This pairs a keto base with an amino base, a purine with a pyrimidine. This third H-bond in the G:C base pair is between the additional exocyclic amino group on G and the C2 keto group on C.
The pyrimidine C2 keto group is not involved in hydrogen bonding in the A:T base pair. These are the complementary base pairs. This will be encountered during recombination in Chapter 8. Rather the two strands are coiled around the same helical axis and are intertwined with themselves which is referred to as a plectonemic coil. One consequence of this intertwining is that the two strands cannot be separated without the DNA rotating, one turn of the DNA for every "untwisting" of the two strands.
The N7 and C6 groups of purines and the C4 and C5 groups of pyrimidines face into the major groove, thus they can make specific contacts with amino acids in DNA-binding proteins.
Base pairs stack, with some rotation between them. Three different forms of duplex nucleic acid have been described. The most common form, present in most DNA at neutral pH and physiological salt concentrations, is B-form. That is the classic, right-handed double helical structure we have been discussing. As the temperature rises, defects grow in size and increase in number. The G. C-rich segments are the last to go.
Thus, if the temperature dependence of the fraction of denatured DNA is carefully measured, the resulting curve will feature sharp peaks corresponding to the partially melted structures Fig. The denaturation process ends in the strands coming fully apart. This can be demonstrated by direct methods, such as measurement of the molecular weight in denatured DNA it is roughly half as high as in the native one , electron microscopy, separation of strands if they differ in buoyant density by ultracentrifugation in the cesium salt density gradient, and so on.
In the case of DNA, the above-mentioned linear relationship between T m , of helical double-stranded polynucleotides and the content of G.
C pairs in their molecules has practical ramifications. Indeed, by experimentally determining T m of DNA in a solution of a particular ionic strength one can find its overall nucleotide composition from a plot e. Since T m of helical double-stranded polynucleotides is also related linearly to the logarithm of salt concentration, the content of G - C pairs in DNA can be determined using a more general relation:. Alkaline denaturation is often used for full separation of DNA strands.
To this end, the pH value of the DNA solution is brought to Of particular interest is the process of DNA renaturation, whereby DNA molecules pass from the denatured into the original native state with more or less complete restoration of the secondary structure.
Renaturation of DNA calls for the same conditions as formation of helical complexes from synthetic complementary polynucleotides; that is, the temperature must be below T m and the ionic strength of the solution must be sufficiently high. However, in this case, too, the renaturation of DNA proceeds in a much more complex manner by virtue of the heterogeneous nature of its polynucleotide chains.
If partially denatured DNA molecules i. But if renaturation takes place after complete separation of the strands, their recombination and restoration of the original structure are hampered by formation of intermediate structures through pairing of relatively short complementary segments in the same or different polynucleotide chains. Therefore, the renaturation of DNA is conducted in such a way that the emerging spurious imperfect structures could fall apart again.
A typical example is so-called renaturation by "annealing" when the DNA solution is very slowly cooled after denaturation. It is clear that the greater the number of repeating complementary sequences in a DNA molecule and the longer they are, the lower the yield of renatured molecules will be. For instance, renaturation of animal DNAs in which such segments are numerous is slow and with a very low yield.
On the contrary, in the case of bacteriophage DNAs practically free of long repeating sequences, renaturation proceeds with a high yield. Melting temperature of DNAs versus content of G.
C pairs. The dots on the straight line correspond to different DNAs whose nucleotide composition has been determined by direct chemical methods. For a solution with ionic strength of 0. Renaturation of DNAs is used to solve quite a few biological problems. A case in point is experiments aimed at isolating so-called "individual" genes.
Although such experiments are merely of historical interest, they provide a vivid illustration of the possibilities of the DNA denaturation-renaturation method. Very briefly, these experiments boil down to the following. Two totally different usually viral DNA molecules were selected. These DNAs differed in nucleotide sequence over the entire length with the exception of the segment encoding this common protein.
Both types of DNA molecules were mixed, denatured, then placed under conditions optimal for their renaturation. Among fully renatured molecules and those remaining in the denatured state there appeared molecules formed by strands of different DNAs, which had paired over a complementary segment the latter being much longer than the duplex helical segments in imperfect intermediate structures.
The reaction mixture was then treated with DNase specifically hydrolysing single-stranded polynucleotides. Such treatment leaves fully renatured starting DNAs in the mixture along with double-stranded DNA fragments containing "individual" genes; both differ widely in molecular weight and can easily be separated from each other Fig. It has become routine in molecular biology to study the kinetics of the process of renaturation or reassociation of DNAs as well as DNAs and complementary RNAs the latter process is normally referred to as molecular hybridization.
Such studies are usually conducted with a view to determining the degree of similarity or, in the case of RNA-DNA hybridization, complementarity of the nucleotide sequences of two nucleic acids. While studying the DNA reassociation kinetics one can also glean interesting information about the principles on which the genetic material is organized.
In addition to the usual parameters ionic strength and temperature , the rate of reassociation of a denatured DNA is also dependent on its concentration and size. Therefore, DNA molecules are broken down in advance to fragments of a more or less the same size with a relatively small molecular weight. After denaturation, the DNA is placed under conditions optimal for renaturation ionic strength of about 0.
The degree of DNA reassociation at a given point in time is determined in different ways: by the hypochromic effect, by estimating the single-stranded DNA fraction hydrolysed with nucleases specific toward such DNA, and by chromatographic methods making it possible to separate the native renatured and denatured DNAs.
Scheme illustrating how an individual DNA fragment is obtained by the denaturation-renaturation method. Since the DNA reassociation process is a bimolecular reaction, the rate at which the amount of single-stranded DNA decreases is given by the equation. As can be seen from this equation, the amount of denatured DNA not yet reassociated by instant t is a function of C 0 t. The shape of the reassociation curve depends on the number and size of the recurring nucleotide sequences in the nucleic acid molecule.
In the case of viral and bacterial DNAs, the curve in its entirety fits the two logarithmic intervals C 0 t Fig.
This plot also shows an association curve for complementary polynucleotides that may be regarded as nucleic acids containing only one pair of nucleotides. Idealized reassociation curve for denatured DNA chains. Reassociation curves for denatured polynucleotides of different origin. Around the same time, researchers James Watson and Francis Crick were pursuing a definitive model for the stable structure of DNA inside cell nuclei. Watson and Crick ultimately used Franklin's images, along with their own evidence for the double-stranded nature of DNA, to argue that DNA actually takes the form of a double helix , a ladder-like structure that is twisted along its entire length Figure 6.
Franklin, Watson, and Crick all published articles describing their related findings in the same issue of Nature in Most cells are incredibly small. For instance, one human alone consists of approximately trillion cells. Yet, if all of the DNA within just one of these cells were arranged into a single straight piece, that DNA would be nearly two meters long!
So, how can this much DNA be made to fit within a cell? The answer to this question lies in the process known as DNA packaging , which is the phenomenon of fitting DNA into dense compact forms Figure 7. During DNA packaging, long pieces of double-stranded DNA are tightly looped, coiled, and folded so that they fit easily within the cell. Eukaryotes accomplish this feat by wrapping their DNA around special proteins called histones , thereby compacting it enough to fit inside the nucleus Figure 8.
Together, eukaryotic DNA and the histone proteins that hold it together in a coiled form is called chromatin. It is impossible for researchers to see double-stranded DNA with the naked eye — unless, that is, they have a large amount of it. Modern laboratory techniques allow scientists to extract DNA from tissue samples, thereby pooling together miniscule amounts of DNA from thousands of individual cells. When this DNA is collected and purified, the result is a whitish, sticky substance that is somewhat translucent.
To actually visualize the double-helical structure of DNA, researchers require special imaging technology, such as the X-ray diffraction used by Rosalind Franklin. However, it is possible to see chromosomes with a standard light microscope, as long as the chromosomes are in their most condensed form. To see chromosomes in this way, scientists must first use a chemical process that attaches the chromosomes to a glass slide and stains or "paints" them.
Staining makes the chromosomes easier to see under the microscope. In addition, the banding patterns that appear on individual chromosomes as a result of the staining process are unique to each pair of chromosomes, so they allow researchers to distinguish different chromosomes from one another. Then, after a scientist has visualized all of the chromosomes within a cell and captured images of them, he or she can arrange these images to make a composite picture called a karyotype Figure This page appears in the following eBook.
Aa Aa Aa. What components make up DNA? Figure 1: A single nucleotide contains a nitrogenous base red , a deoxyribose sugar molecule gray , and a phosphate group attached to the 5' side of the sugar indicated by light gray.
Opposite to the 5' side of the sugar molecule is the 3' side dark gray , which has a free hydroxyl group attached not shown. Figure 2: The four nitrogenous bases that compose DNA nucleotides are shown in bright colors: adenine A, green , thymine T, red , cytosine C, orange , and guanine G, blue. Although nucleotides derive their names from the nitrogenous bases they contain, they owe much of their structure and bonding capabilities to their deoxyribose molecule. The central portion of this molecule contains five carbon atoms arranged in the shape of a ring, and each carbon in the ring is referred to by a number followed by the prime symbol '.
Of these carbons, the 5' carbon atom is particularly notable, because it is the site at which the phosphate group is attached to the nucleotide. Appropriately, the area surrounding this carbon atom is known as the 5' end of the nucleotide.
Opposite the 5' carbon, on the other side of the deoxyribose ring, is the 3' carbon, which is not attached to a phosphate group. This portion of the nucleotide is typically referred to as the 3' end Figure 1. When nucleotides join together in a series, they form a structure known as a polynucleotide.
At each point of juncture within a polynucleotide, the 5' end of one nucleotide attaches to the 3' end of the adjacent nucleotide through a connection called a phosphodiester bond Figure 3.
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