MB 16.1

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What is our genetic endowment?

Chemically speaking, your genetic endowment is the DNA you inherited from your parents. DNA, the substance of inheritance is the most celebrated molecule of our time.

Why are nucleic acids unique?

Of all nature's molecules, nucleic acids are unique in their ability to direct their own replication from monomers. Indeed, the resemblance of offspring to their parents has its basis in the precise replication of DNA and its transmission from one generation to the next. Hereditary information is encoded in the chemical language of DNA and reproduced in all the cells of your body.

Why is DNA so damn important?

It is this DNA program that directs the development of your biochemical, anatomical, physiological, and to some extent, behavioral traits.

What will you learn in this chapter?

In this chapter, you will discover how biologists deduced that DNA is the genetic material and how Watson and Crick worked out its structure. You will also learn about DNA replication, the process by which a DNA molecule is copied, and how cells repair their DNA. Finally, you will explore how a molecule of DNA is packaged together with proteins in a chromosome.

Why was the case for proteins being the candidate for genetic material stronger than DNA before the 1940s?

Until the 1940s, the case for proteins seemed stronger, especially since biochemists had identified them as a class of macromolecules with great heterogeneity and specificity of function, essential requirements for the hereditary material.

Moreover, little was known about nucleic acids, whose physical and chemical properties seemed far too uniform to account for the multitude of specific inherited traits exhibited by every organism.

How did the view change to DNA becoming the stronger candidate for genetic material?

The view gradually changed as experiments with microorganisms yielded unexpected results. As with the work of Mendel and Morgan, a key factor in determining the identity of the genetic material was the choice of appropriate experimental organisms.

When was the role of DNA in hereditary first worked out?

It was worked out while studying bacteria and the viruses that infect them, which are far simpler than pea plants, fruit flies or humans.

In what year was the discovery of the genetic role of DNA made and by whom?

The discovery of the genetic role of DNA dates back to 1928. While attempting to develop a vaccine against pneumonia, a British medical officer named Frederick Griffith was studying Streptococcus pneumoniae, a bacterium that causes pneumonia in mammals.

Griffith had two strains (varieties) of the bacterium, one pathogenic (disease-causing) and one nonpathogenic (harmless). He was surprised to find that when he mixed the killed pathogenic bacteria with heat and then mixed the cell remains with living bacteria of the nonpathogenic strain, some of the living cells became pathogenic. Furthermore, this newly acquired trait of pathogenicity was inherited by all the descendants of the transformed bacteria.

How was Frederick's Griffith experiment set up?

Figure 16.2
Experiment: Frederick Griffith studied two strains of the bacterium Streptococcus pneumoniae. Bacteria of the S (smooth) strain can cause pneumonia in mice; they are pathogenic because they have an outer capsule that protects them from an animal's defense system. Bacteria of the R (rough) strain lack a capsule and are nonpathogenic. To test for the trait of pathogenicity, Griffith injected mice with the two strains.

What did Griffith conclude from his experiment in 1928?

He concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells that allowed the R cells to make capsules.

Define the word transformation.

Transformation is defined as a change in genotype and phenotype due to the assimilation of external DNA by a cell. (This use of the word transformation should not be confused with the conversion of a normal animal cell to a cancerous one.)

What were the three main candidates for the transformation substance that was discovered in Frederick's Griffith experiment, did the bacteriologist Oswald Avery focus on?

Avery focused on DNA, RNA (the other nucleic acid in cells), and protein. Avery broke open the heat-killed pathogenic bacteria and extracted the cellular contents. He treated each of three samples with an agent that inactivated one type of molecule, then tested the sample for its ability to transform live nonpathogenic bacteria. Only when DNA was allowed to remain active did transformation occur.

Why was Oswald Avery's discovery of DNA being the transforming agent met with skepticism?

Their discovery was greeted with interest but considerable skepticism, in part because of the lingering belief that proteins were better for the genetic material. Moreover, many biologists were not convinced that the genes of bacteria would be similar in composition and function to those of more complex organisms. But the major reason for the continued doubt was that so little was known about DNA.

What are bacteriophages (phages for short)?

They are viruses that infect bacteria.

What is a virus?

A virus is a little more than DNA (or sometimes RNA) enclosed by a protective coat, which is often simply protein.

How does a virus reproduce?

To produce more viruses, a virus must infect a cell and take over the cell's metabolic machinery.

What did Alfred Hershey and Martha Chase do in 1952?

They performed experiments showing that DNA is the genetic material of a phage known as T2.

What organisms does the virus T2 infect?

T2 is one of many phages that infect E.coli, a bacterium that normally lives in the intestines of mammals and is a model organism for molecular biologists. A T2 phage could quickly turn an E.coli cell into a T2-producing factory that released many copies when the cell ruptured. Somehow, T2 could reprogram its host cell to produce viruses. But which viral component - protein or DNA - was responsible?

What 2 chemicals did Alfred Hershey and Martha chase use to trace the fates of protein and DNA of the T2 phages that infected bacterial cells?

Alfred Hershey and Martha Chase used radioactive sulfur and phosphorous to trace the fates of protein and DNA, respectively, of T2 phages that infected bacterial cells. They wanted to see which of these molecules entered the cells and could reprogram them make more phages.

What was the protocol that Hershey and Chase used for their experiment?

Batch 1: Phages were grown with radioactive sulfur, which was incorporated into phage protein (pink).

Batch 2: Phages were grown with radioactive phosphorus, which was incorporated into phage DNA (blue).

1. Mixed radioactively phages with bacteria. The phages infected the bacterial cells.

2. Agitated the mixture in a blender to free phage parts outside the bacteria from the cells.

3. Centrifuged the mixture so that bacteria formed a pellet at the bottom of the test tube; free phages and phage parts, which are lighter, remained suspended in the liquid.

4. Measured the radioactivity in the pellet and the liquid. The radioactivity (phage protein) was found in the liquid.

What were the results of Hershey and Chase's experiment?

When the proteins were labeled (batch 1), radioactivity remained outside the cells; but when the DNA was labeled (batch 2), radioactivity was found inside the cells. Bacterial cells with radioactive phage DNA released new phages with some radioactive phosphorus.

The researchers concluded that Phage DNA entered bacterial cells, but phage proteins did not. Hershey and Chase concluded that DNA, not protein, functions as the genetic material of phage T2.

The Hershey-Chase experiment was a landmark study because it provided powerful evidence that nucleic acids, rather than proteins, are the hereditary material, at least for viruses.

What are nucleotides made up of?

It was already known that DNA is a polymer of nucleotides, each consisting of three components: a nitrogenous (nitrogen-containing) base, a pentose sugar called deoxyribose, and a phosphate group. The base can be adenine (A), thymine (T), guanine (G), or cytosine (C).

How did Erwin Chargaff provide further evidence that DNA is the genetic material of life?

Chargaff analyzed the base composition of DNA from a number of different organisms. In 1950 he reported that the base composition of DNA varies from one species to another. For example, 30.4% of human DNA nucleotides have the base A, whereas DNA from the bacterium E.coli has only 24.7% A. This evidence of molecular diversity among species, which had been presumed absent from DNA, made DNA a more credible candidate for the genetic material.

What is the percentage of the four bases A,C,T,G in human DNA?

In the DNA of each species Chargaff studied, the number of adenines approximately equaled the number of thymines, and the number of guanines approximately equaled the number of cytosines. In human DNA for example, the four bases are present in these percentages: A = 30.4% and T = 30.1%; G = 19.6% and C = 19.9%.

What are Chargaff's rules?

1. The base composition varies between species

2. Within a species, the number of A and T bases are equal and the number of G and C bases are equal.

The basis for these rules remained unexplained until the discovery of the double helix.

Describe the structure of a DNA strand.

Each DNA nucleotide monomer consists of a nitrogenous base (A, T, C, or G), the sugar deoxyribose, and a phosphate group. The phosphate group of one nucleotide is attached to the sugar of the next, forming a "backbone" of alternating phosphates and sugars from which the bases project.

The polynucleotide strand has a directionality, from the 5' end (with the phosphate group) to the 3' end (with the OH group of the sugar). 5' and 3' refer to the numbers assigned to the carbons in the sugar ring.

What was the challenge for biology, once most biologists were convinced that DNA was the genetic material?

Once most biologists were convinced that DNA was the genetic material, the challenge was to determine how the structure of DNA could account for its role in inheritance. By the early 1950s, the arrangement of covalent bonds in a nucleic acid polymer was well established, and researchers focused on discovering the three-dimensional structure of DNA.

How did Crick and Watson's partnership begin?

The brief but celebrated partnership that solved the puzzle of DNA structure began soon after Watson journeyed to Cambridge University, where Crick was studying protein structure with a technique called X-ray crystallography.

What did Watson see while in the Wilkin's Lab?

While visiting the laboratory of Maurice Wilkins, Watson saw an X-ray diffraction image of DNA produced by Wilkin's accomplished colleague Rosalind Franklin. Images produced by the X-crystallography are not actually pictures of molecules. The spots and smudges were produced by X-rays that were diffracted (deflected) as they passed through aligned fibers of purified DNA.

How did Watson figure out that DNA was helical in shape?

Watson was familiar with the type of X-ray diffraction pattern that helical molecules produce, and an examination of the photo that Wilkins showed him confirmed that DNA was helical in shape.

It also augmented earlier data obtained by Franklin and others suggesting the width of the helix and the space of the nitrogenous bases along it. The pattern in this photo implied that the helix was made up of two strands, contrary to a three-stranded model that Linus Pauling had proposed a short time earlier. The presence of two strands accounts for the now-familiar term double helix.

What role did Rosalind Franklin play in the discovery of DNA?

Franklin, a very accomplished X-ray crystallographer, conducted critical experiments resulting in the photograph that allowed Watson and Crick to deduce the double-helical structure of DNA.

What are the key features of the DNA structure?

The helix is "right handed", curving up to the right. The two strands are held together by hydrogen bonds between the nitrogenous bases, which are paired in the interior of the double helix.

What is the partial chemical structure of DNA?

Strong covalent bonds link the units of each strand, while weaker hydrogen bonds hold one strand to the other. Notice that the strands are antiparallel, meaning that they are oriented in opposite directions.

What role do Van der Waals forces play in DNA?

Van der Waals interactions between the stacked pairs play a major role in holding the molecule together.

How did Watson and Crick go about building models of a double helix?

Watson and Crick began building models of a double helix that would conform to the X-ray measurements and what was then known about the chemistry of DNA, including Chargaff's rule of base equivalences.

Having also read an unpublished annual report summarizing Franklin's work, they knew she had concluded that the sugar-phosphate backbones were on the outside of the DNA model, contrary to their working model.

Why was Rosalind Franklin's arrangement of the bases appealing?

Franklin's arrangement was appealing because it put the relatively hydrophobic nitrogenous bases in the molecules interior, away from the surrounding aqueous solution, and the negatively charged phosphate groups wouldn't be forced together in the interior.

Watson constructed a model with the nitrogenous bases facing the interior of the double helix. In this model, the two sugar-phosphate backbones are antiparallel - that is, their subunits run in opposite directions.

How is the DNA double similar to a rope ladder?

You can imagine the overall arrangement as a rope ladder with rigid rungs. The side ropes represent the sugar phosphate backbones, and the rungs represent pairs of nitrogenous bases.

Now imagine holding one end of the ladder and twisting the other, forming a spiral. Franklin's X-ray data indicated that the helix makes one full turn every 3.4 nm along its length. With the basses stacked just 0.34 nm apart, there are ten layers of base rungs, or rungs of the ladder, in each full turn of the helix.

How did Watson and Crick discover that the bases A and T paired together and that C and G paired together?

The nitrogenous bases of the double helix are paired in specific combinations : A with T and C with G. It was mainly by trial and error that Watson and Crick arrived at this key feature of DNA. At first Watson imagined that the bases paired like with like, for example A with A and C with C. But this model did not fit the X-ray data, which suggested that the double helix had a uniform diameter.

What are purines?

Purines are nitrogenous bases with two organic rings. Adenine and guanine are purines.

What are pyrimidines?

They are nitrogenous bases that have a single ring. Cytosine and thymine are pyrimidines.

Why did like with like pairing of the bases not work?

Purines are twice as wide as pyrimidines. A purine-purine pair is too wide and a pyrimidine-pyrimidine pair is too narrow for the 2-nm diameter of the double helix. Always pairing a purine with a pyrimidine, however, results in a uniform diameter.

How many hydrogen bonds are formed between the bases A and T? C and G?

Watson and Crick reasoned that there must be additional specificity of pairing dictated by the structure of the bases. Each base has chemical side groups that can form hydrogen bonds with its appropriate partner. Adenine can form two hydrogen bonds with thymine and only thymine; guanine forms three hydrogen bonds with cytosine and only cytosine. In shorthand A pairs with T, and G pairs with C.

How did Watson and Crick's model of DNA explain Chargaff's ratios?

The Watson-Crick model took into account Chargaff's ratios and ultimately explained them. Wherever one strand of a DNA molecule has an A, the partner strand has a T. And a G in one strand is always paired with a C in the complementary strand.

Therefore, in the DNA of any organism, the amount of A equals the amount of T, and the amount of G equals the amount of C. Although the base pairing rules dictate the combinations of nitrogenous bases that form the "rungs" of the double helix, they do not restrict the sequence of nucleotides along each DNA strand. The linear sequence of the four bases can be varied in countless ways, and each gene has a unique order, or base sequence.

When did Watson and Crick turn in their paper to Nature?

In April 1953, Watson and Crick surprised the scientific world with a succinct, one-page paper in the journal Nature. The paper reported their molecular model for DNA: the double helix, which has since become the symbol of molecular biology.

Watson and Crick, along with Maurice Wilkins, were awarded the Nobel Prize in 1962 for this work. (Sadly, Rosalind Franklin died at the age of 38, in 1958, and was thus ineligible for the prize.) The beauty of the double helix model was that the structure of DNA suggested the basic mechanism of its replication.

A fly has the following percentages of nucleotides in its DNA: A = 27.3, T = 27.6, G = 22.5, C = 22.5. How do these numbers demonstrate Chargaff's rule about base ratios?

Chargaff's rule about base ratios states that in DNA, the percentages of A and T are essentially the same, as are those of G and C. The fly data are consistent with that rule. (Slight variations are most likely due to limitations of analytical technique.)

Given a polynucleotide sequence such as GAATTC, can you tell which is the 5' end? If not, what further information do you need to identify the ends?

You can't tell which is the 5' end. You need to know which end has a phosphate group on the 5' end, or which end has an OH group on the 3' end.

Griffith did not expect transformation to occur in his experiment. What results was he expecting? Explain.

He was expecting that the mouse injected with the mixture of heat-killed S cells and living R cells would survive, since neither type of cell alone would kill the mouse.

How would the results of the Chase-Hershey experiment differed if proteins carried the genetic information?

The radioactivity would have been found in the pellet when proteins were labeled because the proteins would have had to enter the bacterial cells to program them with genetic instructions.

Its hard for us to imagine now, but the DNA might have played a structural role that allowed some of the proteins to be projected while it remained outside the bacterial cells.

How did this experiment rule out the possibility that the R cells could have simply used the capsules of the dead S cells to become pathogenic?

The living S cells found in the blood cell were able to reproduce to yield more S cells, indicating that the S trait is a permanent, heritable change, rather than just a one-time use of the dead S cell's capsules.

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