The discovery of structure of DNA involved several people and highly interdisciplinary involvements. Rosalind Franklin and Wilkins along with Gosling acquired X-ray diffraction images, James Watson and Crick, developed the models based on the available information and finally deciphered the complete structure. Yet a very important conclusion about the structure of DNA was made on the foundations laid by Erwin Chargaff. Today what we know as Watson-Crick pairing in DNA that is represented as A-T or G-C pairing was only possible after the enormous efforts of an Ukranian scientist named Erwin Chargaff, who spend several decades of his life contributing the basic science and understanding the base pair composition of DNA in various species. In this blog I will try to bring you closer to the discovery made by him and understand the rules of parity in more detail.
Who was Erwin Chargaff?
Erwin Chargaff (11 August 1905 – 20 June 2002) was born in Czernowitz, a provincial capital of the Austro-Hungarian empire (now in Ukraine). He studied chemistry at the University of Vienna, where he obtained his PhD in 1928, and then spent two years at Yale, studying the tuberculosis bacterium and devising methods of isolating some of the unusual fatty molecules it contained. Later he worked at University of Colombia for several years. He believed that human knowledge will always be limited in relation to the complexity of the natural world, and that it is simply dangerous when humans believe that the world is a machine, even assuming that humans can have full knowledge of its workings.
He also believed that in a world that functions as a complex system of interdependency and interconnectedness, genetic engineering of life will inevitably have unforeseen consequences. Honours awarded to him include the Pasteur Medal (1949) and the National Medal of Science (1974), He was also nominated for the Nobel Prize. He was best known for his work in genetics, involving research into the chemical composition of DNA. Chargaff's data, along with that of Rosalind Franklin's X-ray diffraction pictures of DNA, provided the groundwork for the greatest discovery of 20th-century biology, by James Watson and Francis Crick, when they solved the riddle of heredity and showed how genetic inheritance could pass from one generation to the next through the double-helix structure of DNA. (Image credit - Wikipedia - creative commons)
He once said:
“We have created a mechanism that makes it practically impossible for a real genius to appear. In my own field the biochemist Fritz Lipmann or the much-maligned Linus Pauling were very talented people. But generally, geniuses everywhere seem to have died out by 1914. Today, most are mediocrities blown up by the winds of the time.”
DNA in those days was not double helix !
The knowledge about the DNA was not the same as present, In those days deoxyribose sugar was also named as ‘desoxypentose’ and also the model of DNA which was proposed by Levene et al, was known to have tetra nucleotide structure. The hypothesis of tetra-nucleotide structure of DNA was originally put forth by Phoebus Levene who believed that DNA was composed of a large number of repeats of a GACT tetramer. However, with the work of Chargaff, this hypothesis gradually faded away and experiments of Wilkin’s and Franklin’s supported by Watson and Crick analysis finally elucidated the double helical model of DNA.
Chargaff’s leading discoveries turned into what is known today as Chargaff’s rules. There are two parity rules based on his experiments. One of them forms the basis of Watson -Crick pairing in DNA and the other is used in characterizing a species.
First Parity Rule
First parity rule states that in any double-stranded DNA the number of guanine units equals the number of cytosine units and the number of adenine units equals the number of thymine units.
This rules establishes the pair of A with T and G with C, thus enabled Watson and Crick to finally complete the DNA double helix model or
% A = % T and %G = % C.
You might have witnessed several problems in exams such as determine the percentage of adenine if the total cytosine content is 20%. This is solved by using first parity rule, which implies that if C is 20% then G will also be 20% and remaining 60% will be A and T pair, hence each one of them would be 30% each.
This also implies that the A+G and T+C ratio for an organism shall be nearly equal to 1 or exactly 1 in most cases. The same is indicated from the table below adapted from one of the leading research articles of Chargaff.
Table 1. Base composition of various organisms based on the studies of Erwin Chargaff. It indicates that % A= % T and also A+G /T+C = 1.
In summary, the first parity rule can be indicated as
For a double stranded DNA,
· % A= % T and %G = % C
· or A+G = T+ C
· or A+G/T+C = 1
· or % A/T = % G/C
· or Percentage of Purines /Percentage of Pyrimidines = 1
Second Parity Rule
The second parity rule states that holds that for each of the DNA strands observed following approximate equality: %A ~ %T and %G ~ %C. In other words, it was stated that the composition of DNA varies from one species to another. For the DNA types in which Erwin Chargaff worked, the percentage of A was found to be equal to T and also percentage of G was found to be equal to C , suggesting that that AT content was nearly equal to C. However this notion was disproved by Szybalski.
In the own words of Chargaff:
"DNA is in its composition characteristic of the species from which it is derived. This can ... be demonstrated by determining the ratios in which the individual purines and pyrimidines occur .... There appear to exist two main groups of DNA, namely the ' AT type,' in which adenine and thymine predominate, and the ' GC type,' in which guanine and cytosine are the major constituents."
(Adapted from Erwin Chargaff, 1951).
This, later turned out to be GC rule, and represent that GC content of the DNA isolated from an organism (a species) remains constant and therefore the ratio A+T/G+C is a constant property, a characteristic of species. The rule itself has consequences. In most bacterial genomes (which are generally 80-90% coding) genes are arranged in such a fashion that approximately 50% of the coding sequence lies on either strand.
Table 2. GC content of various species based on the studies of Erwin Chargaff.
Hence, the second parity rule can be summarized as:
For a one of the strand of the double stranded DNA,
· % A= % T and %G = % C (approximated statement – not true for all organisms)
· % A ~ % T and %G ~ % C (original statement based on Chargaff’s observations– not true for all organisms)
· % A + T < % G + C (for bacteriophage – as pointed out by Szybalski)
· %(G+C)/ (A+T+G+C) = constant value for a species.
Exceptions to Chargaff’s rules and Other Rules of Sequence Parity
Szybalski’s rule: Wacław Szybalski, in the 1960s, showed that in bacteriophage coding sequences purines (A and G) exceed pyrimidines (C and T). This rule has since been confirmed in other organisms and should probably be now termed "Szybalski's rule". While Szybalski's rule generally holds, exceptions are known to exist. The biological basis for Szybalski's rule, like Chargaff's, is not yet known. Since the second parity rule was an empirical observation, the basis for this rule is still not yet validated completely. It was shown that it does not apply to organellar genomes (mitochondria and plastids) smaller than ~20-30 kbp, single stranded DNA (viral) genomes or any type of RNA genome.
Chargaff’s cluster rule: Besides DNA base pair parity rules, there were few other rules established in successive studies by Chargaff, one known as Chargaff’ cluster rule, that states that deoxyribonucleic acids of animal and plant contain at least 60% of the pyrimidines as oligonucleotide tracts containing three or more pyrimidines in a row: and a corresponding statement must, owing to the equality relationship [between the two strands], apply also to the purines. The cluster observation was extended by work from Waclaw Szybalski's laboratory in the I960s, which showed that clustering of clusters in microorganisms is most evident in transcriptionally active regions, and that the nature of the clustering of clusters (purine or pyrimidine) relates to transcription direction.
How did they do it?
The original experiments of Chargaff were very tedious and he established the methods of quantification and extraction of nucleotides from various type of samples. He initially used calf thymus and beef spleen as samples for DNA analysis (as much as 1.8 kg of beef spleen was used in each experiment). These tissues were hydrolyzed (hence he used the term hydrolyaste) and processed by using more than one approaches. The presence of purines and pyrimidine in the finally prepared solutions of nucleic acids were confirmed using spectrophotometry as each nucleotide had slight difference in their absorbance maxima. The separation and determination of quantities of purines and pyrimidines was performed using chromatography and the chromatograms were used to decipher the percentage of each of the nucelotides in sample. N-butanol-morpholine-diethylene glycol-water was used as a solvent system for purines and n-Butanol-water was used as a solvent for separation of pyrimidines. Over time, Chargaff improved on his initial quantification methods by introducing formic acid hydrolysis for the simultaneous liberation of all nitrogenous constituents and by using a UV lamp to demonstrate the separated adsorption zones on the filter strip. These improvements permitted him to rapidly analyse DNA from a variety of species and sample types such as human sperm, microbes etc.
The findings of Erwin Chargaff were published in a series of papers which were highly cited by the scientific community. Some of the most influential research articles of Chargaff are listed below: