The Breakthrough of 1944

A model of the structure of DNA. It is DNA that carries the hereditary information for all of life. DNA is located in the chromosomes of cells but chromosomes also consists of proteins. Before 1944, was it protein or DNA that carried genetic information?(Enzymlogic)

A model of the structure of DNA. It is DNA that carries the hereditary information for all of life. DNA is located in the chromosomes of cells but chromosomes also consists of proteins. Before 1944, was it protein or DNA that carried genetic information?(Enzymlogic)

DNA or Deoxyribonucleic acid is the biochemical that carries genetic information and has the ability to replicate itself because of its molecular structure which is a double helix. DNA is composed of monomers or building blocks called nucleotides and each nucleotide is composed of a sugar, deoxyribose, a phosphate, and a nitrogen compound called a base and there are four bases adenine, thymine, guanine, and cytosine. The variable sequences of the bases is what determines the biological information and it does that because of the genetic code which is a universal code found in all life forms where up to three bases code for one or more amino acids or the monomers of protein molecules. Information flows from DNA to a related molecule RNA or ribonucleic acid which is composed of ribose, a different form of sugar, and four bases but with the base uracil instead of thymine along with adenine, cytosine, and guanine. RNA carries the information of the DNA molecule or gene and with the information present in RNA it goes through a cellular structure called a ribosome and along with information with two other RNA molecules ribosomal RNA and transfer RNA is where the process of protein synthesis occurs. The ribosome reads each three bases and it takes up to three bases or codons to code one or more amino acids and as the ribosome reads the messenger RNA or the RNA that carries information on polypeptide sequences or long sequences of proteins composed of amino acids as specified by the gene, each codon is read in combination with the ribosomal RNA and transfer RNA which actually carries the amino acids and along with the ribosome, an amino acid is linked to the next amino acid until the ribosome reaches the end of the messenger RNA indicating STOP and the polypeptide chain or protein molecule is formed. This process of protein synthesis along with the genetic code is universal in all life forms of this biosphere and this is made possible by the nucleic acids, DNA which is the genetic molecule which carries information off all kinds of proteins along with the ability to copy that information from one generation to the next and RNA which are the actual functioning nucleic acid molecules that are involved in protein synthesis.

 

This is the definition of how life functions in the biosphere and the genetic information in DNA is present in all life forms on earth. In fact it is textbook knowledge that for most of us we seldom don’t give much thought of how that fact was arrived but I think we really should for it illustrates how the scientific method, when carefully applied to this one problem in biology and that is the chemical basis of heredity, was arrived which lead to the facts as genes composed of DNA. Before 1944, it was known that cell nuclei consisted of chromosomes and that chromosomes are made out of DNA and proteins and it was established, through research with fruit flies, that genes carry the necessary hereditary information which are located on chromosomes. If chromosomes carry genes which are composed of DNA and protein then what was carrying the genetic information? Was it DNA or was it protein?

 

The thinking was that proteins are what composed genes while DNA was only the scaffolding, providing support for the genes and the reason why is that proteins are composed of 20 amino acids and any protein is composed of various sequences of amino acids while DNA is composed of just four bases. This was the kind of thinking where protein molecules since they can do any biochemical function then it was not unreasonable to think otherwise until a three man team that did research combining biochemistry and microbiology challenged that and proved that DNA carries hereditary information.

 

At first, biochemists and geneticists could not accept the fact that DNA is the hereditary substance but eventually as the results were repeated at first to find any flaws in the experimental procedure but when carefully done and later other independent experiments confirmed the same results that DNA carries biological information and can be passed from one generation to the next which lead to further investigation into nucleic acids culminating in the discovery of the double helix.

 

This experiment carried out by biologists, Oswald Avery, Colin McCleod , and Maclyn McCarty was the experiment that provided evidence that deoxyribonucleic acid is the molecule central to genetics was the most crucial in the history of biology and in my opinion, important in the history of science and when studying this experiment, reveals a balance of observation, experiment, and sceptisism along with repeated confirmations which are all part of the scientific method and in also how out of competing explanations, this one narrowed nucleic acids as those responsible for heredity and thus set molecular biology on the path while changing the concept of the gene and previously I’ve written in a blog “The Changing Concept of the Gene” how a discovery like that has given us the definition of gene as that composed of nucleic acids.

 

To understand the full implications of the Avery experiment, we need to understand this in a much broader context and we start from the initial isolation of nucleic acids in 1869 together with the formation of what is called the tetranucleotide hypothesis which assumed that nucleic acids only act to support genes as protein molecules to its slow but total demise after the 1944 experiment.

 

The Isolation of Nucleic Acids

 

Nucleic acid was originally isolated by the Swiss biochemists Friedrich Miescher in 1869 and was called “nuclein” for the obvious fact it was isolated from cell nuclei. Investigations of nucleic acids revealed that in some respects, it was different than proteins( and indeed they are) in that it was composed of four basic building blocks that are different than amino acids where a an amino acid consists of a central carbon bonded to a carboxyl group and an amino group with a hydrogen and a variable side chain but the monomers of nucleic acids or nucleotides are composed of sugar, phosphate, and base. Later as genes were identified as units of hereditary and were located on specific regions of chromosomes, biochemical analysis revealed that chromosomes consisted of both proteins and nucleic acids and this lead to an the question: If chromosomes carry genes and if a section of a chromosome carries a specific region of heritable information which is the genes then are genes composed of nucleic acids or proteins? It was assumed that if all of life are composed of proteins then it stood to reason to assume that genes are composed of proteins until later evidence proved otherwise and the reason for the belief of genes as protein molecules was mainly due to a hypothesis that was later proven to be false by this biochemist named Phoebus Levene. His hypothesis which denied nucleic acids the role as genes but for the sake of understanding how scientists were led to the fact of genes as nucleic acids which was only performed by the Avery research team, it is worth considering the hypothesis (now discredited) tetranucleotide hypothesis which was developed after the structure of nucleotides was established but also lead to the belief that nucleic acids had a passive role instead of active role in heredity since this will illustrate when one hypothesis is formed to explained given results such as the biochemical aspects of heredity can new findings modify or even a working hypothesis if the new findings can give a new explanation and thus a new hypothesis in place of an old.

 

The Tetranucleotide Hypothesis

 

What was the tetranucleotide hypothesis? It basically states that nucleic acids are composed of nucleotides which consists of sugar phosphate and base and since there are four bases in DNA which are adenine, thymine, guanine, and cytosine while in RNA the sugar is riboses, unlike deoxyribose in DNA and uracil in place of thymine. Because of the four bases, it was determined by Levene that a molecule of nucleic acid consisted of four bases all bonded to one another or that with the four bases, a DNA or RNA molecule would consist of only four bases along with the sugar and phosphate forming a four structure molecule or “tetramer”. It was Levene, who did some significant work in biochemistry in not only determining the structure of nucleic acids as composed of four bases, a deoxyribose or ribose sugar, and phosphate, which defines the nucleotide, but in other works on the biologically important monomers such as sugars, proteins, and lipids. His investigations into the molecules of life could not be overlooked for he did major important contributions to biochemistry so his investigations in biochemistry were crucial yet when it came to moving from chemistry to genetics, his tetranucleotide hypothesis was in some respects something of an obstacle since if his hypothesis was proven true, then if nucleic acids only had this sequence of bases in this order

 

ATCGATCGATCG…..

 

clearly this  would not code for any biological information, at least if DNA or RNA was a tetramer. Also one had to consider the fact that chromosomes both contain proteins and nucleic acids but what was the carrier of hereditary information? As the tetranucleotide hypothesis was being formulated by Levene and was gaining acceptance for the time being, then it was proteins that were candidates for genes. How did scientists accepted the opposite conclusion that DNA are the genes? Before 1944, and because of the tetranucleotide hypothesis, DNA would be the supporting material where genetic and biochemical activities were the result of proteins. Further biochemical research confirmed that enzymes which are proteins speed up biochemical reactions; they can make as well as break but to confirm the truth or falsity of the tetranucleotide required experiments using model organisms and one such model organism turned out to be bacteria. They reproduce quickly when given the right nutrients and temperature and they could be broken down into their constituent biomolecules and through specific techniques the molecules can be separated from one another and can be characterized.

 

The path that lead to the demise of the tetranucleotide started with an experiment with a strain of bacteria that caused pneumonia, which was a deadly disease back then and by the 1920’s  few scientists were attempting to understand the bacteria that caused pneumonia and along the way, vaccines were being developed to combat pneumonia. It was during an investigation of bacterial infections in mice that bacteria and in life in general all shared a common hereditary substances but investigations with bacteria could easily give the clue since bacteria are simple to study than multicellular organisms and it resulted of a curious observation in mice that became the foundation for the 1944 discovery.

 

                                                                                                                                                                                                             Studies of Pneumonia Paves the Way

 

 

Pneumonia, which is a respiratory disease of the lungs is caused by a bacterium Streptococcus pneuominae, and since it was discovered that this species of bacterium causes pneumonia, what was it about this bacterium that results in pneumonia? When studying this bacterium on how it causes diseases, how to prevent from causing pneumonia is also important and before antibiotics became available, vaccines for each bacterial disease such as pneumonia were being developed. Studies of Streptococcus pneuominae revealed that there are two forms or strains of this bacterium and it is evident when it is grown in a petri dish of agar. One strain called the smooth strain, so called because when grown as a colony on the agar surface, the appearance of the colonies has a smooth shape and is called S for smooth and it is the smooth form that is virulent or disease causing. Another strain forms colonies that look rough and are called R strain and R strain tend to be less virulent or non virulent in comparison to the S strain.

 

Around 1928, a microbiologist named Frederick Griffith was in charged of developing vaccines for pneumonia and he distinguished the two strains S and R, the disease causing and the non disease causing strains of Streptococcus pneuominae respectively. He was able to do this by using these two strains taken from patients infected with pneumonia and using laboratory mice to distinguish between the S and R strains. When S strains are injected into mice, the mice develop the signs and symptoms of pneumonia and die but when mice are injected with the R strains they survive since the R strains are non virulent. Autopsy of the mice revealed that the dead mice infected with S strains are identical to the S strains of Streptococcus pneuominae prior to injection and likewise autopsy of killed mice (intentionally by scientists but not the disease of course!) revealed that R strains are identical to the R strains prior to injection.

 

What would happen if using the S strains, the bacteria that form the S strains are subjected to heat for a period of time? When bacteria from the S strain are heated, the thermal energy destroys the S strain making it non virulent, like the R strain, and since Griffith was concerned about techniques for producing vaccines, and one such kind of vaccine, the inactivated vaccine is where a bacterium or virus is rendered useless either by heat or by addition of certain chemicals and once a virus or bacterium is injected into an organism, the immune system or system that defends the organisms against infectious agents like bacteria, produce proteins called antibodies which recognize and destroy the invading foreign microbes. Vaccines like an inactivated vaccine can do just that and that was the goals of Griffith’s experiment. Back to what happens when heat killed S bacteria are injected into live mice. If a mouse receives heat killed S strain, the mouse after injection does not die from pneumonia but survives and that is because the heat destroys the the outermost cells of S strain bacteria and S strain are virulent because of a specific kind of carbohydrate that surrounds the cell and it is this cell wall that causes the disease of pneumonia by damaging the surrounding lung tissues.

 

When S bacteria are heated, the thermal energy destroys the cell wall and so is unable to destroy lung tissue and that is what happened when heat killed S bacteria were injected into healthy mice. The mice remained healthy more or less because of the heat damaged cell walls the S bacteria had. Also recall that bacteria of R strain are non virulent and that is because they lacked that carbohydrate coat so if R strain bacteria were injected into mice, the mice survive.

 

In each separate experiment, R strains cause no pneumonia in mice and likewise heat killed S strain also caused no pneumonia either in mice in comparison to the deadly S forms. What would happen if a mixture of R strains and heat killed strains were injected into mice? The goal as you know is to outline procedures for making vaccines and based on the previous experiments, R strain bacteria are non virulent and so are the heat killed S strain. If a mixture of R and heat killed S strain is injected into mice then the hypothesis is that after injection the mice would survive and when the experiment was carried out, something unexpected happen.

 

When mice were injected with a mixture of heat killed S strain and R strain, the mice did not survive and in fact died from this mixture! The mice with the mixture developed pneumonia and died and examination of the blood of the dead mice revealed live S strain bacteria. How was that possible?

 

Somehow the harmless R strain had to pick up a substance from the destroyed S cells and in doing so, the R strain , the only living cells in the mixture, turned into the virulent S strains in a process that Griffiths (1928) called “transformation” since whatever it was caused the transformation from R to S resulting in pneumonia in mice.

 

Just what was the chemical nature of this substance that is responsible for transformation? Not surprisingly Griffiths believed that the transforming substance was protein and as summarized in regards to his experiments, Griffiths clearly stated (1928) ” By S substance, I mean that specific protein structure of the virulent pneumococcus which enables it to manufacture a specific soluble carbohydrate. This protein seems to be necessary as material enabling the R form to build up into the S form.” Griffith in a way was almost on the right track in identifying what would be the genes or carrier of information for turning live R forms into S forms. Of course as biologists believed in the validity of the tetranucleotide hypothesis, it was also no surprise that Griffith though that the “transforming principle” as it was then called was composed of proteins.

 

The phenomenon of transformation in bacteria is nonetheless real and some versions of Griffith’s experiment were repeated in order to confirm the fact that R strains can turned into S strains and Dawson and Sia (1930) confirmed transformation of pneumococcus from R to S but in test tubes and not using mice.

 

Of course these kinds of experiments do not prove that the transforming principle or genes are composed of nucleic acids or proteins for these experiments reveal that in specific conditions a strain can mutate into another strain but tells us nothing about the kind of molecule that does the transformation.

 

What is needed was to do another version of this experiment and such an experiment was performed by Oswald Avery, Colid Macleod, and Maclyn McCarty and their experiment was a different version of the Griffith’s experiment but without the use of lab mice and this experiment combined microbiology, genetics, and biochemistry and it was the technique of biochemistry that could characterize what the transforming principle was and when the experiment was done, the results were at first so surprising that few biologists accepted the results yet it paved for the overthrow of the tetranucleotide hypothesis and slowly was replaced by a new paradigm that is now accepted today and that is DNA is the genetic material of life.

 

                                                                                                                                                                                                           The Avery Experiment and its Conclusions

 

For 10 years, Oswald Avery and his colleagues, Colin Macleod and Maclyn McCarty not only confirmed Griffith’s experiment in vitro or observation of biochemical reactions in lab glassware instead of in vivo or using live organisms to test the effects of a biochemical reaction or in the Griffith’s experiment, how strains of pneumonia will either kill or not live mice. Not only the transformation phenomenon of bacteria notably Pneumococcus was observed but additional techniques were used to determine just what specific biochemical was responsible for the transformation.

 

The first step in the isolation of the biochemical compound that is the transforming principle was to isolate that particular substance in the first place. This was no easy task since a bacterial cell such as Pneumococcus is composed of proteins, nucleic acids, lipids, and carbohydrates and in varying amounts.

 

The substance that was isolated was nucleic acids and at first because of the thinking that proteins are genes, it did not seem possible that the nucleic acids could cause the transformations but proving that nucleic acids could cause the transformation was possible and one hand to consider the observations of R strains turning into S strains and to prove that DNA is the transforming principle, required the use of enzymes or protein molecules which can break up various biological polymers. Enzymes for breaking down carbohydrates, lipids, proteins, and nucleic acids were used in this one particular experiment.

 

Enzymes that break down polymers are also specific for the kinds of molecules that they break down. For example, enzymes that break down carbohydrates will only break down carbohydrates, enzymes that break down proteins, only proteins and so on. The use of enzymes proved to be very invaluable and it was through enzymes that the chemical nature of the transforming substance could finally be pinned downed precisely.

 

Since the S strains consists of a carbohydrate coat while the R strain lacks this coat, and if the transforming principle or the gene is located inside the cell that results in turning a R form into a S form, and since the inside of a cell is mostly protein then using an enzyme to break down proteins, then fragmented proteins would includes fragmented genes if the genes are composed of proteins so according to this hypothesis, if genes and hence the transforming principle are proteins then using a specific set of enzymes to break down protein molecules which were trypsin and chymotrypsin, the destroyed protein molecules would prevent transformation from R to S.

 

First in order to observe transformation in vitro, first a colony of S strain bacteria was grown in liquid nutrient media which favors the growth of S bacteria, which in this case is Streptococcus pneumonia. Next, the culture of S Streptococcus was broken down into its submolecular components consisting of proteins, DNA, RNA, carbohydrates, and lipids. All the cells were broken down into its molecules and at this point can the enzyme for destroying the transforming principle be applied. If trypsin and chymotrypsin were added, these enzymes will destroy all proteins, including the transforming principle if it is protein and after the addition of those enzymes the fluid was transferred to a colony of R strain Streptococcus the destroyed protein molecules should not turn the R into S bacteria and this was the hypothesis that if the transforming principle is protein, then trypsin and chymotrypsin would prevent the genes from transforming R into S. After that step of the experiment was done and the treated fluid was added to the R bacteria, it turned out that transformation did occur and the R bacteria turned into S bacteria!

 

How could this be? It came as a surprise that the transforming principle was anything but proteins so if proteins are not the transforming principle then the choice was narrowed down to the nucleic acids which are RNA and DNA. Suppose RNA is the transforming principle then treating RNA with enzymes that break down RNA or ribonuclease was added to a separate flask of liquefied S bacteria and if genes are RNA then the destroyed RNA would stop transformation. The opposite occurred and R was transformed into S. This was narrowed to the last molecule which is DNA and using an enzyme called deoxyribonucleodepolymerase or DNase for short, then either adding DNase which will break down DNA will either prevent transformation or if possible won’t make a difference to the transformations.

 

After the solution with destroyed DNA was added to R strains, suddenly no transformation occurred. Unlike the other treatments where trypsin and chymotrypsin which destroys proteins and ribonucleases which destroys RNA but allowed transformation to go unabated, the addition of DNase prevented transformation. What could this mean?

 

To prove that it is not any other impurities that are responsible for transformation, the experiment was repeated by Avery and colleagues under the same experimental conditions and the results were the same: DNases is the only enzyme that destroys the DNA molecule which when added to R strain Streptococci does not allow transformation but treatment of other enzymes does not stop transformation from occurring so it was likely according to Avery that DNA or deoxyribonucleic acid is the transforming principle. You may think that this was something of an Eureka moment but Avery and his colleagues were careful experimenters and Avery himself approached his results and interpretations with skepticism (indeed skepticism is a crucial thing in the scientific method). It was checking the results numerous times that the evidence favored DNA as the transforming principle.

 

If DNA is the transforming principle then are the other tests to confirm that it is only DNA itself and not other biomolecules that are responsible for transformation? One way to tell is to do chemical and physical analyses of the isolated molecule. It was known that all life forms are composed of proteins and nucleic acids and the properties of those molecules were studied such as their chemical composition and other physical properties such as sedimentation or when a biological fluid of molecules is subjected to high speeds in a machine called a centrifuge, the molecules will sediment at different rates, each molecule sediments differently and before the results were published, there were data about the sedimentation of each molecule. Comparison of the DNA from Streptococci did reveal a similar match between DNA from other organisms and it was DNA that when destroyed with DNase , prevents transformation.

 

Another set of experiments involved examining the ultraviolet spectra of biomolecules. DNA has a different characteristic that sets it apart from other molecules in terms of absorbing ultraviolet and when DNA was isolated from Streptococci and exposed to ultraviolet light and using data of ultraviolet spectra of biomolecules, the absorption spectra matched well with recorded spectra of DNA from other sources so both ultraviolet spectra studies and centrifugation were used to prove that DNA is indeed the transforming substance and together with the enzyme breakdown experiments.

 

Of course, when the paper was published in 1944, not every biologist were quick to accept the fact but in addition to skepticism, checking the results as described by Avery and colleagues were done by other researchers and further checks no doubt convinced scientists that DNA is not only the transforming principle but it is the genes which are inherited from one generation to the next and therefore is the genetic material. By the early 1950’s experiments like this and other experiments finally convinced biologists that DNA is the hereditary molecule so the next step was to determine the molecular structure of DNA which was accomplished in 1953.

 

From this, you can see that with careful experiments of a specific kind together with repeated experiments and with close scrutiny with skepticism how scientists reach their conclusions and in each generation what was the dominant paradigm or thinking such as the tetranucleotide hypothesis and genes as proteins was slowly but surely replaced with the Avery experiment of 1944 with DNA as genes and with additional research into nucleic acids did a new and more fruitful paradigm was used while the old hypothesis was finally obsolete. This shows that no hypothesis is something that is set in stone but changes along with new forms of thinkings resulting in new investigations along with the modification, discarding of old hypothesis and with new hypothesis and new sets of questions to be answered. This was the case for molecular biology and in the investigations of DNA . The rest is history.

 

 

 

References:

 

Aldridge, S (1996) The Thread of Life: The Story of Genes and Genetic Engineering Cambridge England: Cambridge University Press

 

Avery, O, Macleod, M, McCarty, M (1944) Studies of the Chemical Nature of the Substance Inducing Transformation in Pneumococcal Types Journal of Experimental Medicine79 137-156 . doi:10.1084/jem.79.2.137

 

Avery-Macleod-McCarty Experiment (n.d). Retrieved Oct. 10, 2015, from https://en.wikipedia.org/wiki/Avery%E2%80%93MacLeod%E2%80%93McCarty_experiment

 

Dawson, H.M, Sia, R (1930) The Transformation of Pneumococcal Types in Vitro. Proceedings of the Society for Experimental Biology and Medicine, 27, 989-990

 

DNA (n.d). Retrieved Oct 10, 2015, from https://en.wikipedia.org/wiki/DNA#History_of_DNA_research

 

Griffith, F.(1928). The Significance of Pneumococcal Types. Journal of Hygiene, 2, 113-159. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2167760/

 

Griffith’s Experiment. (n.d). Retrieved Oct 7 , 2015 https://en.wikipedia.org/wiki/Griffith%27s_experiment

 

Hargittai, I. (2009). The Tetranucleotide hypothesis: A Centennial. Structural Chemistry, 20, 753-756. doi:10.1007/s11224-009-9497-x

 

Martinez, A. (2015, Sept. 05). The Ever Changing Concept of the Gene. Retrieved from  http://unityoflifeblog.com/the-ever-chang…pt-of-the-gene

 

Pierce, B. (2012). Genetics: A Conceptual Approach (4th ed.)  New York, NY W.H Freeman and Company

 

Tetranucleotide Hypothesis. (2015). Memorial University of Newfoundland. Retrieved from http://www.mun.ca/biology/scarr/Tetranucleotide_Hypothesis.html

 

Vaccine. (n.d.). Retrieved Oct. 6, 2015 from https://en.wikipedia.org/wiki/Vaccine

 

Photos:

 

Enzymlogic DNA https://www.flickr.com/photos/101755654@N08/9735192821/in/photolist-ae1zYZ-7TqgUV-qZChgN  CC BY-SA 2.

 

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