are simply referred as Phage, a kind of which contain DNA as core and surrounded by the protein coat. The electron micrograph of T2 phage is shown in Fig.8.

Fig.8. Electron micrograph of a typical phage T2.

The life cycle of phage T2 is briefly described in Fig.9. T2 Phages were first adsorbed on the bacterial cell surface and subsequently injected its chromosome inside the bacterial cell. Inside bacterial cell, degradation of the chromosome was initiated by phage specific enzymes. Following the infection step, the viral information “commandeers” the cellular machinery of the host and directs viral reproduction. Within a very short time, many new phages emerged from a single bacterial cell and the bacterial cell is lysed. It seemed that some molecular components of the phage (whether DNA, or protein or both) enter inside the bacterial cell and directs the viral reproduction.

As it is well known fact that DNA contains phosphorus (P) and not sulphur(S), whereas in proteins only sulphur (S) is present, Hershey and Chase strategically designed their experiments where they had used radioisotope 32P and 35S to label DNA and protein respectively. This was a key determining

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STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry INTRODUCTION TO NUCLEIC ACIDS-HISTORICAL PERSPECTIVE

point in their experiments. They grew E. Coli in two separate radioactive medium containing 32P and 35S and phages were allowed to attack the radiolabeled E. Coli bacteria.

Fig.9. Life cycle of phage T2

As a result, the progeny of Phages were also become radiolabeled. In some of the phages, progeny DNA gets labelled and some phages protein coat gets labelled with their respective radioisotope. When these radiolabeled phases were allowed to attack on general without radiolabeled E. coli bacteria, the progeny showed a remarkable result in that phases whose DNA was radiolabeled got a radiolabeled progeny but the phages whose protein was labelled got a non-radiolabeled progeny. This ultimately confirms that none other than the DNA serves as hereditary genetic material.

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STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry INTRODUCTION TO NUCLEIC ACIDS-HISTORICAL PERSPECTIVE

Fig.10. Brief summary of Hershey & Chase experiment

3.3 Timeline of DNA discovery

Apart from the above mentioned three classical experiments, several researchers have also contributed significantly in the search of DNA and nucleic acid. Some of them are listed in Table-2.

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STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry INTRODUCTION TO NUCLEIC ACIDS-HISTORICAL PERSPECTIVE

Table 1: Discovery of DNA: Timeline

Year Scientist Scientist’s contribution 1861-1870 1865 Gregor Mendel Conducted breeding experiments on pea to discover that the hereditary characteristics are transmitted in units and that traits were inherited based on specific Mendel’s laws 1866 Ernst Haeckel Proposed that within the cell, it is the nucleus which contains factors responsible for transfer of genetic traits. 1869 Friedrich Miescher DNA molecules were isolated from cells for the first time. 1871-1880 1871 Friedrich Miescher, Felix Described an acidic substance from cell’s nuclei, Hoppe-Seyler, and P. Plo´ sz called “nuclein”. First publication describing nuclein.

1881-1890 1882 Walther Flemming Described chromosomes behaviour during cell division; described the process of mitosis. 1884– Oscar Hertwig, Albrecht von Provided the evidence that basis for inheritance is 1885 Kflliker, Eduard Strasburger, contained within the cell’s nucleus. and August Weismann 1889 Richard Altmann “nuclein” was renamed as “nucleic acid” 1900 Carl Correns, Hugo de Vries, Rediscovery of Mendel’s work and Erich von Tschermak 1901-1910 1902 Walter Sutton Gave chromosome theory. He observed that during meiosis, the segregation pattern of chromosomes was similar to that of Mendel’s. 1902 Theodor Boveri and Walter Postulated that heredity units are positioned on the Sutton chromosomes. 1902– Archibald Garrod Proposed that genetic defects lead to enzymatic 1909 losses and hereditary metabolic diseases. 1909 Wilhelm Johannsen The word “gene” is coined 1910 and He and his group studied heredity using fruit flies students (Drosophila) as a model. They show that genes are

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STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry INTRODUCTION TO NUCLEIC ACIDS-HISTORICAL PERSPECTIVE

carried by chromosomes and discover the genetic linkage 1911-1920 1913 Alfred Sturtevant and Thomas Produced the first genetic linkage map (for the fruit Hunt Morgan fly Drosophila) 1921-1930 1928 Frederick Griffith Postulated the “transforming principle”- hereditary material were permitted to be transferred from one type of bacteria to another . 1929 Discovered components of the DNA and the four bases adenine (A), cytosine (C), guanine (G), and thymine (T). 1941-1950 1941 and Edward Gave “one gene-one enzyme” hypothesis-one gene is Tatum responsible for the production of one enzyme. 1943 First pictures of DNA taken through X-Ray diffraction. Shows that DNA has a periodic structure. 1944 Oswald T. Avery, Colin Demonstrated that DNA, and not protein forms the MacLeod, and Maclyn McCarty basis of Griffith’s “transforming principle”. Also showed that hereditary information is transferred through DNA. 1944 Barbara McClintock Discovered “jumping genes”- that genes are able to change position on chromosomes 1949 Colette and Roger Vendrely and Discovered that the amount of DNA in the nuclei of Andre´ Boivin germ cells is half of that is found somatic cells. This parallels the decrease in the number of chromosomes during gametogenesis and provides evidence that DNA is the genetic material. 1950 Demonstrated that within a given species, the bases in DNA are always present in fixed ratios (A=T; G=C) 1951-1960 1952 Alfred Hershey and Martha Confirmed that DNA and not proteins is the genetic Chase material. Used bacteriophage to demonstrate that during infection, viral DNA and not viral proteins enters the bacteria, thus leading to the presence of viral DNA in progeny of virus particles also. 1953 and Maurice Used X-Ray diffraction to establish that DNA has a 14

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry INTRODUCTION TO NUCLEIC ACIDS-HISTORICAL PERSPECTIVE

Wilkins repeating helical structure. DNA crystals were photographed using X-Ray. 1953 and Discovered the double helical structure of DNA 1955 Jo Hin Tjio Showed humans have 46 chromosomes 1956 Discovered the enzyme (DNA polymerase) responsible for replication of DNA 1957 Francis Crick Proposed the “central dogma” (DNA to RNA to protein) and speculated that three bases in DNA will specify one amino acid in a protein. 1958 Matthew Meselson and Discovered the semi conservative replication in DNA 1961-1970 1961– Robert W. Holley, Har Gobind Discovery of the genetic code 1966 Khorana, Heinrich Matthaei, Marshall W. Nirenberg, and colleagues. 1968– , Hamilton Smith, Described the first restriction enzyme 1970 and 1971-1980 1972 Paul Berg Established the concept of recombinant DNA. Used restriction enzymes to insert piece of DNA of one species into another to produce the first DNA recombinant molecule. 1973 First animal gene cloned 1977 Frederick Sanger, Allan Maxam, Developed methods for sequencing the DNA. and Walter Gilbert 1977 Richard Robert and Phil Sharp First introns (interruptions in eukaryotic genes) discovered 1981-1990 1981 Scientists successfully insert inherited genes to laboratory animals (for. e.g., transgenic mice) 1982 Genebank database is formed that is open to public. 1982 First recombinant drug, human insulin, appears in the market. 1983 Mapping of the first disease gene (for Huntington’s disease) 1983 Kary Mullis Invented polymerase chain reaction (PCR) method for amplification of the DNA in vitro. 1987 First comprehensive genetic map is discovered by digesting DNA with restriction enzymes 15

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry INTRODUCTION TO NUCLEIC ACIDS-HISTORICAL PERSPECTIVE

1989 Discovery of microsatellites, which are repetitive DNA sequences used as genetic landmarks to distinguish between people 1990 Sequencing of the human genome initiated 1991-2000 1995 Publication of the complete sequence of the genome of bacterium Haemophilus influenza, first free-living organism 1996 Publication of the complete genome sequence of the yeast S. cerevisiae, first eukaryotic organism. 1996 Mouse genetic map completed 1997 The first animal clone, Dolly, the sheep 1997 Sequencing of the E. coli genome 1998 Publication of the complete genome sequence of the first roundworm Caenorhabditis elegans, a multicellular organism. 1998 M. bacterium sequenced 1999 Publication of the sequence of the first human chromosome 22). 2000 Publication of the complete sequences of the genomes of the fruit fly Drosophila and the first plant, Arabidopsis. 2001 onwards 2001 Publication of the complete sequence of the human genome. 2002 Publication of the complete genome sequence of the first mammalian model organism, the mouse. 2003 Completion of the Human Genome Project.

Summary:

In this lecture we learnt about

 The initial discovery of nuclein  How different scientist proved that protein is not containing the genetic information  How DNA was discovered as genetic material  The timeline of DNA related discoveries

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STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry INTRODUCTION TO NUCLEIC ACIDS-HISTORICAL PERSPECTIVE