Overview
Most people look somewhat like a mixture of their parents. They may have their father's nose or their mother's eyes, but in general, certain traits are passed on from one generation to the next. This is also the case with unicellular organisms. This "genetic information" (the information which determines the traits which are passed down) must be stored somewhere in the cell, but just how or where eluded biologists for a long time.
Eventually, microscopists noticed that during cell division, the sister chromatid pairs split, and each corresponding chromosome became part of a different daughter cell. This phenomenon suggested to biologists that the chromosomes must in some way contain the genetic information, especially since cells which obtained an abnormal number of chromosomes failed to function. However, chemical analysis showed that the chromosomes were made of two major components: DNA (deoxyribonucleic acid) and protein.
DNA was, at the time, thought to be a much simpler molecule than proteins, which scientists knew came in many varieties and combinations. So, it seemed logical that they hypothesized that protein was the genetic material. But by the late 1920s and 1930s, experimental evidence began to mount that DNA was in fact the bearer of genetic information.
While the period from the early 1900s to World War II has been considered the "golden age" of genetics, scientists still had not determined that DNA, and not protein, was the hereditary material. However, during this time a great many genetic discoveries were made and the link between genetics and evolution was made.
Friedrich Meischer in 1869 isolated DNA from fish sperm and the pus of open wounds. Since it came from nuclei, Meischer named this new chemical, nuclein. Subsequently the name was changed to nucleic acid and lastly to deoxyribonucleic acid (DNA). Robert Feulgen, in 1914, discovered that fuchsin dye stained DNA. DNA was then found in the nucleus of all eukaryotic cells.
During the 1920s, biochemist P.A. Levene analyzed the components of the DNA molecule. He found it contained four nitrogenous bases: cytosine, thymine, adenine, and guanine; deoxyribose sugar; and a phosphate group. He concluded that the basic unit (nucleotide) was composed of a base attached to a sugar and that the phosphate also attached to the sugar. He (unfortunately) also erroneously concluded that the proportions of bases were equal and that there was a tetranucleotide that was the repeating structure of the molecule. The nucleotide, however, remains as the fundemantal unit (monomer) of the nucleic acid polymer. There are four nucleotides: those with cytosine (C), those with guanine (G), those with adenine (A), and those with thymine (T).
During the early 1900s, the study of genetics began in earnest: the link between Mendel's work and that of cell biologists resulted in the chromosomal theory of inheritance; Garrod proposed the link between genes and "inborn errors of metabolism"; and the question was formed: what is a gene?
The answer came from the study of a deadly infectious disease: pneumonia. During the 1920s Frederick Griffith studied the difference between a disease-causing strain of the pneumonia causing bacteria (Streptococcus peumoniae) and a strain that did not cause pneumonia. The pneumonia-causing strain (the S strain) was surrounded by a capsule. The other strain (the R strain) did not have a capsule and also did not cause pneumonia.
Frederick Griffith (1928) was able to induce a nonpathogenic strain of the bacterium Streptococcus pneumoniae to become pathogenic. Griffith referred to a transforming factor that caused the non-pathogenic bacteria to become pathogenic. Griffith injected the different strains of bacteria into mice. The S strain killed the mice; the R strain did not. He further noted that if heat killed S strain was injected into a mouse, it did not cause pneumonia. When he combined heat-killed S with Live R and injected the mixture into a mouse (remember neither alone will kill the mouse) that the mouse developed pneumonia and died. Bacteria recovered from the mouse had a capsule and killed other mice when injected into them!
Hypotheses:
1. The dead S strain had been reanimated/resurrected.
2. The Live R had been transformed into Live S by some "transforming factor".
Further experiments led Griffith to conclude that number 2 was correct.
In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty revisited Griffith's experiment and concluded the transforming factor was DNA. Their evidence was strong but not totally conclusive. The then-current favorite for the hereditary material was protein; DNA was not considered by many scientists to be a strong candidate.
The breakthrough in the quest to determine the hereditary material came from the work of Max Delbruck and Salvador Luria in the 1940s. Bacteriophage are a type of virus that attacks bacteria, the viruses that Delbruck and Luria worked with were those attacking Escherichia coli, a bacterium found in human intestines. Bacteriophages consist of protein coats covering DNA. Bacteriophages infect a cell by injecting DNA into the host cell. This viral DNA then "disappears" while taking over the bacterial machinery and beginning to make new virus instead of new bacteria. After 25 minutes the host cell bursts, releasing hundreds of new bacteriophage. Phages have DNA and protein, making them ideal to resolve the nature of the hereditary material.
In 1952, Alfred D. Hershey and Martha Chase conducted a series of experiments to determine whether protein or DNA was the hereditary material. By labeling the DNA and protein with different (and mutually exclusive) radioisotopes, they would be able to determine which chemical (DNA or protein) was getting into the bacteria. Such material must be the hereditary material (Griffith's transforming agent). Since DNA contains Phosphorous (P) but no Sulfur (S), they tagged the DNA with radioactive Phosphorous-32. Conversely, protein lacks P but does have S, thus it could be tagged with radioactive Sulfur-35. Hershey and Chase found that the radioactive S remained outside the cell while the radioactive P was found inside the cell, indicating that DNA was the physical carrier of heredity.
Many scientists were interested in deciphering the structure of DNA, among them were Francis Crick, James Watson, Rosalind Franklin, and Maurice Wilkens. Watson and Crick gathered all available data in an attempt to develop a model of DNA structure. Franklin took X-ray diffraction photomicrographs of crystalline DNA extract, the key to the puzzle. The data known at the time was that DNA was a long molecule, proteins were helically coiled (as determined by the work of Linus Pauling), Chargaff's base data, and the x-ray diffraction data of Franklin and Wilkens.
DNA is a double helix, with bases to the center (like rungs on a ladder) and sugar-phosphate units along the sides of the helix (like the sides of a twisted ladder). The strands are complementary (deduced by Watson and Crick from Chargaff's data, A pairs with T and C pairs with G, the pairs held together by hydrogen bonds). Notice that a double-ringed purine is always bonded to a single ring pyrimidine. Purines are Adenine (A) and Guanine (G). We have encountered Adenosine triphosphate (ATP) before, although in that case the sugar was ribose, whereas in DNA it is deoxyribose. Pyrimidines are Cytosine (C) and Thymine (T). The bases are complementary, with A on one side of the molecule you only get T on the other side, similarly with G and C. If we know the base sequence of one strand we know its complement.
Chemical analyses by scientists revealed the general chemical composition of nucleic acids (DNA and RNA): they are composed of nucleotides. A nucleotide consists of a phosphate group, a five-carbon sugar (deoxyribose in DNA and ribose in RNA), and a nitrogenous base bonded together.
Each nucleotide in a DNA molecule has one of four nitrogenous bases: adenine, guanine, thymine, and cytosine. The first two are called purine bases because their structure consists of two rings of atoms. The latter two are known as pyrimidine bases, since they have a single ring of atoms. RNA has three of the same nucleotides, but instead of thymine, RNA has uracil, another pyrimidine base. For this section, we will discuss only DNA.
Knowing what DNA is composed of is only half of the mystery, as scientists still could not work out the physical structure of the molecule. In the 1940s, Erwin Chargaff made an important discovery which had significant implications regarding the structure of DNA. He found that a DNA molecule contains about the same amount of adenine as thymine, and about the same amount of cytosine as guanine. This countered an earlier suggestion that the four bases existed in equal amounts in the DNA molecule.
So, if one side of the DNA molecule reads TTGACTA (we have abbreviated the names of the bases), then the other strand must read AACTGAT. One final point about DNA's structure is that the opposite sides of the helix are said to be antiparallel. That means that they run in opposite directions. At the very end of each side of the molecule is, of course, a nucleotide. At one end, the phosphate group is the very last molecule, while at the other end, the sugar molecule is the last one. Scientists have dubbed the end with the phosphate group the 5' end (read "five prime"), and the end with the sugar the 3' end. Since the sides of the helix are antiparallel, the 3' end on one side of the ladder is opposite the 5' end on the other side.
Many people believe that American biologist James Watson and English physicist Francis Crick discovered DNA in the 1950s. In reality, this is not the case. Rather, DNA was first identified in the late 1860s by Swiss chemist Friedrich Miescher. Then, in the decades following Miescher's discovery, other scientists--notably, Phoebus Levene and Erwin Chargaff--carried out a series of research efforts that revealed additional details about the DNA molecule, including its primary chemical components and the ways in which they joined with one another. Without the scientific foundation provided by these pioneers, Watson and Crick may never have reached their groundbreaking conclusion of 1953: that the DNA molecule exists in the form of a three-dimensional double helix.
Although scientists have made some minor changes to the Watson and Crick model, or have elaborated upon it, since its inception in 1953, the model's four major features remain the same yet today. These features are as follows:
One of the ways that scientists have elaborated on Watson and Crick's model is through the identification of three different conformations of the DNA double helix. In other words, the precise geometries and dimensions of the double helix can vary. The most common conformation in most living cells (which is the one depicted in most diagrams of the double helix, and the one proposed by Watson and Crick) is known as B-DNA. There are also two other confirmations: A-DNA, a shorter and wider form that has been found in dehydrated samples of DNA and rarely under normal physiological circumstances; and Z-DNA, a left-handed confirmation. Z-DNA is a transient form of DNA, only occasionally existing in response to certain types of biological activity (Figure 5). Z-DNA was first discovered in 1979, but its existence was largely ignored until recently. Scientists have since discovered that certain proteins bind very strongly to Z-DNA, suggesting that Z-DNA plays an important biological role in protection against viral disease (Rich & Zhang, 2003).
DNA Experiments
While the period from the early 1900s to World War II has been considered the "golden age" of genetics, scientists still had not determined that DNA, and not protein, was the hereditary material. However, during this time a great many genetic discoveries were made and the link between genetics and evolution was made.
Friedrich Meischer in 1869 isolated DNA from fish sperm and the pus of open wounds. Since it came from nuclei, Meischer named this new chemical, nuclein. Subsequently the name was changed to nucleic acid and lastly to deoxyribonucleic acid (DNA). Robert Feulgen, in 1914, discovered that fuchsin dye stained DNA. DNA was then found in the nucleus of all eukaryotic cells.
During the 1920s, biochemist P.A. Levene analyzed the components of the DNA molecule. He found it contained four nitrogenous bases: cytosine, thymine, adenine, and guanine; deoxyribose sugar; and a phosphate group. He concluded that the basic unit (nucleotide) was composed of a base attached to a sugar and that the phosphate also attached to the sugar. He (unfortunately) also erroneously concluded that the proportions of bases were equal and that there was a tetranucleotide that was the repeating structure of the molecule. The nucleotide, however, remains as the fundemantal unit (monomer) of the nucleic acid polymer. There are four nucleotides: those with cytosine (C), those with guanine (G), those with adenine (A), and those with thymine (T).
During the early 1900s, the study of genetics began in earnest: the link between Mendel's work and that of cell biologists resulted in the chromosomal theory of inheritance; Garrod proposed the link between genes and "inborn errors of metabolism"; and the question was formed: what is a gene?
The answer came from the study of a deadly infectious disease: pneumonia. During the 1920s Frederick Griffith studied the difference between a disease-causing strain of the pneumonia causing bacteria (Streptococcus peumoniae) and a strain that did not cause pneumonia. The pneumonia-causing strain (the S strain) was surrounded by a capsule. The other strain (the R strain) did not have a capsule and also did not cause pneumonia.
Frederick Griffith (1928) was able to induce a nonpathogenic strain of the bacterium Streptococcus pneumoniae to become pathogenic. Griffith referred to a transforming factor that caused the non-pathogenic bacteria to become pathogenic. Griffith injected the different strains of bacteria into mice. The S strain killed the mice; the R strain did not. He further noted that if heat killed S strain was injected into a mouse, it did not cause pneumonia. When he combined heat-killed S with Live R and injected the mixture into a mouse (remember neither alone will kill the mouse) that the mouse developed pneumonia and died. Bacteria recovered from the mouse had a capsule and killed other mice when injected into them!
Hypotheses:
1. The dead S strain had been reanimated/resurrected.
2. The Live R had been transformed into Live S by some "transforming factor".
Further experiments led Griffith to conclude that number 2 was correct.
In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty revisited Griffith's experiment and concluded the transforming factor was DNA. Their evidence was strong but not totally conclusive. The then-current favorite for the hereditary material was protein; DNA was not considered by many scientists to be a strong candidate.
The breakthrough in the quest to determine the hereditary material came from the work of Max Delbruck and Salvador Luria in the 1940s. Bacteriophage are a type of virus that attacks bacteria, the viruses that Delbruck and Luria worked with were those attacking Escherichia coli, a bacterium found in human intestines. Bacteriophages consist of protein coats covering DNA. Bacteriophages infect a cell by injecting DNA into the host cell. This viral DNA then "disappears" while taking over the bacterial machinery and beginning to make new virus instead of new bacteria. After 25 minutes the host cell bursts, releasing hundreds of new bacteriophage. Phages have DNA and protein, making them ideal to resolve the nature of the hereditary material.
In 1952, Alfred D. Hershey and Martha Chase conducted a series of experiments to determine whether protein or DNA was the hereditary material. By labeling the DNA and protein with different (and mutually exclusive) radioisotopes, they would be able to determine which chemical (DNA or protein) was getting into the bacteria. Such material must be the hereditary material (Griffith's transforming agent). Since DNA contains Phosphorous (P) but no Sulfur (S), they tagged the DNA with radioactive Phosphorous-32. Conversely, protein lacks P but does have S, thus it could be tagged with radioactive Sulfur-35. Hershey and Chase found that the radioactive S remained outside the cell while the radioactive P was found inside the cell, indicating that DNA was the physical carrier of heredity.
DNA Structure
Erwin Chargaff analyzed the nitrogenous bases in many different forms of life, concluding that the amount of purines does not always equal the amount of pyrimidines (as proposed by Levene). DNA had been proven as the genetic material by the Hershey-Chase experiments, but how DNA served as genes was not yet certain. DNA must carry information from parent cell to daughter cell. It must contain information for replicating itself. It must be chemically stable, relatively unchanging. However, it must be capable of mutational change. Without mutations there would be no process of evolution.
Many scientists were interested in deciphering the structure of DNA, among them were Francis Crick, James Watson, Rosalind Franklin, and Maurice Wilkens. Watson and Crick gathered all available data in an attempt to develop a model of DNA structure. Franklin took X-ray diffraction photomicrographs of crystalline DNA extract, the key to the puzzle. The data known at the time was that DNA was a long molecule, proteins were helically coiled (as determined by the work of Linus Pauling), Chargaff's base data, and the x-ray diffraction data of Franklin and Wilkens.
DNA is a double helix, with bases to the center (like rungs on a ladder) and sugar-phosphate units along the sides of the helix (like the sides of a twisted ladder). The strands are complementary (deduced by Watson and Crick from Chargaff's data, A pairs with T and C pairs with G, the pairs held together by hydrogen bonds). Notice that a double-ringed purine is always bonded to a single ring pyrimidine. Purines are Adenine (A) and Guanine (G). We have encountered Adenosine triphosphate (ATP) before, although in that case the sugar was ribose, whereas in DNA it is deoxyribose. Pyrimidines are Cytosine (C) and Thymine (T). The bases are complementary, with A on one side of the molecule you only get T on the other side, similarly with G and C. If we know the base sequence of one strand we know its complement.
Further recommended reading
Chemical analyses by scientists revealed the general chemical composition of nucleic acids (DNA and RNA): they are composed of nucleotides. A nucleotide consists of a phosphate group, a five-carbon sugar (deoxyribose in DNA and ribose in RNA), and a nitrogenous base bonded together.
Each nucleotide in a DNA molecule has one of four nitrogenous bases: adenine, guanine, thymine, and cytosine. The first two are called purine bases because their structure consists of two rings of atoms. The latter two are known as pyrimidine bases, since they have a single ring of atoms. RNA has three of the same nucleotides, but instead of thymine, RNA has uracil, another pyrimidine base. For this section, we will discuss only DNA.
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So, if one side of the DNA molecule reads TTGACTA (we have abbreviated the names of the bases), then the other strand must read AACTGAT. One final point about DNA's structure is that the opposite sides of the helix are said to be antiparallel. That means that they run in opposite directions. At the very end of each side of the molecule is, of course, a nucleotide. At one end, the phosphate group is the very last molecule, while at the other end, the sugar molecule is the last one. Scientists have dubbed the end with the phosphate group the 5' end (read "five prime"), and the end with the sugar the 3' end. Since the sides of the helix are antiparallel, the 3' end on one side of the ladder is opposite the 5' end on the other side. Discovery of DNA Structure and Function: Watson and Crick
By: Leslie A. Pray, Ph.D. © 2008 Nature Education
Citation: Pray, L. (2008) Discovery of DNA structure and function: Watson and Crick. Nature Education 1(1)
The landmark ideas of Watson and Crick relied heavily on the work of other scientists. What did the duo actually discover?
Many people believe that American biologist James Watson and English physicist Francis Crick discovered DNA in the 1950s. In reality, this is not the case. Rather, DNA was first identified in the late 1860s by Swiss chemist Friedrich Miescher. Then, in the decades following Miescher's discovery, other scientists--notably, Phoebus Levene and Erwin Chargaff--carried out a series of research efforts that revealed additional details about the DNA molecule, including its primary chemical components and the ways in which they joined with one another. Without the scientific foundation provided by these pioneers, Watson and Crick may never have reached their groundbreaking conclusion of 1953: that the DNA molecule exists in the form of a three-dimensional double helix.
The First Piece of the Puzzle: Miescher Discovers DNA
Although few people realize it, 1869 was a landmark year in genetic research, because it was the year in which Swiss physiological chemist Friedrich Miescher first identified what he called "nuclein" inside the nuclei of human white blood cells. (The term "nuclein" was later changed to "nucleic acid" and eventually to "deoxyribonucleic acid," or "DNA.") Miescher's plan was to isolate and characterize not the nuclein (which nobody at that time realized existed) but instead the protein components of leukocytes (white blood cells). Miescher thus made arrangements for a local surgical clinic to send him used, pus-coated patient bandages; once he received the bandages, he planned to wash them, filter out the leukocytes, and extract and identify the various proteins within the white blood cells. But when he came across a substance from the cell nuclei that had chemical properties unlike any protein, including a much higher phosphorous content and resistance to proteolysis (protein digestion), Miescher realized that he had discovered a new substance (Dahm, 2008). Sensing the importance of his findings, Miescher wrote, "It seems probable to me that a whole family of such slightly varying phosphorous-containing substances will appear, as a group of nucleins, equivalent to proteins" (Wolf, 2003).
More than 50 years passed before the significance of Miescher's discovery of nucleic acids was widely appreciated by the scientific community. For instance, in a 1971 essay on the history of nucleic acid research, Erwin Chargaff noted that in a 1961 historical account of nineteenth-century science, Charles Darwin was mentioned 31 times, Thomas Huxley 14 times, but Miescher not even once. This omission is all the more remarkable given that, as Chargaff also noted, Miescher's discovery of nucleic acids was unique among the discoveries of the four major cellular components (i.e., proteins, lipids, polysaccharides, and nucleic acids) in that it could be "dated precisely... [to] one man, one place, one date."
More than 50 years passed before the significance of Miescher's discovery of nucleic acids was widely appreciated by the scientific community. For instance, in a 1971 essay on the history of nucleic acid research, Erwin Chargaff noted that in a 1961 historical account of nineteenth-century science, Charles Darwin was mentioned 31 times, Thomas Huxley 14 times, but Miescher not even once. This omission is all the more remarkable given that, as Chargaff also noted, Miescher's discovery of nucleic acids was unique among the discoveries of the four major cellular components (i.e., proteins, lipids, polysaccharides, and nucleic acids) in that it could be "dated precisely... [to] one man, one place, one date."
Laying the Groundwork: Levene Investigates the Structure of DNA
Meanwhile, even as Miescher's name fell into obscurity by the twentieth century, other scientists continued to investigate the chemical nature of the molecule formerly known as nuclein. One of these other scientists was Russian biochemist Phoebus Levene. A physician turned chemist, Levene was a prolific researcher, publishing more than 700 papers on the chemistry of biological molecules over the course of his career. Levene is credited with many firsts. For instance, he was the first to discover the order of the three major components of a single nucleotide (phosphate-sugar-base); the first to discover the carbohydrate component of RNA (ribose); the first to discover the carbohydrate component of DNA (deoxyribose); and the first to correctly identify the way RNA and DNA molecules are put together.
During the early years of Levene's career, neither Levene nor any other scientist of the time knew how the individual nucleotide components of DNA were arranged in space; discovery of the sugar-phosphate backbone of the DNA molecule was still years away. The large number of molecular groups made available for binding by each nucleotide component meant that there were numerous alternate ways that the components could combine. Several scientists put forth suggestions for how this might occur, but it was Levene's "polynucleotide" model that proved to be the correct one. Based upon years of work using hydrolysis to break down and analyze yeast nucleic acids, Levene proposed that nucleic acids were composed of a series of nucleotides, and that each nucleotide was in turn composed of just one of four nitrogen-containing bases, a sugar molecule, and a phosphate group. Levene made his initial proposal in 1919, discrediting other suggestions that had been put forth about the structure of nucleic acids. In Levene's own words, "New facts and new evidence may cause its alteration, but there is no doubt as to the polynucleotide structure of the yeast nucleic acid" (1919).
Indeed, many new facts and much new evidence soon emerged and caused alterations to Levene's proposal. One key discovery during this period involved the way in which nucleotides are ordered. Levene proposed what he called a tetranucleotide structure, in which the nucleotides were always linked in the same order (i.e., G-C-T-A-G-C-T-A and so on). However, scientists eventually realized that Levene's proposed tetranucleotide structure was overly simplistic and that the order of nucleotides along a stretch of DNA (or RNA) is, in fact, highly variable. Despite this realization, Levene's proposed polynucleotide structure was accurate in many regards. For example, we now know that DNA is in fact composed of a series of nucleotides and that each nucleotide has three components: a phosphate group; either a ribose (in the case of RNA) or a deoxyribose (in the case of DNA) sugar; and a single nitrogen-containing base. We also know that there are two basic categories of nitrogenous bases: the purines (adenine [A] and guanine [G]), each with two fused rings, and the pyrimidines (cytosine [C], thymine [T], and uracil [U]), each with a single ring. Furthermore, it is now widely accepted that RNA contains only A, G, C, and U (no T), whereas DNA contains only A, G, C, and T (no U) (Figure 1).
During the early years of Levene's career, neither Levene nor any other scientist of the time knew how the individual nucleotide components of DNA were arranged in space; discovery of the sugar-phosphate backbone of the DNA molecule was still years away. The large number of molecular groups made available for binding by each nucleotide component meant that there were numerous alternate ways that the components could combine. Several scientists put forth suggestions for how this might occur, but it was Levene's "polynucleotide" model that proved to be the correct one. Based upon years of work using hydrolysis to break down and analyze yeast nucleic acids, Levene proposed that nucleic acids were composed of a series of nucleotides, and that each nucleotide was in turn composed of just one of four nitrogen-containing bases, a sugar molecule, and a phosphate group. Levene made his initial proposal in 1919, discrediting other suggestions that had been put forth about the structure of nucleic acids. In Levene's own words, "New facts and new evidence may cause its alteration, but there is no doubt as to the polynucleotide structure of the yeast nucleic acid" (1919).
Indeed, many new facts and much new evidence soon emerged and caused alterations to Levene's proposal. One key discovery during this period involved the way in which nucleotides are ordered. Levene proposed what he called a tetranucleotide structure, in which the nucleotides were always linked in the same order (i.e., G-C-T-A-G-C-T-A and so on). However, scientists eventually realized that Levene's proposed tetranucleotide structure was overly simplistic and that the order of nucleotides along a stretch of DNA (or RNA) is, in fact, highly variable. Despite this realization, Levene's proposed polynucleotide structure was accurate in many regards. For example, we now know that DNA is in fact composed of a series of nucleotides and that each nucleotide has three components: a phosphate group; either a ribose (in the case of RNA) or a deoxyribose (in the case of DNA) sugar; and a single nitrogen-containing base. We also know that there are two basic categories of nitrogenous bases: the purines (adenine [A] and guanine [G]), each with two fused rings, and the pyrimidines (cytosine [C], thymine [T], and uracil [U]), each with a single ring. Furthermore, it is now widely accepted that RNA contains only A, G, C, and U (no T), whereas DNA contains only A, G, C, and T (no U) (Figure 1).
Figure 1: Nucleotides have three components.
A nucleotide consists of a phosphate group, a pentose sugar (ribose or deoxyribose), and a nitrogen-containing base, all linked together by covalent bonds. The nitrogenous bases have two different chemical forms: purines have two fused rings, and the smaller pyrimidines have a single ring.
© 2008 by Sinauer Associates, Inc. All rights reserved. Used with permission.
Strengthening the Foundation: Chargaff Formulates His "Rules"
Erwin Chargaff was one of a handful of scientists who expanded on Levene's work by uncovering additional details of the structure of DNA, thus further paving the way for Watson and Crick. Chargaff, an Austrian biochemist, had read the famous 1944 paper by Oswald Avery and his colleagues at Rockefeller University, which demonstrated that hereditary units, or genes, are composed of DNA. This paper had a profound impact on Chargaff, inspiring him to launch a research program that revolved around the chemistry of nucleic acids. Of Avery's work, Chargaff (1971) wrote the following:
"This discovery, almost abruptly, appeared to foreshadow a chemistry of heredity and, moreover, made probable the nucleic acid character of the gene... Avery gave us the first text of a new language, or rather he showed us where to look for it. I resolved to search for this text."
As his first step in this search, Chargaff set out to see whether there were any differences in DNA among different species. After developing a new paper chromatography method for separating and identifying small amounts of organic material, Chargaff reached two major conclusions (Chargaff, 1950). First, he noted that the nucleotide composition of DNA varies among species. In other words, the same nucleotides do not repeat in the same order, as proposed by Levene. Second, Chargaff concluded that almost all DNA--no matter what organism or tissue type it comes from--maintains certain properties, even as its composition varies. In particular, the amount of adenine (A) is usually similar to the amount of thymine (T), and the amount of guanine (G) usually approximates the amount of cytosine (C). In other words, the total amount of purines (A + G) and the total amount of pyrimidines (C + T) are usually nearly equal. (This second major conclusion is now known as "Chargaff's rule.") Chargaff's research was vital to the later work of Watson and Crick, but Chargaff himself could not imagine the explanation of these relationships--specifically, that A bound to T and C bound to G within the molecular structure of DNA (Figure 2).
"This discovery, almost abruptly, appeared to foreshadow a chemistry of heredity and, moreover, made probable the nucleic acid character of the gene... Avery gave us the first text of a new language, or rather he showed us where to look for it. I resolved to search for this text."
As his first step in this search, Chargaff set out to see whether there were any differences in DNA among different species. After developing a new paper chromatography method for separating and identifying small amounts of organic material, Chargaff reached two major conclusions (Chargaff, 1950). First, he noted that the nucleotide composition of DNA varies among species. In other words, the same nucleotides do not repeat in the same order, as proposed by Levene. Second, Chargaff concluded that almost all DNA--no matter what organism or tissue type it comes from--maintains certain properties, even as its composition varies. In particular, the amount of adenine (A) is usually similar to the amount of thymine (T), and the amount of guanine (G) usually approximates the amount of cytosine (C). In other words, the total amount of purines (A + G) and the total amount of pyrimidines (C + T) are usually nearly equal. (This second major conclusion is now known as "Chargaff's rule.") Chargaff's research was vital to the later work of Watson and Crick, but Chargaff himself could not imagine the explanation of these relationships--specifically, that A bound to T and C bound to G within the molecular structure of DNA (Figure 2).
Putting the Evidence Together: Watson and Crick Propose the Double Helix
Chargaff's realization that A = T and C = G, combined with some crucially important X-ray crystallography work by English researchers Rosalind Franklin and Maurice Wilkins, contributed to Watson and Crick's derivation of the three-dimensional, double-helical model for the structure of DNA. Watson and Crick's discovery was also made possible by recent advances in model building, or the assembly of possible three-dimensional structures based upon known molecular distances and bond angles, a technique advanced by American biochemist Linus Pauling. In fact, Watson and Crick were worried that they would be "scooped" by Pauling, who proposed a different model for the three-dimensional structure of DNA just months before they did. In the end, however, Pauling's prediction was incorrect.
Using cardboard cutouts representing the individual chemical components of the four bases and other nucleotide subunits, Watson and Crick shifted molecules around on their desktops, as though putting together a puzzle. They were misled for a while by an erroneous understanding of how the different elements in thymine and guanine (specifically, the carbon, nitrogen, hydrogen, and oxygen rings) were configured. Only upon the suggestion of American scientist Jerry Donohue did Watson decide to make new cardboard cutouts of the two bases, to see if perhaps a different atomic configuration would make a difference. It did. Not only did the complementary bases now fit together perfectly (i.e., A with T and C with G), with each pair held together by hydrogen bonds, but the structure also reflected Chargaff's rule (Figure 3).
Using cardboard cutouts representing the individual chemical components of the four bases and other nucleotide subunits, Watson and Crick shifted molecules around on their desktops, as though putting together a puzzle. They were misled for a while by an erroneous understanding of how the different elements in thymine and guanine (specifically, the carbon, nitrogen, hydrogen, and oxygen rings) were configured. Only upon the suggestion of American scientist Jerry Donohue did Watson decide to make new cardboard cutouts of the two bases, to see if perhaps a different atomic configuration would make a difference. It did. Not only did the complementary bases now fit together perfectly (i.e., A with T and C with G), with each pair held together by hydrogen bonds, but the structure also reflected Chargaff's rule (Figure 3).
Figure 3: DNA is a double helix.
(A) Francis Crick (left) and James Watson (right) proposed that the DNA molecule has a double-helical structure. (B) Biochemists can now pinpoint the position of every atom in a DNA molecule. To see that the essential features of the original Watson-Crick model have been verified, follow with your eyes the double-helical chains of sugar-phosphate groups and note the horizontal rungs of the bases.
© 2008 by Sinauer Associates, Inc. All rights reserved. Used with permission.
Although scientists have made some minor changes to the Watson and Crick model, or have elaborated upon it, since its inception in 1953, the model's four major features remain the same yet today. These features are as follows:
- DNA is a double-stranded helix, with the two strands connected by hydrogen bonds. A bases are always paired with Ts, and Cs are always paired with Gs, which is consistent with and accounts for Chargaff's rule.
- Most DNA double helices are right-handed; that is, if you were to hold your right hand out, with your thumb pointed up and your fingers curled around your thumb, your thumb would represent the axis of the helix and your fingers would represent the sugar-phosphate backbone. Only one type of DNA, called Z-DNA, is left-handed.
- The DNA double helix is anti-parallel, which means that the 5' end of one strand is paired with the 3' end of its complementary strand (and vice versa). As shown in Figure 4, nucleotides are linked to each other by their phosphate groups, which bind the 3' end of one sugar to the 5' end of the next sugar.
- Not only are the DNA base pairs connected via hydrogen bonding, but the outer edges of the nitrogen-containing bases are exposed and available for potential hydrogen bonding as well. These hydrogen bonds provide easy access to the DNA for other molecules, including the proteins that play vital roles in the replication and expression of DNA (Figure 4).
One of the ways that scientists have elaborated on Watson and Crick's model is through the identification of three different conformations of the DNA double helix. In other words, the precise geometries and dimensions of the double helix can vary. The most common conformation in most living cells (which is the one depicted in most diagrams of the double helix, and the one proposed by Watson and Crick) is known as B-DNA. There are also two other confirmations: A-DNA, a shorter and wider form that has been found in dehydrated samples of DNA and rarely under normal physiological circumstances; and Z-DNA, a left-handed confirmation. Z-DNA is a transient form of DNA, only occasionally existing in response to certain types of biological activity (Figure 5). Z-DNA was first discovered in 1979, but its existence was largely ignored until recently. Scientists have since discovered that certain proteins bind very strongly to Z-DNA, suggesting that Z-DNA plays an important biological role in protection against viral disease (Rich & Zhang, 2003).
Summary
Watson and Crick were not the discoverers of DNA, but rather the first scientists to formulate an accurate description of this molecule's complex, double-helical structure. Moreover, Watson and Crick's work was directly dependent on the research of numerous scientists before them, including Friedrich Miescher, Phoebus Levene, and Erwin Chargaff. Thanks to researchers such as these, we now know a great deal about genetic structure, and we continue to make great strides in understanding the human genome and the importance of DNA to life and health.
