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In the early 1900's many people thought that protein must be the genetic material responsible for inherited characteristics. One of the reasons behind this belief was the knowledge that proteins were quite complex molecules and therefore, they must be specified by molecules of equal or greater complexity (i.e. other proteins). DNA was known to be a relatively simple molecule, in comparison to proteins, and therefore it was hard to understand how a complex molecule (a protein) could be determined by a simpler molecule (DNA). What were the key experiments which identified DNA as the primary genetic material?
1928 F. Griffith
Diplococcus pneumoniae, or pneumococcus, is a nasty little bacteria which, when injected into mice, will cause pneumonia and death in the mouse. The bacteria contains a capsular polysaccharide on its surface which protects the bacteria from host defenses. Occasionally, variants (mutants) of the bacteria arise which have a defect in the production of the capsular polysaccharide. The mutants have two characteristics: 1) They are avirulent, meaning that without proper capsular polysaccharide they are unable to mount an infection in the host (they are destroyed by the host defenses), and 2) Due to the lack of capsular polysaccharide the surface of the mutant bacteria appears rough under the microscope and can be distinguished from the wild type bacteria (whose surface appears smooth).
Figure 1.1.1: Wild type vs. Mutant type pneumococcus
The virulent smooth wild type pneumococcus can be heat treated and rendered avirulent (still appears smooth under the microscope however). Finally, there are several different subtypes of pneumococcus capsular polysaccharide (subtypes I, II and III). These subtypes are readily distinguishable from one another, and each can give rise to mutants lacking capsular polysaccharide (i.e. the avirulent rough type).
- w.t. (smooth) + mouse = dead mouse
- mutant (rough) + mouse = live mouse
- heat treated w.t. (smooth) + mouse = live mouse
- heat treated w.t. (smooth) + mutant (rough) + mouse = dead mouse
In this case when the bacteria were recovered from the cold lifeless mouse they were smooth virulent pneumococcus (i.e. indistinguishable from wild type).
A closer look at what is going on, by keeping using, and keeping track of, different subtypes
- heat treated w.t. (smooth) type I + mutant(rough) type II + mouse = dead mouse
In this case when the bacteria were isolated from the cold lifeless mouse they were smooth virulent type I pneumococcus.
The overall conclusions from these experiments was that there was a "transforming agent" in the the heat treated type I bacteria which transfomed the live mutant (rough) type II bacteria to be able to produce type I capsule polysaccharide.
Was the "transforming agent" protein or DNA, or what?
1944 O.T. Avery
The experiment of Griffith could not be taken further until methods were developed to separate and purify DNA and protein cellular components. Avery utilized methods to extract relatively pure DNA from pneumococcus to determine whether it was the "transforming agent" observed in Griffith's experiments.
- w.t. (smooth) type I -> extract the DNA component
- mutant (rough) type II + type I DNA + mouse = dead mouse
Isolation of bacteria from the dead mouse showed that they were type I w.t. (smooth) bacteria
A more sophisticated experiment:
Purified type I DNA was divided into two aliquots. One aliquot was treated with DNAse - an enzyme which non-specifically degrades DNA. The other aliquot was treated with Trypsin - a protease which (relatively) non-specfically degrades proteins.
- Type I DNA + DNAse + mutant (rough) type II + mouse = live mouse
- Type I DNA + Trypsin + mutant (rough) type II + mouse = dead mouse
The work of Avery provided strong evidence that the "transforming agent" was in fact DNA (and not protein). However, not everyone was convinced. Some people felt that a residual amount of protein might remain in the purified DNA, even after Trypsin treatment, and could be the "transforming agent".
1952 A.D. Hershey and M. Chase
T2 is a virus which attacks the bacteria E. coli. The virus, or phage, looks like a tiny lunar landing module:
Figure 1.1.2: T2 phage
The viral particles adsorb to the surface of the E. coli cells. It was known that some material then leaves the phage and enters the cell. The "empty" phage particles on the surface cells can be physically removed by putting the cells into a blender and whipping them up. In any case, some 20 minutes after the phage adsorb to the surface of the bacteria the bacteria bursts open (lysis) and releases a multitude of progeny virus.
If the media in which the bacteria grew (and were infected) included 32P labeled ATP, progeny phage could be recovered with this isotope incorporated into its DNA (normal proteins contain only hydrogen, nitrogen, carbon, oxygen, and sulfur atoms). Likewise, if the media contained 35S labeled methionine the resulting progeny phage could be recovered with this isotope present only in its protein components (normal DNA contains only hydrogen, nitrogen, carbon, oxygen, and phosphorous atoms).
Phage were grown in the presence of either 32P or 35S isotopic labels.
1) E. coli were infected with 35S labeled phage. After infection, but prior to cell lysis, the bacteria were whipped up in a blender and the phage particles were separated from the bacterial cells. The isolated bacterial cells were cultured further until lysis occurred. The released progeny phage were isolated.
Where the 35S label went:
- Adsorbed phage shells 85%
- Infected cells (prior to lysis) 15%
- Lysed cell debris 15%
- Progeny phage <1%
2) E. coli were infected with 32P labeled phage. The same steps as in 1) above were performed.
Where the 32P label went:
- Adsorbed phage shells 30%
- Infected cells (prior to lysis) 70%
- Lysed cell debris 40%
- Progeny phage 30%
The material which was being transferred from the phage to the bacteria during infection appeared to be mainly DNA. Although the results were not entirely unambiguous they provided additional support for the view that DNA was the "stuff" of genetic inheritance.
DNA as the genetic material
Hershey and Chase studied bacteriophage (phage=eater). Phage are bacterial viruses that infect bacteria and cause lysis of the cells. They have a very simple structure of a proteinaceous head/collar/tail and a DNA core. It was known that bacteria infected with phage were resistant to additional infection. In 1952 Hershey and Chase grew bacteriophage in conditions that would specifically label either the DNA or the protein with radioactivity. They subsequently took phage with radiolabeled DNA and infected bacteria. In parallel, they took phage with radiolabeled protein and infected another set of bacteria. After just enough time for infection, the bacterial cultures were placed into a blender to separate the bacteriophage from the bacteria.
Solutions were centrifuged to isolate bacteria from the phage. Bacteria were radioactive only when the phage grown in conditions to radiolabel DNA infected the bacteria to indicate that DNA might be the transforming agent.
Understanding Genetics: A New York, Mid-Atlantic Guide for Patients and Health Professionals.
Almost every human trait and disease has a genetic component, whether inherited or influenced by behavioral factors such as exercise. Genetic components can also modify the body’s response to environmental factors such as toxins. Understanding the underlying concepts of human genetics and the role of genes, behavior, and the environment is important for appropriately collecting and applying genetic and genomic information and technologies during clinical care. It is important in improving disease diagnosis and treatment as well. This chapter provides fundamental information about basic genetics concepts, including cell structure, the molecular and biochemical basis of disease, major types of genetic disease, laws of inheritance, and the impact of genetic variation.
Why DNA is considered as genetic material? | Criteria of genetic material | Characteristics of RNA
RNA (or ribonucleic acid) is a single stranded structure which consists of uracil as a nitrogenous base instead of thymine and ribose sugar.
RNA is also the genetic material of some of the viruses, such as: Tobacco Mosaic viruses, QB bacteriophage, etc. Also, in all organisms RNA performs the dynamic functions of messenger and adapter. Most of the essential life processes (such as: metabolism, splicing, translation, etc.) are performed by RNA, but still DNA is considered as the genetic material instead of RNA because of the following reasons.
For being genetic material, a molecule must have to fulfill the following criteria:
(i) It should be able to generate (or make) its replica.
(ii) It should always be chemically and structurally stable.
(iii) It should provide scope for slow changes (or mutation) essential for evolution.
(iv) It should be capable to express itself in the form of 'Mendelian Characters’.
On examining all these criteria one by one, it is found that:
> On the basis of the rule of base pairing and complementarity both DNA and RNA have the capability to make their replica (or perform duplications). While, proteins fail to fulfill this criteria (or first criteria).
> The two strands of DNA being complementary to each other, if denatured (or separated by heating) come together (or renature) when favourable conditions are returned back.
> Moreover, due to the presence of 2'-OH group at every nucleotide of RNA is a reactive group and this makes RNA labile and easily degradable. Meanwhile, RNA is also known to be catalytic in nature, therefore it is reactive.
Hence, DNA is chemically less reactive and structurally more stable than that of RNA. Therefore, DNA is chemically and structurally more stable. Thus, DNA fulfils the second criteria.
Also, the presence of thymine at the position of uracil in DNA provides additional stability to DNA.
Hence, except the stability criteria both DNA and RNA can function as genetic material, but DNA being more stable is preferred most for storing genetic information. But, for the transmission of genetic information, RNA is most preferable.
Therefore, with the exception of certain viruses, DNA rather than RNA carries the hereditary genetic code in every forms of biological life on Earth.
DNA is also more resilient and more easily repaired than RNA. As a result, DNA serves as a more stable carrier of the genetic information which is very essential for survival and reproduction in every living forms.
Thus, DNA is considered as a genetic material instead of RNA and protein.
DNA: as Hereditary Material and Properties of Genetic Material (DNA versus RNA)| Biology
Principles of inheritance given by Mendel and discovery of nuclein (nucleic acids) by Meischer (1871) almost coincided but for claiming that DNA acts as a genetic material took long time. Earlier discoveries made by Mendel, Walter Sutton, T.H. Morgan and others had narrowed the search for genetic material to chromosomes.
Chromosomes are made up of nucleic acids and proteins and are known as hereditary vehicles. In the first instance it appeared that proteins would be hereditary material, until experiments were performed to prove that nucleic acids act as genetic material.
DNA (deoxyribose nucleic acid) has been found to be a genetic material in all the living beings except few plant viruses where RNA is the genetic material because DNA is not found in such viruses.
A. Evidences for DNA as Hereditary Material:
The concept that DNA is the genetic material has been supported by the following evidences:
1. Bacterial Transformation or Transforming Principle (Griffith Effect):
In 1928, Frederick Griffith, a British Medical Officer encountered a phenomenon, now called as bacterial transformation. His observations involved the bacterium Streptococcus pneumoniae (Fig. 6.12) which is associated with certain type of pneumonia. During the course of this experiment, a living organism (bacteria) had changed into living form.
This bacterium is found in two forms:
Whose cells produce a capsule of polysaccharides (mucous), causing the colonies on agar to be smooth and rather shiny? This strain is virulent (pathogenic) and causes pneumonia.
In this case, cells lack capsule and produce dull rough (R) colonies.
Presence or absence of capsule is known to be genetically determined.
Both S and R strains are found in several types and are known as S-I, S-II, S-III etc. and R-I, R-II and R-III etc. respectively.
Mutations from smooth to rough occur spontaneously with a frequency of about one cell in 10 7 though, the reverse is much less frequent.
Griffith performed his experiment by injecting the above bacteria into mice and found the following results:
(a) S-III (virulent) bacteria were injected into mice the mice developed pneumonia and finally died.
(b) R-II (non-virulent) bacteria were injected into mice the mice suffered no illness because R-II strain was non-pathogenic.
(c) When Griffith injected heat killed S-III bacteria into mice, they did not suffer from pneumonia and thus survived.
(d) A mixture of R-II (non-virulent) and heat killed S-III bacteria were injected into mice the mice developed pneumonia and died. By postmortoming the dead mice, it was noticed that their heart blood had both R-II and S-III strains of bacteria.
Thus some genetic factor from dead S-III cells converted the live R-II cells into live S-III cells and the latter produced the disease. In short, living R-II cells were somehow transformed. So the Griffith effect gradually became known as transformation and turned out to be the first step in the identification of genetic material.
Biochemical Characterization of Transforming Principle:
Identification of Transforming Genetic Substance:
In 1944, sixteen years after Griffith’s experiment, Oswald Avery, Colin MacLeod and Maclyn McCarty (1933-1944) reported successfully repetition of bacterial transformation, but in vitro. They were able to identify the transforming genetic material. They tested fractions of heat killed cells for transforming ability. Their findings were as under.
(i) DNA alone from S bacteria caused R bacteria to become transformed.
(ii) They found that proteases (protein digesting enzymes) and RNAse (RNA digesting enzymes) did not affect transformation.
(iii) Digestion with DNAase did inhibit transformation.
Thus they finally concluded that DNA is the hereditary material.
Mixture injected into healthy mice
1. R-U type living cells + Capsule of heat killed S-III type.
Mice did not develop pneumonia.
2. R-II type living cells + Cell wall of heat killed S-III type.
3. R-II type living cells + Cytoplasm of heat killed S-III type (without DNA)
4. R-II type living cells + DNA of heat killed S-III type.
Mice developed pneumonia and died.
5. R-II type living cells + DNA of heat killed S-III type + DNAase
Mice did not develop pneumonia.
Therefore, it is now beyond any reasonable doubt that DNA is the hereditary material.
2. Bacteriophage Infection:
Viral infecting agent is DNA. By using radioactive tracers, Alferd Hershey and Maratha Chase (1952) gave evidence that DNA is the hereditary material in certain bacteriophages (bacterial viruses).
Structure of T2 bacteriophage:
This bacterial virus contains an outer non-genetic protein shell and inner core of genetic material (DNA). The T2 phages are of tadpole shape differentiated into head and tail region. Head is an elongated, bipyramidal, six sided structure composed of several proteins.
Within the head (Fig. 6.13) is a closed, non-ending DNA molecule. The dimensions of head is such that it is able to pack DNA molecule tightly inside it. The tail is a hollow cylinder. The tail bears 24 helical striations.
(ii) Some other bacteriophages were grown in bacteria having 32P. This radioactive 32P was restricted to DNA of phage particles.
Six tail fibres appear from an hexagonal plate at the distal end of plate. Tail is formed of proteins only. Proteinaceous outer shell contains sulphur (S) but no phosphorus (P), whereas DNA contains phosphorus but no sulphur.
Hershey and Chase (1952) conducted their experiment on T2 phage which attacks the bacterium Escherichia coli.
The phage particles were prepared by using radio isotopes of 35 S and 32 P in the following steps:
(i) Few bacteriophages were grown in bacteria containing 35 S.This radioactive was 35 S incorporated into the cysteine and methionine amino acids of proteins and thus these amino acids with 35 S formed the proteins of phage.
(ii) Some other bacteriophages were grown in bacteria having 32 P. This radioactive 32 P was restricted to DNA of phage particles.
These two radioactive phage preparations (one with radioactive proteins and another with radioactive DNA) were allowed to infect the culture of E. coli. The protein coats were separated from the bacterial cell walls by shaking and centrifugation.
The heavier infected bacterial cells during centrifugation pelleted to bottom (Fig. 6.14). The supernatant had the lighter phage particles and other components that failed to infect bacteria.
It was observed that bacteriophages with radioactive DNA gave rise to radioactive pellets with 32 P in DNA. However in the phage particles with radioactive protein (with 35 S) the bacterial pellets have almost nil radioactivities indicating that proteins have failed to migrate into bacterial cell.
So, it can be safely concluded that during infection by bacteriophage T2, it was DNA which entered the bacteria. It was followed by an eclipse period during which phage DNA replicates numerous times within the bacterial cell (Fig. 6.15).
Towards the end of eclipse period phage DNA directs the production of protein coats assembly of newly formed phage particles. Lysozyme (an enzyme) brings about the lysis of host cell and releases the newly formed bacteriophages.
The above experiment clearly suggests that it is phage DNA and not protein which contains the genetic information for the production of new bacteriophages. However, in some plant viruses (like TMV), RNA acts as hereditary material (being DNA absent).
B. Properties of Genetic Material (DNA versus RNA):
DNA is the genetic material RNA has been found to be genetic material in TMV (Tobacco mosaic virus), ф β bacteriophage etc. DNA is major hereditary material in most of the organisms. RNA mainly performs the functions of messenger and adapter. This is mainly due to differences between chemical structure of DNA and RNA.
Required properties of genetic material:
This refers to duplication of its genetic material by faithful replication which is shown by both DNA and RNA. Proteins and other molecules present in living being do not exhibit this property.
Stability of genetic material should exist. It should not change its structure easily with changed stages of life, age of physiology of living beings. Even in Griffith’s experiment of ‘transforming principle’, DNA survived in heat killed bacteria. Both the strands of DNA which are complementary can be separated.
RNA is liable and easily degradable due to presence of 2’—OH group present in each nucleotide. As RNA is catalytic, it has become reactive. Because DNA is more stable than RNA, it is said to be better genetic material. Presence of thymine instead of uracil is another reason which leads to stability of DNA.
Genetic material should be able to undergo mutation and such a change should be stably inherited. Both nucleic acids DNA and RNA have the capacity to mutate. RNA mutates at a faster rate when compared with DNA. Virus with RNA genome show mutation and evolution at a faster rate and thus has shorter life span.
Table 6.6. Types of nucleic acids:
Macromolecule in shape of double helix with many thousands of sub- units.
Mainly in nucleus, also in mitochondria and chloroplasts.
Acts as store of coded instruc­tions for synthesis of all proteins required by the cell.
Messenger ribonucleic acid.
Single-stranded polymer with hundreds of sub-units.
In nucleus and cytoplasm especially ribosomes.
Made on the DNA template it carries coded instructions for synthesis of one or more proteins from nucleus to ribosomes.
Ribosomal ribonucleic acid.
Molecule very closely bound to protein fraction.
Forms part of ribosome structure. Helps in locating mRNA correctly on ribosome surface.
Transfer ribonucleic acid.
Single-stranded polymer of less than one hundred sub- units.
Many kinds of tRNA act as amino acid carriers. Take specific amino acid from cytoplasm to mRNA tem­plate on ribosome.
4. Genetic expression:
RNA expresses easily the characters in the form of proteins. DNA requires RNA for formation of proteins. DNA being more stable is considered better than RNA for storage of genetic information. However, for transmission of genetic characters, RNA gives better results.
Stahl, like his two older sisters, graduated from the public schools of Needham, a Boston suburb. In 1951, he was awarded an AB degree in biology from Harvard College, and matriculated in the biology department of the University of Rochester. His interest in genetics was cemented in 1952 by his introduction to bacterial viruses (phages) in a course taught by A. H. (Gus) Doermann at the Cold Spring Harbor Biological Laboratory. In 1956, he received a PhD in biology for his work with Doermann on the genetics of T4 phage. In 1955, he undertook postdoctoral studies with Giuseppe Bertani (in the Phage group) at Caltech (Pasadena) with the aim of learning some bacterial genetics. He subsequently turned his attentions to collaborations with Charley Steinberg and Matt Meselson. With Steinberg, he undertook mathematical analyses of T4 growth, mutation, and genetic recombination. With Meselson, he studied DNA replication in Escherichia coli. That study produced strong support for the semiconservative model proposed by Jim Watson and Francis Crick. 
For one year, Stahl served on the zoology faculty at the University of Missouri in Columbia, Missouri before accepting, in 1959, a position in the new Institute of Molecular Biology at the University of Oregon in Eugene. In the succeeding years, his research involved the phages T4 and Lambda and the budding yeast, Saccharomyces cerevisiae, with his primary focus on genetic recombination. He taught various genetics courses at Oregon and presented phage courses in America, Italy and India. He undertook sabbatical studies in Cambridge, UK, Edinburgh, Jerusalem, and Cambridge, Massachusetts. 
Stahl's research was undertaken in association with numerous colleagues, especially his long-tem associates Jean M. Crasemann (1921–1992), Mary M. Stahl (1935–1996), and Henriette (Jette) M. Foss (1937–date).  Since his retirement in 2001, he lives with Jette and four llamas in Eugene, where he continues to submit research papers and participates in University of Oregon governance.
Stahl and his wife Mary (married in 1955) raised two boys and a girl. Surviving are Andy Stahl, a forester and political activist, and Emily Morgan, a hairdresser and shop owner. With his partner, Jette, he shares five children (plus spouses) and eight grandchildren, of whom five are adopted. 
Historical Background and Scientific Foundations
The study of heredity began as a response to Jean-Baptiste Lamarck's (1744–1829) theory of acquired characteristics. This proposed that traits acquired by an individual during its lifetime, whether in response to environmental conditions or caused by its own habits, would be inherited by its offspring. In 1856 an Austrian monk named Gregor Mendel (1822–1884) began to test Lamarck's theories with experiments in hybridizing pea plants.
In contrast to Lamarckian predictions, he found that particular characteristics, such as flower color or seed shape, were passed on to offspring by special cells, which he called “factors,” that determined how those traits were expressed. Through his work, Mendel was able to link an organism's phenotype (its external appearance) to its inherited genotype (its genetic inheritance) and illustrate the effects of dominant and recessive genes. He showed that these factors were inherited in predictable mathematical ratios.
Because Mendel worked on the margins of the scientific community, his findings remained unknown until 1900, when German botanist and geneticist Carl Correns (1864–1933), Dutch botanist Hugo de Vries (1848–1935), and Austrian botanist Erich von Tschermak-Seysenegg (1871–1962) independently discovered Mendel's achievements. While crediting de Vries, Correns connected earlier discoveries about meiosis (the process by which sex cells—egg and sperm—reproduce) and Mendel's laws of inheritance to propose the chromosome theory of heredity, concluding that “each germ cell must receive one or other of the factors that Mendel held responsible for dominant or recessive traits.” Within the cell, genes were strung together on the chromosomes like beads in a necklace, copying themselves during meiosis and separating as the cell divided.
The Discovery of DNA: Johann Friedrich Miescher and Nuclein
Scientists still didn't understand how genetic information in the old chromosome was transferred to the new, however. In 1871, Johann Friedrich Miescher, a Swiss professor of pathology at the University of Basel, identified DNA as one biochemical component of chromosomes. Miescher extracted cell nuclei from white blood cells or lymphocytes that he got from used hospital bandages, since they were filled with pus. While pursuing his research, Miescher realized that the cells' nuclei contained something other than proteins. This strange material did not react with the digestive enzyme pepsin, showing it was not protein in nature. This substance, which he called “nuclein”—renamed nucleic acid by German pathologist Richard Altmann (1852–1900) in 1889—was present in a large variety of cells. He found that it contained phosphorus in addition to carbon, hydrogen, nitrogen, and oxygen. This substance turned out to be DNA. Though Miescher speculated that his “nuclein” could be involved in fertilization in some way, he did not take his hypotheses any further.
Despite the discovery of nuclein, many biologists continued to believe that genetic material was protein in nature. Chromosomes do contain a good deal of protein, and proteins seemed good candidates to be the molecule that contained the genetic code as they were made of up to 20 different amino acids. DNA, on the other hand, contained only four nucleotides, so it was considered too simple to contain the genetic code.
The Work of Oswald Avery, Colin Macleod, and Maclyn McCarty in the 1940s
Miescher's work was largely ignored until the 1940s, with proteins deemed the source of genetic material. As Johns Hopkins biology professor Bentley Glass commented,
“The dismal blindness of scientists to the significance of a chemical substance [nucleic acid] so uniquely limited to the nucleus, and indeed to the very chromosomes themselves, endured until 1944, when the work of Avery, Macleod and McCarty on the transformation phenomenon in Pneumococcus at last reawakened geneticists to the importance of DNA.”
What were their experiments? British microbiologist Frederick Griffith (1881–1941), working with two types of pneumococcus, discovered that the bacteria could be made to change immunological specificity. One was the virulent S strain, which had a smooth cell membrane or “smooth polysaccharide coat.” The other was the considerably milder, rough-coated R strain its Petri dish colonies actually had ragged edges.
When Griffith injected mice with the S-type bacteria, they died quickly injecting another group with the R-strain did not affect their health. He then took the virulent S-bacteria and subjected it to intense heat, which seemed to destroy its ability to infect mice. But when he mixed the heat-treated S-type with the milder rough-coated strain and injected this mixture into mice, the mice died in a few days. Blood samples from the dead mice showed high levels of virulent pneumococcus, leading him to conclude that something caused the mild strain to adopt a new smooth polysaccharide coat and become lethal. The source of this genetic transformation was unknown, however.
In 1943, though he was near retirement, Canadian-born American bacteriologist Oswald Avery (1877–1955), a physician and researcher at the Rockefeller Institute, decided to investigate which agent caused this bacterial transformation. Using centrifugation, he and his coworkers Maclyn McCarty (1911–) and Colin MacLeod (1909–1972) used centrifugation to remove the large cellular structures from S-type bacteria, which they treated with proteases, enzymes that removed the proteins from the cells, then mixed the S and R strains together.
The R-bacteria were transformed into a virulent strain, so proteins could not carry the genes. The rest of the S bacteria were treated with an enzyme that removed the DNA when this was mixed with the R bacteria there was no change. This meant that DNA contained cellular hereditary information. Avery, McCarty, and MacLeod thus demonstrated that DNA, not proteins, contained a cell's genes. DNA's physical structure, however, was still a mystery.
Determining the Structure of DNA
Before World War II (1939–1945), two chromatographers, Irish crystallographer John Desmond Bernal (1901–1971) at Cambridge and English physicist William Astbury (1898–1961) at Leeds, used x-ray diffraction to determine crystals' molecular structure. The crystal's atomic planes caused entering x-ray beams to interfere with one another as they left the crystal. The interference patterns revealed the molecule's structure.
Using hundreds of x-ray diffraction pictures, Astbury built a DNA model to see how its sugar and phosphate bases fit together. The results convinced him that the molecule's bases were stacked on each other like a pile of pennies, spaced 3.4 Angstroms apart (an Angstrom [Å], equals one ten-billionth of a meter).
The remaining question of DNA's structure would be partially answered by Erwin Chargaff (1905–2002), an Austrian biochemist and war refugee working at Colombia University. Having read Avery's paper about bacterial transformation in 1944, he turned his attention to the study of DNA and its four chemical bases, or nucleotides: adenine (A), cytosine (C), guanine (G), and thymine (T). Using chromatography to analyze plant and animal tissue, Chargaff realized that the proportions of A and T were almost the same as those of C and G.
The total amount of the pyrimidines molecules (T and C) were also equal to the total amount of the purines (A and G). Although Chargaff sensed that this 1:1 ratio was important, he did not understand how it related to DNA's overall structure.
The Question of Rosalind Franklin
Rosalind Franklin (1920–1958) was an x-ray crystallographer who until 1951 was known primarily for her groundbreaking work with the crystalline structure of coal and other carbons. Educated at Cambridge, she received her doctorate in 1945, and worked at the Laboratoire Central des Services Chimique de L'Etat in Paris. In 1951 Franklin left coal research to go to King's College, London, to study the structure of DNA with Maurice Wilkins (1916–2004).
Franklin discovered that there were two different forms of DNA, A and B, which had to be separated to get clear x-ray diffraction images of each. Franklin even developed a special camera to improve the DNA-fiber orientation in the x-ray beam to photograph the B form. One picture from this apparatus, photograph 51, verified Astbury's work, showing that each helical curve in B-form DNA was 34Å long and contained 10 base pairs.
From her data, Franklin proposed a double-helix structure for the DNA molecule, with sugar phosphates composing its external backbone, and hydrophobic (water-hating) base pairs of purines and pyrimidines on the inside. In 1951, James Watson attended a talk she gave on her work. The following year, he was also given a copy of a report of Franklin's work in this area by one of Franklin' co-workers. This proved instrumental to his and Crick's later discovery of DNA's structure.
James Watson and Francis Crick
Watson and Crick, based at Cambridge University, were also working on the DNA problem. Crick, trained as a physicist, had designed circuits for acoustic and magnetic mines for the British Admiralty during World War II. Watson, 12 years younger, had studied zoology and microbiology at Indiana University. After meeting Maurice Wilkins at a symposium and seeing the x-ray diffraction pattern of crystalline DNA, Watson changed research directions. In October 1951 he took a fellowship to work on DNA with Crick at the Cavendish laboratory at Cambridge.
Their first attempts in November 1951 were not successful, based as they were on the ideas of American biochemist Linus Pauling (1901–1994), who was also close to solving the structure. Pauling had discovered in 1948 that a large number of proteins were shaped like a spring coil, termed the alpha-helix. Watson and Crick, taking Pauling's idea of a helix but misinterpreting Franklin's data, initially proposed a model in which the nucleotide bases were on the outside of the helix. So unpromising was their initial model that laboratory chiefs told Watson and Crick to abandon their efforts. Pauling incorrectly proposed a triple-helical structure in January 1952, and Watson persuaded his bosses to let the model building resume at Cambridge before Pauling saw his error and corrected it.
The data from Franklin's 1951 lecture had convinced Watson and Crick that the nucleotide bases belonged on the inside of the DNA molecule. Chargaff, who consulted with the duo, later recalled in his work Heraclitean Fire: Sketches from a Life before Nature.
So far as I could make out, they wanted, unencumbered by any knowledge of the chemistry involved, to fit DNA into a helix. The main reason seemed to be Pauling's alpha-helix model of a protein. I told them all I knew. If they had heard before about the pairing rules, they concealed it. But as they did not seem to know much about anything, I was not unduly surprised. I mentioned our early attempts to explain the complementarity relationships by the assumption that, in the nucleic acid chain, adenylic was always next to thymidylic acid and cytidylic next to guanylic acid. I believe that the double-stranded model of DNA came about as a consequence of our conversation but such things are only susceptible of a later judgment.
In 1953, Watson visited King's College, London, where Franklin and Wilkins worked. Without her knowledge, Wilkins showed Franklin's photograph 51 to Watson. Franklin's colleague and member of a research oversight committee, Austrian-born chemist Max Perutz (1914–2002), also gave Watson a copy of Franklin's 1952 MRC Report. Watson later admitted, “Rosy [Franklin], of course, did not directly give us her data. For that matter, no one at King's realized they were in our hands.”
After seeing photograph 51 and the MRC report and consulting with Chargaff, Watson and Crick had all the pieces of the DNA puzzle. Crick realized, as Franklin did not, that while the molecule consisted of two spiral chains in a helix, the strands ran parallel to each other—in opposite directions. Watson, now knowing
the base pairs were inside the molecule, made cardboard cutouts of the four DNA bases, and put them together in various ways. When he noticed that an A—T pair matched the shape of a G—C pair, he realized that in the DNA double helix, a base on one side is matched by its opposite on the other. When separated, each half of the chain becomes the template for a new, identical sequence.
Watson, Crick, and Wilkins shared the 1962 Nobel Prize for medicine or physiology for their discovery. Like Marie Curie before her, however, Rosalind Franklin's work had exposed her to high doses of radiation before its dangers were known she died of ovarian cancer in 1958. Despite her enormous contributions to the research, she was not mentioned by the prize committee, as the Nobel is not awarded posthumously. Cambridge University later named a building in Newnham College in her honor.
After DNA: Deciphering the Genetic Code
Once the structure of DNA had been determined, the challenge in the late 1950s and early 1960s was to determine how its information expressed genetic traits. How do the four nucleic acids code for specific proteins that comprise the cell's working parts? As Crick noted in his Nobel lecture,
It now seems certain that the amino acid sequence of any protein is determined by the sequence of bases in some region of a particular nucleic acid molecule. Twenty different kinds of amino acid are commonly found in protein, and four main kinds of base occur in nucleic acid. The genetic code describes the way in which a sequence of twenty or more things is determined by a sequence of four things of a different type.
The main issue in cracking the genetic code was 1) to determine how many base pairs would code for the 20 amino acids that composed proteins, and 2) discover what series of bases specified which amino acids. Simple mathematics indicated that because there were 20 possible amino acids, and there were four bases (A, T, C, G), there had to be at least three bases, a codon, to code for each protein. As Crick again noted,
This can hardly be done by a pair of bases, as from four different things we can only form 4 × 4 = 16 different pairs, whereas we need at least twenty and probably one or two more to act as spaces or for other purposes. However, triplets of bases would give us 64 possibilities. It is convenient to have a word for a set of bases which codes one amino acid and I shall use the word “codon” for this.
But could this theoretical supposition be proven in the laboratory? In a 1961 experiment, Crick and his colleague, South African-born biologist Sydney Brenner (1927–), created genetic mutations in the DNA of a bacteriophage, a virus that infects bacteria. Crick and Brenner induced the mutations using a chemical called proflavine to change individual bases in the DNA, destroying the function of a particular and crucial phage gene. They found that if there were two or four mutations together, the gene was still inactive, but if three mutations were put together in the same gene, the gene started working. In other experiments, Crick and Brenner used proflavine to delete bases one by one in the DNA, demonstrating that only after three bases next to each other were eliminated did the DNA transcribing system come back to its correct phase, and the gene would begin to work again.
Their results indicated that the genetic code was one in which three bases coded for an amino acid. As DNA unwinds during replication, it is read by a molecule called RNA polymerase this, in turn, makes a template for protein synthesis called messenger RNA (mRNA), a process called transcription. An organelle called a ribosome then “reads” each codon in the mRNA and another carrier molecule called transfer RNA (tRNA) brings the appropriate amino acid called for by the
codon. The amino acids are then linked together to make the protein by the ribosome. This process is called translation.
After Crick and Brenner's discovery, a key question remained: Which particular triplet of nucleotides (codon) coded for which particular amino acid? In 1961 American zoologist Marshall Nirenberg (1927–) and German biochemist Heinrich Matthaei, working at the National Institutes of Health in Bethesda, Maryland, finally cracked the genetic code. Nirenberg took Escherichia coli (E. coli) bacteria and ground them up with a mortar and pestle to release their cytoplasm, which contained all the organelles, such as ribosomes, needed for protein synthesis.
Nirenberg then made a synthetic RNA made of just the base uracil and combined it with the E. coli cytoplasm to see which amino acid would be created. This created a protein chain made only of phenyalanine, so Nirenberg realized the codon coded for phenylalaline. Similar experiments revealed which codons coded for the nineteen other amino acids. By 1965, Nirenberg understood the language of DNA.
Molecular markers used in forensic genetics
Forensic genetics is a field that has become subject to increasing interest in recent years. Both the technology and the markers used for forensic purposes have changed since the 1980s. The minisatellite sequences used in the famous Pitchfork case introduced genetics to the forensic sciences. Minisatellite sequences have now been replaced by more sensitive microsatellite markers, which have become the basis for the creation of genetic profile databases. Modern molecular methods also exploit single nucleotide polymorphisms, which are often the only way to identify degraded DNA samples. The same type of variation is taken into consideration in attempting to establish the ethnicity of a perpetrator and to determine phenotypic traits such as the eye or hair colour of the individual who is the source of the genetic material. This paper contains a review of the techniques and molecular markers used in human and animal forensic genetics, and also presents the potential trends in forensic genetics such as phenotyping.
Keywords: Forensic genetics SNP STR forensic DNA phenotyping.
Dad’s DNA butts in
It’s not clear why mtDNA prefers being exclusively maternal, but a higher rate of mutation in paternal mtDNA may have something to do with it. With the huge array of mechanisms that different species have evolved to prevent interloping paternal contributions, it seems that evolution is holding males' contribution at arm’s length. And while some species have been found to have “leaking” paternal DNA, including mice and sheep, reports in humans have been very limited. Aside from the case in Denmark, a review of other reports argued that they could all be “ascribed solely to contamination and sample mix-up.”
Taosheng Huang and his colleagues were keen to avoid that kind of problem, so when they found weird patterns in a patient’s mitochondrial DNA, they sent fresh samples to be resequenced. The results came back the same: the four-year-old boy had both paternal and maternal mtDNA, and so did his two sisters.
The detective work was just beginning. Huang and his colleagues sequenced the mtDNA of 11 people in the family, finding a pattern of paternal contributions. When they looked at two other families, both with a family member with suspected mitochondrial disease, they found similar results. Altogether, they found 17 people across the three families with mixed mtDNA. In all cases, there was a backup check: the whole procedure was “repeated independently in at least two different laboratories by different laboratory technicians with newly obtained blood samples,” the researchers write.
Because the researchers explored the genomes of whole families, they were able to work out the pattern of transmission across generations. Some people in the families weren’t affected they just had typical maternal mtDNA. It seemed that if a mother had mixed mtDNA, she passed that mixture directly to the kids—the kids would inherit the same mixture she had, essentially getting male mtDNA from further up in the family tree. But if a father had mixed mtDNA, he passed some of his own mtDNA on to his kids.
All of this pointed to the males in the family as the likely source of the escape hatch to the normal paternal dead-end. The pattern suggests that there could be a gene running in the families that allows paternal mtDNA to hitch a ride into the egg with their sperm, and then persist there—and that gene is probably in the normal, nuclear genome rather than the mtDNA itself. That genetic trait could then get passed down, giving every male that inherits it the capacity to pass their mtDNA to their offspring.
More women than men have added their DNA to the human gene pool
More women than men have contributed to the gene pool of humanity since the first modern humans walked out of Africa around 70,000 years ago, according to a study.
Though the laws of biology state that male and female DNA should contribute roughly equally to the next generation, they are silent on how many of each sex must be involved in the business.
Researchers in Germany found that throughout human history, mothers have regularly outnumbered fathers, meaning that more women have passed on their DNA than men.
There might have been more women than men around in the past, an imbalance that could help to explain the results. But the researchers point to cultural biases, whereby relatively few men got to mate with multiple women and women tended to move home to live with their partners.
“Imagine a population of 100 females and 100 males,” said Mark Stoneking, who led the study at the Max Planck Institute for Evolutionary Anthropology. “If all the females but only one of the males reproduced, then while the males and females contribute 50:50 to the next generation, the male contribution is all from just one male.” The next generation would all have the same Y chromosome but 100 different sets of mitochondrial DNA, which is passed solely down the maternal line.
Stoneking’s team gathered together the genomes of 623 men from 51 populations around the world. They then compared the genetic diversity of the male Y chromosomes with the diversity of the men’s mitochondrial DNA.
They found that genetic differences between human populations were almost always larger for the Y chromosome than for mitochondrial DNA. The only exception was East Asia.
Using computer models, the researchers showed that the differences in genetic diversity arose if more women than men were breeding throughout human history. According to their simulation, an ancestral population of 60 women and 30 men were breeding in Africa before humans left the continent. The numbers fell to around 25 women and 15 men breeding at the time of the first migration of Homo sapiens, around 70,000 years ago. The whole population would have been larger, but the extras were not contributing to the gene pool.
As modern humans moved into Europe more than 45,000 years ago, the number of mothers may have outnumbered fathers by around 100 to 30, according to Stoneking. His study appears in the journal, Investigative Genetics.
In static populations, genetic diversity falls over time because some people do not have children, so their genetic quirks die out. But the tradition of women moving to be with their partners helped to counter the genetic decline by importing fresh DNA.
“What we’ve found is that there are significant differences in the history of human males and females in different parts of the world. Understanding why that’s the case and what are the social historical processes that led to those differences are what we want to investigate now,” said Stoneking.