Friedrich Miescher, a Swiss biologist, was investigating the ways of isolating ‘material’ from cell nuclei accompanied by least possible contamination from cytoplasm components. The ideal cells for this study were the ones with a large nucleus and with easy procurement in sufficient quantities. White blood cells (WBCs), serving the purpose of the required cell features, were acquired from the pus in surgical bandages.
Procedure followed for isolating ‘material’ from WBC nuclei:
Cells are subjected to dilute HCl
The extracts (containing pepsin for protein digestion) from pigs’ stomach were added to the mix.
The precipitate obtained constituted of cell nuclei which was then treated with dilute alkali for extraction.
The alkali soluble content was finally purified via precipitation with dilute acid and reextraction with dilute alkali.
Miescher hypothesized the ‘material’ to be proteins. To validate this presumption, he carried out biochemical test for the ‘material’. The test uncovered the presence of carbon, hydrogen, oxygen, nitrogen and phosphorous. Since phosphorous is not a protein component, the ‘material’ was unlikely to be protein. Furthermore, there was no substance then known that had such properties. Hence, he identified the material as ‘nuclein’. Miescher continued his investigation by replicating the same study in sperm cells (nucleus makes up 90% of the cell volume). The sperm nuclei contained an elevated percent of phosphorous in comparison to WBCs nuclei. This indicated less protein contamination in the ‘nuclein’ preparation. These preparations are now thought to be as first preparations of comparatively pure DNA.
THE HURDLE: Chromosomes consists of 2 components: Proteins and DNA. Many believed proteins to be the genetic material since proteins are complex (are composed of 20 amino acids) and are highly specific in nature (enzymes). Contrastingly, DNA was labelled as ‘too simple’ as it is composed of only 4 nucleotides in monotonous repeats.
Streptococcus pneumoniae occurs in three immunologically discrete types: I, II and III. Each of the three types can exist as R or S form giving rise to characteristically and functionally distinct colonies. The S strain is virulent, shiny and smooth due to the presence of gelatinous capsule whereas R strain is rough and avirulent due to the absence of the capsule. The two forms are found to have type-specific interconvertibility. This means that if III-S form converts to III-R form then this III-R form can convert back specifically to III-S and not to I-S or II-S form.
Griffiths transformation experiment:
Animal model system used: mice
Method followed: Injecting mice with different pneumococcus strains.
Observations:
Mice injected with II-R derived from II-S strain --> mice survived
Mice injected with live III-S strain --> mice died and III-S was later isolated
Mice injected with heat killed III-S strain --> mice survived.
Conclusion: Live S strain is virulent and as a consequence kills mice.
Mice injected with II-R strain + heat killed III-S strain --> mice died and live III-S strain was isolated.
Conclusion: The live III-S was not derived from II-R form as II-R can mutate back only to II-S form not to III-S form. Hence, it is only possible that II-R form interacted with heat killed III-S form and ‘transformed’ to III-S. An unknown agent then named as the ‘transforming principle’ (wrongly assumed to be a protein) was involved.
Their work was focused on identifying the ‘transforming principle’.
The workflow followed: III-S cells were lysed --> centrifuged --> the cellular debris was incubated with live II-R strain culture --> cells plated on a medium in Petri dish.
Observation: III-S colonies were observed in the plate.
Inference: Since III-S colonies grew, the extract did consist of the ‘transforming principle’.
This ‘transforming principle’ could either be a protein, carbohydrate, RNA or DNA. In other words, it could be any one of the macromolecules.
Methodology: To identify the ‘transforming principle’ the III-S extracts were subjected to enzymes that degraded one or more of these macromolecules. This enzyme treated extract was mixed with II-R cells to see if transformation occurred.
Criteria for understanding if transformation occurred:
If the mix was plated:
III-S colonies formation --> transformation did occur.
No III-S colonies formed --> no transformation occurred.
If the mix is injected into the mice:
Mice dies and III-S strain was then isolated --> transformation did occur.
Mice survives --> no transformation occurred.
Findings: It was seen that only when the extract was treated with DNase (i.e. DNA degrading enzyme) individually or in combination with other enzymes, no transformation occurred. In other words, only DNA degradation prohibited transformation. Hence, it was concluded that DNA alone is the ‘transforming principle’.
A story of rejection for Bob: A particular event emphasises on the low impact of the paper published by Avery, MacLeod and McCarty in 1944. McCarty was one of the speakers at Johns Hopkins University alongside Leslie Gay. During Gay's turn the hall was packed and soon after the Q&A session it was McCarty's turn. However, when the host of the event was introducing McCarty, majority of the crowd was rushing out so the announcements were barely audible. McCarty recalls this event and mentions that only 35 individuals did not leave either because they wanted to listen about his work or maybe out of courtesy. The paper did evidently point towards DNA as the genetic material however, the majority of the science community opposed it.
The T2 bacteriophage (viruses targeting E.coli) invades (infects) the bacterial cell, then injects its genetic material leading to the formation of the virus particles in the infected cell. The infected cell ultimately ruptures (lysis) to release the progeny phages (phage lysate). This entire process is known as lytic cycle. The bacteriophage contains only two molecules: protein and DNA. Hence, the genetic material has to be one of the two. With the aid of electron microscopy, it was observed that during infection the bacteriophage left its outer shell outside in attachment with the bacterial cell surface. This left out outer shell is referred as ‘phage ghost’.
To establish DNA as the genetic material the following procedure was employed:
E.coli was grown in the medium that consisted of either radioactive isotope of phosphorus (32P) or a radioactive isotope of sulfur (35S).
NOTE 1: Phosphorus is DNA component not found in protein. Sulfur is protein component not found in DNA.
NOTE 2: The two radioisotopes were labelled in a distinguishable manner.
These bacteria were then infected with T2 and the phage lysate was collected.
Product: One batch of phage lysate contained 32P hence, in this batch the DNA was radioactively labelled. The other batch contained 35S hence, in this case the proteins were radioactively labelled.
These batches were then used individually to infect separate E. coli cultures.
Lastly, the amount of radioactivity found within the bacterial cell was compared to the amount found in phage ghost.
Findings:
BATCH 1: In cells infected with 32P bacteriophage (DNA is radioactively labelled):
Majority of radioactivity was found within the bacterial cells.
Minute amounts was found in phage ghost (this was analyzed by detaching the phage ghost from bacterial cells via a blender).
Furthermore, after lytic cycle completion some radioactivity (~30%) was indicated in phage progeny.
BATCH 2: In cells infected with 35S bacteriophage (protein is radioactively labelled):
Almost no radioactivity was found within the bacterial cell.
Majority of radioactivity was found in phage ghost.
<1% was indicated in phage progeny.
Inference: The variation in location and amounts of radioactivity between the two batches indicates that DNA entered the cell, not protein since 32P was located within the cell unlike 35S. This evidently proves that DNA is functionally important in T2. Additionally, the presence of 32P in phage progeny, indicates that DNA is also important for T2 reproduction. Only genetic material is passed from one generation to the next, hence DNA is the genetic material and protein isn’t.
Simultaneously, as progress was being made in identifying DNA as the genetic material, the structure of the molecule was also being scrutinized. One of the major contributors was Phoebus Aaron Levene who established that 2-deoxyribose was the sugar component of the DNA. Though he is known for his work on structural understanding of DNA, he was equally known to be a major supporter of the proven wrong: ‘protein is the genetic material’.
The next infodump will focus on understanding the composition of DNA.
References:
Iwasa, J., Marshall, W. F., & Karp, G. (2015). Karps cell and molecular biology. Hoboken:Wiley.
Bose, R. (2010). iGenetics : A Molecular Approach.