By the late 1940s, scientists had hints that DNA was important in conveying genetic information in cells, but they wondered how a repetitive polymer made of only four nucleotides could transmit such genetic information. Erwin Chargaff began “dissecting” DNA using chemical methods available at the time to try to unravel its structure.

He developed methods for isolating, purifying and deconstructing DNA into its individual nucleotides. He then developed methods for separating and quantifying the levels of A, G, C and T. You will explore some of his data below, taken from a paper published in 1950.

Comparing DNA composition of various tissues within a species

1. What do the numbers in the table represent?

2. Look at the three preparation values for adenine in thymus. How do they compare? Do the same for each of the nitrogenous bases.

Adenine

Guanine

Cytosine

Thymine

3. What is meant by “recovery”?

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Empowering Teachers Through ModelingInterrogating the Data – Chargaff’s Rules

Empowering Teachers Through ModelingInterrogating the Data – Chargaff’s Rules

Empowering Teachers Through ModelingInterrogating the Data – Chargaff’s Rules

Empowering Teachers Through ModelingInterrogating the Data – Chargaff’s Rules

Empowering Teachers Through ModelingInterrogating the Data – Chargaff’s Rules

Empowering Teachers Through ModelingInterrogating the Data – Chargaff’s Rules

Empowering Teachers Through ModelingInterrogating the Data – Chargaff’s Rules

Empowering Teachers Through ModelingInterrogating the Data – Chargaff’s Rules

Empowering Teachers Through ModelingInterrogating the Data – Chargaff’s Rules

4. Is it reasonable to include data points from all three preparation in a single analysis? Why or why not?

Total the values for each individual nitrogenous base for ox thymus DNA in the table above, then calculate the percent composition of each base.

Nitrogenous Base Total value, ox thymus DNA preps 1,2 and 3

Per cent of total recovered nitrogenous bases in preps 1, 2 and 3

Total value of nitrogenous base /sum of all nitrogenous bases x 100

AdenineGuanineCytosine ThymineSum of all nitrogenous bases

Make the same calculations, using preps 1 and 2 from ox spleen:

Nitrogenous Base Total value, ox spleen DNA preps 1 and 2

Per cent of total recovered nitrogenous bases in preps 1 and 2

Total value of nitrogenous base /sum of all nitrogenous bases x 100

AdenineGuanineCytosine ThymineSum of all nitrogenous bases

At the time these data were collected, the tetranucleotide hypothesis was commonly accepted. The tetranucleotide hypothesis states that DNA consists of equal molar concentrations of each of the four nucleotides. Remember that the structure of DNA was not yet known.

5. Are the molar concentrations of all nucleotides in DNA the same within an organ? Explain your observations.

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6. Are the molar concentrations of each nucleotide in DNA the same between thymus and spleen in ox? Explain your observations.

Comparing DNA composition of the same tissue in different species

Next, calculate the per cent molar composition of nitrogenous bases in DNA from sperm (combine preps 1 and 2) and thymus in man.

Nitrogenous Base Total value, human sperm DNA preps 1

and 2

Per cent of total recovered

nitrogenous bases in preps 1 and 2,

human spermTotal value of nitrogenous

base/sum of all nitrogenous bases

x 100

Human thymus values

Per cent of total recovered

nitrogenous bases, human thymus Total value of nitrogenous

base /sum of all nitrogenous bases

x 100

AdenineGuanineCytosine ThymineSum of all nitrogenous bases

7. Are the molar concentrations of all nucleotides in DNA the same within human sperm or thymus? Explain your observations.

8. Are the molar concentrations of each nucleotide in DNA the same between human sperm and thymus? Explain your observations.

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9. How do the molar concentrations of each nucleotide in DNA from ox thymus and human thymus compare?

10. Can you say with certainty whether the amount of each nitrogenous base is the same within a particular organ between species? Why or why not?

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DNA Composition in Yeast and Bacteria

Table IV in Chargaff’s 1950 paper compares base composition of DNA in yeast and bacterial cells:

Calculate the per cent molar composition of nitrogenous bases in DNA in yeast and avian tubercle bacilli, a bacterial species.

Nitrogenous Base Total value, yeast DNA

preps 1 and 2

Per cent of total recovered

nitrogenous bases in preps 1 and 2,

yeastTotal value of nitrogenous

base/sum of all nitrogenous bases

x 100

Value avian tubercle bacilli

Per cent of total recovered

nitrogenous bases, avian tubercle

bacilli Total value of nitrogenous

base /sum of all nitrogenous bases

x 100AdenineGuanineCytosine ThymineSum of all nitrogenous bases

11. Compare the molar concentrations of DNA nucleotides from bacteria to those of eukaryotic species. What do you observe?

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Comparing composition of DNA and RNA within yeast cells

Next, calculate the molar concentration of ribonucleotides in yeast. Note that there are three different procedures used to separate ribonucleotides from each of the preparations. Chargaff notes in the paper: “As matters stand now, I consider the values for purines yielded by Procedures 1 and 3 and those for pyrimidines found in Procedures 1 and 2 as quite reliable.”

12. Why should we use only the data from procedure 1 in each of the three preparations to calculate molar percentages of each ribonucleotide?

Nitrogenous Base Total value, yeast RNA preps 1, 2 and 3

Per cent of total recovered nitrogenous bases in preps 1, 2 and 3

Total value of nitrogenous base /sum of all nitrogenous bases x 100

AdenineGuanineCytosine UridineSum of all nitrogenous bases

13. How do the molar concentrations of nucleotides in yeast compare between DNA and RNA? Provide evidence for your answer.

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14. It is important in comparing data to keep as many variables consistent as possible. Why do you think Chargaff chose to conduct this experiment using DNA and RNA from yeast, rather than isolating them from human or ox?

Chargaff continued to develop better methods of isolating, purifying and analyzing molar concentrations of nucleic acids, and he used these methods to measure the composition of DNA and RNA in numerous organs from various species. By 1951, Chargaff had a set of rules that described the proportions of various deoxyribonucleotides across organ types within a species.

15. What do the molar ratios imply? Express these in mathematical equations.

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Over the years, G/C content (percent of DNA that is G or C) has been gathered for a wide range of organisms. This information in summarized in the table below.

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Advanced Extension: Chargaff also compared molar concentrations of ribonucleotides in various tissues of various organs (see original paper for data). He noted:

In the absence of a truly reliable standard method for the isolation of pentose nucleic acid [now called RNA] from animal tissue, generalizations are not yet permitted; but it would appear that pentose nucleic acids from the same organ of different species are more similar to each other, at least in certain respects (e.g. the ratio of guanine to adenine), than are those from different organs of the same species. (Compare the pentose nucleic acids from the liver and the pancreas of pig in Table VIII.)

16. Based on what you now know about the structure and function of DNA and RNA, explain why there are differences in molar concentrations of nucleotides between DNA and RNA.

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