DNA story (7): How was Base Ratios and Chargaff's Rules Discovered?

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Early Life: Avery promoted Erwin Chargaff's shift from TB to nucleic acids.

Chargaff was born on 11 August 1905 in a Jewish family in Czernowitz, Duchy of Bukovina, Austria-Hungary, which is now Chernivtsi, Ukraine. He received his PhD in chemistry from the University of Vienna in 1928. He left Germany and went to the Pasteur Institute in Paris in 1933, because Nazi Germany intended to exclude all Jews from universities. In 1935, Chargaff joined Columbia University and stayed there until retirement.

During the second world war, the military supported some of his research in hope of addressing infections in soldier, including studies on Rickettsia ·and lipid metabolism and virulence of TB. When he discovered that these very small organisms, Rickettsia, also contained nucleic acids, he developed an interest in nucleic acid chemistry. In 1944, Avery announced that the transforming principle responsible for phenotypic changes in pneumococci was DNA. Although this was later recognized as a landmark experiment in biology, it only attracted attention of a few scientists at the time. Avery's report fascinated Chargaff so much that he completely shifted his research field from bacterial lipids to nucleic acid biochemistry. He might even have been the first to reorganize his laboratory to verify Avery’s discovery. Years later he said, "I saw before me in dark contours the beginning of a grammar of biology."

How was Base Ratios and Chargaff's Rules discovered?

Levene’s books and papers on nucleic acids were one of few references about these compounds at the time. Phoebus Levene asserted that the four different nucleotides were uniformly distributed in DNA molecule ((ATCG) n). The simple repetitive structure made it nearly impossible for nucleic acids to be genetic material, but this conflicted with Avery’s experiment. Therefore, Chargaff intended to re-examine the tetranucleotide hypothesis and tried to find a way to accurately measure the proportions of the four nucleotides. The greatest challenge was difficulty of extracting adequate pure DNA from animal tissues. It looked hopeless at the time to do an accurate chemical analysis on the pitifully small amount of DNA.

However, in 1944, Chargaff was inspired by a paper from the Wool Industries Research Association in Leeds that described how to use paper chromatography to separate amino acids. When researchers dipped one end of filter paper into a mixture of amino acids and organic solvents, the mixture slowly seeped upward along paper via capillary action. Since each component was attracted to the cellulose with varying electrostatic forces, they separated from each other. The stronger attraction, the closer they were to the paper bottom, and vice versa. Chargaff realized that if he could find a suitable solvent, he could separate the four nucleotides from nucleic acid hydrolysate. Their relative contents in extract were determined by measuring UV absorbance at the end of paper chromatography.

The first samples tested were from bovine thymus and spleen. Chargaff was astonished that ratio of four nucleotides was not 1:1:1:1. This phenomenon was similar across other organisms. For example, ratio of 4 Nucleotides (A:T:G:C) in human sperm DNA was 29:31:18:18. There was 24:25:14:13 in yeast. After carefully analyzing the data and ruling out experimental errors, they summarized the data as Chargaff’s Rules of Base Pairing. In DNA of any organism, A=T, G=C, that is total purines equal total pyrimidines (A+G=C+T); the ratio of A+T to G+C or the ratio of A to G varies from one species to another, but it is constant in different tissues of same species.

Chargaff's experiment disproved Levene's tetranucleotide hypothesis. There was an essential difference between DNA as a biological macromolecule and proteins. The four nucleotides were randomly distributed in DNA. Nucleotides sequence might represent genetic information, just like letters form words. DNA must have a special structure that pairs A with T, and G with C.

There is no rule for Nucleotide Ratio or base ratio in RNA.

Chargaff extended his research to RNA and revealed its fundamental differences from DNA. All Chargaff's rules failed in RNA. A≠U, G≠C, and total pyrimidines does not equal total purines. Although nucleotide contents of both RNA and DNA exhibited similar species-specific diversity, RNA nucleotide content of the same species even varied from one tissue to another (if you've studied the central dogma, you’ll understand that RNA is the template for protein synthesis. Each tissue has specific RNA to express its unique proteins.) For example, ratio of four nucleotides in pig liver RNA was 10:7.7:16.2:16.1 (A:T:G:C), and it was 10:4.6:22.5:9.8 in pig pancreas.

Frequently Asked Questions

Significance of Chargaff's rules

Chargaff’s experiments proved that Levene's tetranucleotide hypothesis, which had dominated for 40 years, was wrong. More scientists began focusing their work on nucleic acids rather than proteins. More importantly, Chargaff's DNA base pairing rule provided crucial experimental evidence for Watson and Crick's double-helix model. It was unfortunate that Chargaff also considered various models to explain his experimental data, but he failed to propose the correct structure due to a lack of knowledge about hydrogen bonds in these bases. Later, Chargaff became bitter to feel that his contribution to DNA structure was not fully recognized.

Anec  > Biology > Genetic material

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