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19.3: Deoxyribonucleic Acid (DNA) - Biology

19.3: Deoxyribonucleic Acid (DNA) - Biology


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Learning Objectives

  1. State the three basic parts of a deoxyribonucleotide.
  2. State which nitrogenous bases are purines and which are pyrimidines.
  3. Define complementary base pairing.
  4. State why DNA can only be synthesized in a 5' to 3' direction.
  5. Compare the prokaryotic nucleoid with the eukaryotic nucleus in terms of the following:
    1. number of chromosomes
    2. linear or circular chromosomes
    3. presence or absence of a nuclear membrane
    4. presence or absence of nucleosomes
    5. presence or absence of mitosis
    6. presence or absence of meiosis

DNA is a long, double-stranded, helical molecule composed of building blocks called deoxyribonucleotides. Each deoxyribonucleotide is composed of three parts: a molecule of the 5-carbon sugar deoxyribose, a nitrogenous base, and a phosphate group (Figure (PageIndex{1})).

  • Deoxyribose. Deoxyribose is a ringed 5-carbon sugar (Figure (PageIndex{2})). The 5 carbons are numbered sequentially clockwise around the sugar. The first 4 carbons actually form the ring of the sugar with the 5' carbon coming off of the 4' carbon in the ring. The nitrogenous base of the nucleotide is attached to the 1' carbon of the sugar and the phosphate group is bound to the 5' carbon. During DNA synthesis, the phosphate group of a new deoxyribonucleotide is covalently attached by the enzyme DNA polymerase to the 3' carbon of a nucleotide already in the chain.
  • A nitrogenous base. There are four nitrogenous bases found in DNA: adenine, guanine, cytosine, or thymine. Adenine and guanine are known as purine bases while cytosine and thymine are known as pyrimidine bases (Figure (PageIndex{3})).
  • A phosphate group (Figure (PageIndex{4})).

To synthesize the two chains of deoxyribonucleotides during DNA replication, the DNA polymerase enzymes involved are only able to join the phosphate group at the 5' carbon of a new nucleotide to the hydroxyl (OH) group of the 3' carbon of a nucleotide (Figure (PageIndex{2})) already in the chain. The covalent bond that joins the nucleotides is called a phosphodiester bond. Each DNA strand has what is called a 5' end and a 3' end. This means that one end of each DNA strand, called the 5' end , will always have a phosphate group attached to the 5' carbon of its terminal deoxyribonucleotide (Figure (PageIndex{5})). The other end of that strand, called the 3' end, will always have a hydroxyl (OH) on the 3' carbon of its terminal deoxyribonulceotide.

As will be seen in the next section, each parent strand, during DNA replication, acts as a template for the synthesis of the other strand by way of complementary base pairing. Complementary base pairing refers to DNA nucleotides with the base adenine only forming hydrogen bonds with nucleotides having the base thymine (A-T). Likewise, nucleotides with the base guanine can hydrogen bond only with nucleotides having the base cytosine (G-C). (In the case of RNA nucleotides, as will be seen later, adenine nucleotides form hydrogen bonds with nucleotides having the base uracil since thymine is not found in RNA.) As a result of this bonding, the DNA assumes its helical shape. Therefore, the two strands of DNA are said to be complementary. Wherever one strand has an adenine-containing nucleotide, the opposite strand will always have a thymine nucleotide; wherever there is a guanine-containing nucleotide, the opposite strand will always have a cytosine nucleotide (Figure (PageIndex{1})).

While the two strands of DNA are complementary, they are oriented in opposite directions to each other. One strand is said to run 5' to 3'; the opposite DNA strand runs antiparallel, or 3' to 5' (Figure (PageIndex{1})).

We will now briefly compare the genome of prokaryotic cells with that of eukaryotic cells.

The Prokaryotic (Bacterial) Genome

The area within a bacterium where the chromosome can be seen with an electron microscope is called a nucleoid. The chromosome of most prokaryotes is typically one long, single molecule of double stranded, helical, supercoiled DNA which forms a physical and genetic circle. The chromosome is generally around 1000 µm long and frequently contains around 4000 genes (Figure (PageIndex{8})). Escherichia coli, which is 2-3 µm in length has a chromosome approximately 1400 µm long. To enable a macromolecule this large to fit within the bacterium, histone-like proteins bind to the DNA, segregating the DNA molecule into around 50 chromosomal domains and making it more compact. A DNA topoisomerase enzyme called DNA gyrase then supercoils the chromosome into a tight bundle forming a compacted, supercoiled mass of DNA approximately 0.2 µm in diameter.

Bacterial enzymes called DNA topoisomerases are essential in the unwinding, replication, and rewinding of the circular, supercoiled bacterial DNA (Figure (PageIndex{7})). They are also essential in transcription of DNA into RNA, in DNA repair, and in genetic recombination in bacteria.

Figure (PageIndex{7}): Circular, Supercoiled Prokaryotic DNA. To enable the large DNA molecyle to fit within the bacterium, a DNA topoisomerase enzyme called DNA gyrase supercoils the chromosome into a tight bundle forming a compacted, supercoiled mass of DNA approximately 0.2 µm in diameter.

The prokaryotic nucleoid has no nuclear membrane surrounding the DNA and the nuclear body does not divide by mitosis. The cytoplasmic membrane plays a role in DNA separation during bacterial replication. Since bacteria are haploid (have only one chromosome), there is also no meiosis.

The Eukaryotic Genome

Prokaryotic and eukaryotic cells differ a great detail in both the amount and the organization of their molecules of DNA. Eukaryotic cells contain much more DNA than do bacteria, and this DNA is organized as multiple chromosomes located within a nucleus.

The nucleus in eukaryotic cells is surrounded by a nuclear membrane (Figure (PageIndex{7})) and contains linear chromosomes composed of negatively charged DNA associated with positively charged basic proteins called histones to form structures known as nucleosomes. The nucleosomes are part of what is called chromatin, the DNA and proteins that make up the chromosomes. The nucleus divides my mitosis and haploid sex cells are produced from diploid cells by meiosis.

The DNA in eukaryotic cells is packaged in a highly organized way. It consists of a basic unit called a nucleosome, a beadlike structure 11 nm in diameter that consists of 146 base pairs of DNA wrapped around eight histone molecules. The nucleosomes are linked to one another by a segment of DNA approximately 60 base pairs long called linker DNA (Figure (PageIndex{9})). Another histone associated with the linker DNA then packages adjacent nucleotides together to form a nucleosome thread 30nm in diameter. Finally, these packaged nucleosome threads form large coiled loops that are held together by nonhistone scaffolding proteins. These coiled loops on the scaffolding proteins interact to form the condensed chromatin seen in chromosomes during mitosis (Figure (PageIndex{10})).

In recent years its been found that the structural nature of the deoxyribonucleoprotein contributes to whether or not DNA is transcribed into RNA. For example, chemical changes to the chromatin can enable portions of it to condense or relax. When a region is condensed, genes cannot be transcribed. In addition, chemical can attach to or be removed from the histone proteins around which the DNA wraps. The attachment or removal of these chemical groups to the histone determines whether nearby gene expression is amplified or repressed.

The epigenome refers to a variety of chemical compounds that modify the genome typically by adding a methyl (CH3) group to the nucleotide base adenine at specific locations along the DNA molecule. This methylation can, in turn, either repress or activate transcription of specific genes. By basically turning genes on or off, the epigenome enables the genome to interact with and respond to the cell's environment. The epigenome can be inherited just like the genome.

Summary

  1. Deoxyribonucleic acid (DNA) is a long, double-stranded, helical molecule composed of building blocks called deoxyribonucleotides.
  2. A deoxyribonucleotide is composed of 3 parts: a molecule of the 5-carbon sugar deoxyribose, a nitrogenous base, and a phosphate group.
  3. There are four nitrogenous bases found in DNA: adenine, guanine, cytosine, or thymine. Adenine and guanine are known as purine bases while cytosine and thymine are known as pyrimidine bases.
  4. Deoxyribose is a ringed 5-carbon sugar. The nitrogenous base of the nucleotide is attached to the 1' carbon of the sugar and the phosphate group is bound to the 5' carbon.
  5. During DNA synthesis, the enzyme DNA polymerase can only attach the phosphate group of a new deoxyribonucleotide to the 3' carbon of a nucleotide already in the chain.
  6. During DNA replication, each parent strand acts as a template for the synthesis of the other strand by way of complementary base pairing.
  7. Complementary base pairing refers to DNA nucleotides with the base adenine only forming hydrogen bonds with nucleotides having the base thymine (A-T). Likewise, nucleotides with the base guanine can hydrogen bond only with nucleotides having the base cytosine (G-C).
  8. While the two strands of DNA are complementary, they are oriented in opposite directions to each other. One strand is said to run 5' to 3'; the opposite DNA strand runs antiparallel, or 3' to 5'.
  9. In prokaryotic cells there is no nuclear membrane surrounding the DNA. Prokaryotic cells lack mitosis and meiosis.
  10. To enable a macromolecule this large to fit within the bacterium, histone-like proteins bind to the DNA, segregating the DNA molecule into around 50 chromosomal domains and making it more compact. Then an enzyme called DNA gyrase supercoils each domain around itself forming a compacted, supercoiled mass of DNA. A topoisomerase called DNA gyrase catalyzes the negative supercoiling of the circular DNA found in bacteria. Topoisomerase IV, on the other hand, is involved in the relaxation of the supercoiled circular DNA, enabling the separation of the interlinked daughter chromosomes at the end of bacterial DNA replication.
  11. The DNA in eukaryotic cells is packaged in basic units called a nucleosomes, a beadlike structure consisting of DNA wrapped around eight histone molecules. The DNA is organized as multiple chromosomes located within a nucleus surrounded by a nuclear membrane. The nucleus divides by mitosis and gametes are produced by meiosis in eukaryotes reproducing sexually.
  12. The structural nature of the deoxyribonucleoprtein contributes to whether or not DNA is transcribed into RNA. The attachment or removal of these chemical groups to the histone determines whether nearby gene expression is amplified or repressed.

Growth inhibition induced by chronic dexamethasone treatment of foals

Pony and horse foals were given daily intramuscular injections of dexamethasone, beginning at 6 months of age. Ponies were injected with 0 or 0.5 mg/100 kg bodyweight for 3, 8, or 11 months, or with 5.0 mg/100 kg for 11 months. Horses were treated with 0 or 5.0 mg/100 kg for 20 weeks.

Rates of gain were inhibited (P< .01 ) in both ponies and horses after 8 weeks of treatment with either dose. Weight loss occurred in ponies after 40 weeks of treatment and in horse foals after 16 weeks, although feed intakes and dietary energy and nitrogen digestibilities were not effected by treatment (P>. 10). Urinary nitrogen loss increased within 3 months (P <.01), allowing little increase in total body protein during the period of normally rapid growth.

Samples of left femur growth cartilage taken from ponies killed after 3 and 8 months were analyzed. Deoxyribonucleic acid content (ug/ mg dry cartilage) was doubled by treatment (P <.01), lactate dehydrogenase activities per cell and hexosamine and hexuronic acid contents were decreased 80–90% (P <.01), and hydroxyproline content decreased about 50% (P<.01), compared to tissues from untreated ponies.

Growth inhibition appeared to result from the production of abnormal growth cartilage, deficient in collagen and glycosaminoglycans. A metabolic shift from energy production to intra-chondrocyte energy storage, as reflected by decreased lactate dehydrogenase activities, appeared responsible for the decreased production of cartilage components.

The bodyweight data appearing herein were previously presented at the 25th Annual Convention of the American Association of Equine Practitioners, Miami Beach, 1979.


Deoxyribonucleic acid

a nucleic acid of complex molecular structure occurring in cell nuclei as the basic structure of the genes . DNA is present in all body cells of every species, including unicellular organisms and DNA viruses. The structure of DNA was first described in 1953 by J. D. Watson and F. H. C. Crick.

DNA molecules are linear polymers of small molecules called nucleotides, each of which consists of one molecule of the five-carbon sugar deoxyribose bonded to a phosphate group and to one of four heterocyclic nitrogenous compounds referred to as bases. A single strand of DNA is made by linking the nucleotides together in a chain with bonds between the sugar and phosphate groups of adjacent nucleotides. It thus consists of a backbone of alternating sugar and phosphate groups with a base attached to each sugar as a side chain. The four bases are two purines, adenine (A) and guanine (G), and two pyrimidines, cytosine (C) and thymine (T). Single-stranded DNA can be synthesized with any specified sequence of bases, but in living cells the base sequence has a meaning: it specifies the amino acid sequence of all of the polypeptides and proteins made by the cell. And since all of the enzymes that catalyze biochemical reactions are proteins, the DNA contains the specifications for all of the biochemistry and structure of the cell.

The chemical basis of the genetic code lies in the ability of the bases to form hydrogen bonds with each other. Unlike the covalent bonds holding together the atoms of a single strand of DNA, hydrogen bonds are weak and easily broken and reformed. Hydrogen bonding is governed by the base pairing rule: A always bonds with T, and C always bonds with G. A and T (or C and G) are called complementary bases. The genetic information is read and preserved by the matching up of complementary bases.

In cells, the DNA is double-stranded. The configuration of the DNA molecule resembles a ladder in which the sides are the sugar-phosphate backbones, which are antiparallel (they run in opposite directions), and the rungs are hydrogen-bonded complementary bases thus, the entire sequence along the two strands is complementary. This whole structure is twisted so that the two strands form a double helix. Once before each cell division, a group of proteins splits the two strands apart, and as complementary nucleotides bond to the bases of each strand they are joined to form a new strand. This process is called replication. It results in the exact duplication of the DNA molecule, because each strand serves as a template (pattern) for the synthesis of its complementary strand. When the cell divides, one copy goes to each daughter cell. Thus, the genetic information is passed on from generation to generation without change except for rare mutations, which result from copying errors or incorrectly repaired breaks in the DNA molecule that change the base sequence.

The reading of the genetic code involves two processes: transcription and translation. In transcription, a length of DNA is used as a template to make a complementary strand of messenger RNA (mRNA). RNA (ribonucleic acid) is a nucleic acid like DNA. The only differences are that the sugar, ribose, has an extra oxygen atom, and the pyrimidine base, uracil (U), which also pairs with adenine, replaces thymine. In translation, the mRNA molecule is read by a structure called a ribosome, which produces the polypeptide specified by the mRNA message.

The genetic code is a triplet code. Every triplet of bases along the strand specifies a single amino acid. There are 64 possible triplets (codons) that can be formed from the four bases. Each one specifies that one of 20 different amino acids be inserted in a growing polypeptide chain or marks either the start or the end of a chain.

Two other types of RNA are involved in translation. Ribosomal RNA (rRNA) forms a large part of the ribosome. Transfer RNA (tRNA) is the means by which codons are matched with amino acids. tRNAs are small molecules with several self-complementary sections so that they fold up into a compact structure owing to bonding between complementary bases. One end of the molecule is a three-base anticodon, which bonds to its complementary codon on mRNA molecules. The other end is recognized by a specific enzyme that attaches the correct amino acid to it. During translation, the ribosome proceeds along the mRNA molecule and, as each codon is matched by a specific tRNA, the amino acid it carries is transferred to the growing polypeptide chain, and the process is repeated until the &ldquostop&rdquo codon is reached. Like the mRNA molecules, rRNA and tRNA molecules are formed on DNA templates the genetic material contains the information not only for polypeptide sequences but also for rRNA and tRNA sequences.

There is an enormous amount of information stored in the DNA of a cell. The 48 chromosomes of a human cell contain a total length of about 6 billion base pairs of DNA. This is enough to code for the thousands of enzymes and structural proteins in the cell. DNA is the molecule that directs all of the activities of living cells, including its own reproduction and perpetuation in generation after generation.


Biology 171

By the end of this section, you will be able to do the following:

  • Describe nucleic acids’ structure and define the two types of nucleic acids
  • Explain DNA’s structure and role
  • Explain RNA’s structure and roles

Nucleic acids are the most important macromolecules for the continuity of life. They carry the cell’s genetic blueprint and carry instructions for its functioning.

DNA and RNA

The two main types of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) . DNA is the genetic material in all living organisms, ranging from single-celled bacteria to multicellular mammals. It is in the nucleus of eukaryotes and in the organelles, chloroplasts, and mitochondria. In prokaryotes, the DNA is not enclosed in a membranous envelope.

The cell’s entire genetic content is its genome, and the study of genomes is genomics. In eukaryotic cells but not in prokaryotes, DNA forms a complex with histone proteins to form chromatin, the substance of eukaryotic chromosomes. A chromosome may contain tens of thousands of genes. Many genes contain the information to make protein products. Other genes code for RNA products. DNA controls all of the cellular activities by turning the genes “on” or “off.”

The other type of nucleic acid, RNA, is mostly involved in protein synthesis. The DNA molecules never leave the nucleus but instead use an intermediary to communicate with the rest of the cell. This intermediary is the messenger RNA (mRNA) . Other types of RNA—like rRNA, tRNA, and microRNA—are involved in protein synthesis and its regulation.

DNA and RNA are comprised of monomers that scientists call nucleotides . The nucleotides combine with each other to form a polynucleotide , DNA or RNA. Three components comprise each nucleotide: a nitrogenous base, a pentose (five-carbon) sugar, and a phosphate group ((Figure)). Each nitrogenous base in a nucleotide is attached to a sugar molecule, which is attached to one or more phosphate groups.


The nitrogenous bases, important components of nucleotides, are organic molecules and are so named because they contain carbon and nitrogen. They are bases because they contain an amino group that has the potential of binding an extra hydrogen, and thus decreasing the hydrogen ion concentration in its environment, making it more basic. Each nucleotide in DNA contains one of four possible nitrogenous bases: adenine (A), guanine (G) cytosine (C), and thymine (T).

Scientists classify adenine and guanine as purines . The purine’s primary structure is two carbon-nitrogen rings. Scientists classify cytosine, thymine, and uracil as pyrimidines which have a single carbon-nitrogen ring as their primary structure ((Figure)). Each of these basic carbon-nitrogen rings has different functional groups attached to it. In molecular biology shorthand, we know the nitrogenous bases by their symbols A, T, G, C, and U. DNA contains A, T, G, and C whereas, RNA contains A, U, G, and C.

The pentose sugar in DNA is deoxyribose, and in RNA, the sugar is ribose ((Figure)). The difference between the sugars is the presence of the hydroxyl group on the ribose’s second carbon and hydrogen on the deoxyribose’s second carbon. The carbon atoms of the sugar molecule are numbered as 1′, 2′, 3′, 4′, and 5′ (1′ is read as “one prime”). The phosphate residue attaches to the hydroxyl group of the 5′ carbon of one sugar and the hydroxyl group of the 3′ carbon of the sugar of the next nucleotide, which forms a 5′–3′ phosphodiester linkage. A simple dehydration reaction like the other linkages connecting monomers in macromolecules does not form the phosphodiester linkage. Its formation involves removing two phosphate groups. A polynucleotide may have thousands of such phosphodiester linkages.

DNA Double-Helix Structure

DNA has a double-helix structure ((Figure)). The sugar and phosphate lie on the outside of the helix, forming the DNA’s backbone. The nitrogenous bases are stacked in the interior, like a pair of staircase steps. Hydrogen bonds bind the pairs to each other. Every base pair in the double helix is separated from the next base pair by 0.34 nm. The helix’s two strands run in opposite directions, meaning that the 5′ carbon end of one strand will face the 3′ carbon end of its matching strand. (Scientists call this an antiparallel orientation and is important to DNA replication and in many nucleic acid interactions.)


Only certain types of base pairing are allowed. For example, a certain purine can only pair with a certain pyrimidine. This means A can pair with T, and G can pair with C, as (Figure) shows. This is the base complementary rule. In other words, the DNA strands are complementary to each other. If the sequence of one strand is AATTGGCC, the complementary strand would have the sequence TTAACCGG. During DNA replication, each strand copies itself, resulting in a daughter DNA double helix containing one parental DNA strand and a newly synthesized strand.


A mutation occurs, and adenine replaces cytosine. What impact do you think this will have on the DNA structure?

Ribonucleic acid, or RNA, is mainly involved in the process of protein synthesis under the direction of DNA. RNA is usually single-stranded and is comprised of ribonucleotides that are linked by phosphodiester bonds. A ribonucleotide in the RNA chain contains ribose (the pentose sugar), one of the four nitrogenous bases (A, U, G, and C), and the phosphate group.

There are four major types of RNA: messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and microRNA (miRNA). The first, mRNA, carries the message from DNA, which controls all of the cellular activities in a cell. If a cell requires synthesizing a certain protein, the gene for this product turns “on” and the messenger RNA synthesizes in the nucleus. The RNA base sequence is complementary to the DNA’s coding sequence from which it has been copied. However, in RNA, the base T is absent and U is present instead. If the DNA strand has a sequence AATTGCGC, the sequence of the complementary RNA is UUAACGCG. In the cytoplasm, the mRNA interacts with ribosomes and other cellular machinery ((Figure)).


The mRNA is read in sets of three bases known as codons. Each codon codes for a single amino acid. In this way, the mRNA is read and the protein product is made. Ribosomal RNA (rRNA) is a major constituent of ribosomes on which the mRNA binds. The rRNA ensures the proper alignment of the mRNA and the Ribosomes. The ribosome’s rRNA also has an enzymatic activity (peptidyl transferase) and catalyzes peptide bond formation between two aligned amino acids. Transfer RNA (tRNA) is one of the smallest of the four types of RNA, usually 70–90 nucleotides long. It carries the correct amino acid to the protein synthesis site. It is the base pairing between the tRNA and mRNA that allows for the correct amino acid to insert itself in the polypeptide chain. MicroRNAs are the smallest RNA molecules and their role involves regulating gene expression by interfering with the expression of certain mRNA messages. (Figure) summarizes DNA and RNA features.

DNA and RNA Features
DNA RNA
Function Carries genetic information Involved in protein synthesis
Location Remains in the nucleus Leaves the nucleus
Structure Double helix Usually single-stranded
Sugar Deoxyribose Ribose
Pyrimidines Cytosine, thymine Cytosine, uracil
Purines Adenine, guanine Adenine, guanine

Even though the RNA is single stranded, most RNA types show extensive intramolecular base pairing between complementary sequences, creating a predictable three-dimensional structure essential for their function.

As you have learned, information flow in an organism takes place from DNA to RNA to protein. DNA dictates the structure of mRNA in a process scientists call transcription , and RNA dictates the protein’s structure in a process scientists call translation . This is the Central Dogma of Life, which holds true for all organisms however, exceptions to the rule occur in connection with viral infections.

To learn more about DNA, explore the Howard Hughes Medical Institute BioInteractive resources on DNA (videos, animations, interactives).

Section Summary

Nucleic acids are molecules comprised of nucleotides that direct cellular activities such as cell division and protein synthesis. Pentose sugar, a nitrogenous base, and a phosphate group comprise each nucleotide. There are two types of nucleic acids: DNA and RNA. DNA carries the cell’s genetic blueprint and passes it on from parents to offspring (in the form of chromosomes). It has a double-helical structure with the two strands running in opposite directions, connected by hydrogen bonds, and complementary to each other. RNA is a single-stranded polymer composed of linked nucleotides made up of a pentose sugar (ribose), a nitrogenous base, and a phosphate group. RNA is involved in protein synthesis and its regulation. Messenger RNA (mRNA) copies from the DNA, exports itself from the nucleus to the cytoplasm, and contains information for constructing proteins. Ribosomal RNA (rRNA) is a part of the ribosomes at the site of protein synthesis whereas, transfer RNA (tRNA) carries the amino acid to the site of protein synthesis. The microRNA regulates using mRNA for protein synthesis.

Art Connections

(Figure) A mutation occurs, and cytosine is replaced with adenine. What impact do you think this will have on the DNA structure?

(Figure) Adenine is larger than cytosine and will not be able to base pair properly with the guanine on the opposing strand. This will cause the DNA to bulge. DNA repair enzymes may recognize the bulge and replace the incorrect nucleotide.

Free Response

What are the structural differences between RNA and DNA?

DNA has a double-helix structure. The sugar and the phosphate are on the outside of the helix and the nitrogenous bases are in the interior. The monomers of DNA are nucleotides containing deoxyribose, one of the four nitrogenous bases (A, T, G and C), and a phosphate group. RNA is usually single-stranded and is made of ribonucleotides that are linked by phosphodiester linkages. A ribonucleotide contains ribose (the pentose sugar), one of the four nitrogenous bases (A,U, G, and C), and the phosphate group.

What are the four types of RNA and how do they function?

The four types of RNA are messenger RNA, ribosomal RNA, transfer RNA, and microRNA. Messenger RNA carries the information from the DNA that controls all cellular activities. The mRNA binds to the ribosomes that are constructed of proteins and rRNA, and tRNA transfers the correct amino acid to the site of protein synthesis. microRNA regulates the availability of mRNA for translation.

Glossary


PHYSICAL STRUCTURE AND PROPERTIES OF BROWN COAL

F. Woskoboenko , . D. Raisbeck , in The Science of Victorian Brown Coal , 1991

5.2 RHEOLOGY OF SLURRIES

At low solids concentrations (< 15 wt%) aqueous brown coal slurries are Newtonian and are essentially time-independent. They are weakly aggregating systems and the individual particles and floes will settle out unless agitated. However, as the solids concentration increases the floe size increases, until ultimately the system forms a single floe, i.e. a gel is formed.

Concentrated raw coal suspensions have been found to be stable even after one year of storage ( Woskoboenko, 1985 , and Leong, et al., 1987 ). The formation of floes and networks of particles is responsible for the transition from Newtonian to non-Newtonian flow at volumetric solids concentrations well below the maximum packing density of the particles ( Woskoboenko, 1985 ). The degree of structure in the suspension increases with increasing particle concentration and this is reflected as an increase in yield stress. Furthermore, as the yield stress of the suspensions increases the flow properties become time-dependent. Upon storage the concentrated slurries form gels, and, provided that the “true yield stress” exceeds a initial value τy(crit) ( Equation 11 ) not even the largest particles will settle out of suspension ( Woskoboenko, 1985 ) -

where a is the particle radius, ρc and ρs are the densities of the particles and solvent and g is the gravitational constant. When the gel is sheared the weakest particle-particle bonds are ruptured and the gel is broken down into smaller flocs. The apparent viscosity ratio of shear stress to shear rate of brown coal slurries decreases with time at a given shear rate (thixotropy) and it decreases with increasing shear rate (shear thinning).

Brown coals are unusual in that the gels are fragile and upon agitation are rapidly broken down and steady-state flow properties are attained ( Figure 4.17 ). However, reformation of the gel structure is slow and it may take several weeks for total structure recovery. The breakdown and recovery of the gel structure on shearing and ageing respectively varies from seam to seam ( Leong et al., 1987 ) and for different lithotypes. ( Woskoboenko, Hodges and Krnic, 1987a ) owing to differences in the naturally occurring level of flocculation.

Figure 4.17 . Yield stress as a function of time of mixing or rest for Morwell Coal Slurry (32.7% solids).

The more highly flocculated coals (e.g. Loy Yang) are the least thixotropic ( Leong et al., 1987 ) and, owing to the strength of binding within the floes, have more water immobilised within the floes. As the degree of natural flocculation increases, the following changes occur in the rheology of coal/water slurries ( Woskoboenko, Hodges, Krnic, 1987a Leong et al., 1987 ): (1) The solids concentration at which the transition from Newtonian to non-Newtonian flow occurs increases (2) The solids concentration at which a “true” yield stress becomes apparent increases (3) The maximum achievable solids concentration increases.

The extent of structure formation in a slurry is dependent upon the number of particle-particle bonds that are formed. Thus, the yield stress, which is a measure of brown coal slurry structure, increases as the concentration of particles increases or as the particle size decreases ( Woskoboenko, Siemon and Creasy, 1987 ). In fact, the Bingham yield stress can be directly related to the concentration and particle size via the average interparticle spacing (Dc) in a suspension ( Figure 4.18 ). For a particular sample of Morwell coal the relation was found to be -

Figure 4.18 . Variation of yield stress with mean inter-particle spacing. Aged Morwell coal.( Woskoboenko, Siemon and Creasy, 1987 )


RESULTS

DNA damage produced by doxorubicin and the cytoprotective effect of heat shock

We first used the alkaline comet assay to measure the levels of DNA damage induced by doxorubicin. Previous studies have shown significant differences in the distribution of DNA damage among PBMC from each individual, between individuals according to age (Singh et al 1991), and among smokers depending to the extent of smoking (Dhawan et al 2001). Therefore, to diminish the basal level of damage, a group of nonsmoker control subjects (between 23 and 29 years of age) was selected. Figure 1 shows the different kinds of DNA damage induced by heat shock, doxorubicin alone, and doxorubicin after heat shock, in comparison with a control group. Note the significant difference found between test 1 and test 2 in group 4 (HSʽo), which constitutes the first evidence on the relationship between the recovery period after heat shock (test 1, 4 hours test 2, 24 hours) and the cytoprotective effect on PBMC.

Lymphocytes with silver staining to show comets and immunostained to reveal heat shock protein (Hsp)70. (A) Group 1 (control), high proportion of undamaged cells. (B) Positive control (60 μM H2O2). Note the damaged cells, one with score 3 (arrow) and the other with score 4 (arrow head). (C) Group 2 (heat shock) is represented by undamaged cells and cells with a small level of deoxyribonucleic acid (DNA) damage: score 2 (arrow). (D) Group 3 (21 nM doxorubicin) presents a high proportion of cells with score 4 (more than 50% of damage). (E) Group 4 (heat shock and doxorubicin) of test 1 at T24 presents elevated number of damaged cells, as represented by the heterogeneous sizes and intensity of the comet tails. (F) In contrast, group 4 of test 2 at T24 is characterized by a significant reduction of DNA damage. (G) Cytoplasmic immunostaining of Hsp70. (H) Nuclear expression of Hsp70. Original magnification: Fig A𠄿, 220× Fig G–H, 1370×

Figure 2 shows the detailed evaluation of the comets after hyperthermia and doxorubicin treatments. The heat shock caused a modest decrease in the percentage of cells without damage, with a concomitant small increase in cells with high and total damage ( Fig 2A,C,D ). On the other hand, doxorubicin induced a significant DNA damage ( Fig 2A,C,D ). This was observed in the percentage of cells with high damage for test 1 at T0, test 1 at T24, and test 2 at T0 ( Fig 2C ) and in the percentage of cells with total damage for test 1 at T0, test 2 at T0, and test 2 at T24 ( Fig 2D ). At low levels of damage, no differences were found ( Fig 2B ).

Comet assay, deoxyribonucleic acid damage caused by hyperthermia and doxorubicin. Note in group 4 the difference in cells with none (A), high (C), and total (D) damage between test 1 and test 2 at T24 in group 4. Bars represent mean ± standard error (SEM). Significance of the differences are given by *P < 0.05, **P < 0.01, and ***P < 0.001. C: control HS: heat shock Do: doxorubicin HSʽo: heat shock and doxorubicin

The heat shock protected the PBMC from the damage caused by doxorubicin (group 4), but only in test 2 (24-hour recovery period). This is supported by the significant differences found between test 1 and test 2 at T24 in the percentage of cells with no damage, with high damage, and with total damage ( Fig 2A,C,D , respectively). In addition, the mentioned group in test 1 at T24 showed statistically significant differences in comparison with group 1 (control) in the percentage of undamaged cells ( Fig 2A ), cells with high damage ( Fig 2C ), and cells with total damage ( Fig 2D ). Furthermore, there was also an interesting significant difference in the percentage of cells with high damage ( Fig 2C ) in test 2 at T24 between groups 3 (Do) and 4 (HSʽo), suggesting that the heat shock protected from doxorubicin treatment.

Previous studies have shown that the nature of the cytotoxic effects of doxorubicin was mediated by the process of apoptosis. To verify whether the PBMC that presented a total damage (score 5) in the comet assay were really in apoptosis, we assessed the PARP cleavage by immunocytochemistry and by the TUNEL technique. PARP is a Mr 116� nuclear protein involved in the response to DNA damage. Studies of programmed cell death by genotoxic agents have demonstrated that apoptotic cells show a unique PARP cleavage pattern that is considered an early marker of apoptosis (Kaufmann et al 1993). PARP also showed a strong correlation with acridine orange, which is used to visualize the chromatin condensation pattern characteristic of apoptosis (Whitacre and Berger 1997). In the present study, we analyzed by TUNEL and PARP the group with the highest percentage of cells with total damage: group 4 of test 1 at T24 ( Fig 2D ). We found by linear regression a very good correlation between the comet assay and TUNEL (P = 0.0014, r 2 = 0.9363), between the comet assay and PARP (P = 0.0032, r 2 = 0.9052), and between PARP and TUNEL (P = 0.0001, r 2 = 0.9822).

The DNA repair capacity was calculated after a fixed repair time of 24 hours as the percentage of undamaged cells in each group (groups 3 and 4) divided by the percentage of undamaged cells in group 1 (control) × 100 (Schmezer et al 2001). There was a significant difference between test 1 and test 2 in group 4 (HSʽo) only ( Fig 3 ). These data confirm that the PBMC in group 4 (HSʽo) in test 2 repaired more efficiently the DNA damage caused by doxorubicin than in group 3 (Do). In contrast, there was no repair of the damage in group 4 in test 1, which reflects that the function of the chaperones may depend on the recovery period after heat shock.

Deoxyribonucleic acid (DNA) repair capacity after a fixed repair of 24 hours. In group 3 (Do), there was no significant difference between test 1 and test 2. In contrast, in group 4 (HSʽo), the difference between both tests was statistically significant (* P < 0.05), which reflects the importance of the recovery period after the heat shock in the thermotolerance developed in response to doxorubicin. Percentages of DNA repair capacity are reported (±SEM)

Induction of Hsp27, Hsp60, Hsp70, and Hsp90 by hyperthermia and doxorubicin in vitro in PBMC

We measured by immunocytochemistry Hsp27, Hsp60, Hsp70, and Hsp90 expression in all groups of both tests at T0 and at T24. Mammalian cells are known to synthesize Hsps in vitro after brief exposures to temperatures of 3𠄵ଌ above normal (heat shock). The heat shock performed for 1 hour at 42ଌ induced all the Hsps with 4 as well as 24 hours of recovery in group 2 (HS), and in comparison with group 1 (control), there was a significant difference for all the Hsps measured and for test 1 and test 2 at T0 ( Fig 4 ). The mentioned differences observed between groups 1 and 2 remained at T24 in both tests, although they were statistically significant in test 2 at T24 for all Hsps studied and in test 1 at T24 for Hsp60 only ( Fig 4 ).

Induction of heat shock proteins (Hsps) in PBMC by a heat shock at 42ଌ. After the thermal treatment, the lymphocytes had a recovery period of 4 hours (test 1) or 24 hours (test 2). %: Percentage of positive cells for a particular Hsp evaluating at least 200 cells per sample. * P < 0.05 and ** P < 0.01 bars represent mean ± SEM

Doxorubicin (group 3) increased the expression of the Hsps by more than 10%, and the differences with respect to group 1 (control) were significant only in test 2 at T24. The increased production of oxygen free radicals by doxorubicin may explain the increased expression of Hsp ( Fig 4 ).

All Hsps studied increased in group 4 (HSʽo) when compared with group 1 (control). The values obtained in group 4 were very similar to those in group 2 (HS). At T0 there were significant differences with regard to the control group for Hsp27 in test 1 and test 2 ( Fig 4A ) and Hsp60 in test 1 ( Fig 4B ). In test 2, at T24, we found a significant Hsp expression with respect to group 1 (control) for Hsp27 ( Fig 4A ), Hsp60 ( Fig 4B ), Hsp70 ( Fig 4C ), and Hsp90 ( Fig 4D ).

Because the purpose of the experiment was to evaluate the influence of heat treatment on doxorubicin-induced DNA damage, we paid attention to the differences in Hsps induction between group 3 (Do) and group 4 (HSʽo) and between test 1 and test 2. Nevertheless, we found no significant differences between groups 3 and 4 and between tests 1 and 2 in Hsps expression by immunocytochemistry. Because the highest mean levels of Hsps were observed in group 4 of test 2 at T24, and corresponded to Hsp27 and Hsp70, we assessed by Western blot the expression levels of the mentioned Hsps ( Fig 5 ). In group 4 (HSʽo) we noted a higher expression of Hsp27 and Hsp70 in test 2 at T24 than in test 1 at T24. In addition, group 3 (Do) presented a modest decrease in the expression of Hsp27 and Hsp70 than group 4 (HSʽo). These data permit to conclude that the high expression levels of Hsp27 and Hsp70 found in group 4, together with the increased DNA repair capacity, suggest a clear relationship of the Hsps with the DNA repair pathway. The latter observations are consistent with previous in vitro studies in which elevated levels of Hsp27 and Hsp70 were associated with drug resistance.

Western blot analysis of heat shock protein (Hsp)27 and Hsp70. (A) Hsp27 expression in test 1. Note the induction by heat shock at T0 and the reduced expression in group 4 (HSʽo) at T24. (B) Hsp27 in test 2 was more induced by heat shock (group 2) than doxorubicin (group 3). In group 4 at T24 there was an overexpression of the Hsp. (C) Hsp70 expression in test 1. Note the decreased levels in group 4 (HSʽo). (D) Hsp70 expression in test 2. The heat shock induction was more important than in test 1, and there was an overexpression in group 4. Group 1 (control: C) group 2 (heat shock: HS) group 3 (doxorubicin: Do) group 4 (heat shockʽoxorubicin: HSʽo). T0: time 0, harvested immediately after doxorubicin treatment. T24: time 24, harvested 24 hours after postdoxorubicin recovery

Nuclear translocation of Hsp27 and Hsp70

A nuclear translocation of Hsp27 and Hsp70 was found in group 4 (HSʽo) (see Fig 1G,H ). This observation was more evident in test 2 than in test 1 and particularly at T24 ( Fig 6 ). We found a statistically significant difference in test 2 at T24 between control and group 4 for nuclear Hsp70. The differences in the percentages of cytoplasmic Hsp27 and Hsp70 were significant in both tests between group 4 and control (group 1).

Nuclear and cytoplasmic percentages of heat shock protein (Hsp)27 and Hsp70. The graph shows the differences found between group 1 (control: C) and group 4 (heat shock + doxorubicin: HSʽo) and between test 1 (recovery period of 4 hours after heat shock) and test 2 (recovery period of 24 hours after heat shock) at time 24 (T24). Note that the induction of Hsp70 (A) and Hsp27 (B) in test 2 was greater than in test 1, and at the same time in test 2 we observed the largest nuclear translocation of the mentioned Hsps. The latter observation correlates with the increased deoxyribonucleic acid (DNA) repair capacity verified in group 4 of test 2, suggesting that Hsp70 and Hsp27 are accomplishing a role in the DNA repair process. Significant differences in the percentage of cytoplasmic Hsp27 and Hsp70 were found between group 1 and group 4 in both tests. In addition, significant nuclear Hsp70 induction was observed in test 2 at T24 (A) in group 4 compared with group 1. *P < 0.05, **P < 0.01, and ***P < 0.001 bars represent mean ± SEM

Hsc70 was studied in group 4 of test 2, which presented a significant percentage of nuclear Hsp70. However, we verified that the nuclear translocation of Hsp70 was predominantly of inducible Hsp70 (�% calculated by difference between total Hsp70 and Hsc70).

Effect of hyperthermia on the expression of hMLH1, hMSH2, and PCNA

MMR is a postreplicative DNA repair process, and PCNA is undetectable in circulating lymphocytes, therefore, for this study, the PBMC were cultured with a mitogen for 48 hours before the subsequent treatment to bring them into the cell cycle. The MMR proteins hMLH1 and hMSH2 and PCNA were studied by immunocytochemistry ( Fig 7 ). The difference noted between both tests could be related to the increased DNA repair capacity observed in group 4 of test 2. Taken together, the data suggest that the Hsps, particularly the nuclear-translocated Hsp72, are helping certain DNA repair proteins, such as MMR proteins, in damage correction and are inhibiting the apoptotic pathways. We were interested in the action of PCNA because it is required for MMR and because this protein interacts with complexes containing hMSH2 or hMLH1 (Gu et al 1998). However, we did not observe statistically significant changes in PCNA levels between test 1 and test 2, as well as among the 4 studied groups (data not shown).

Expression of the mismatch repair (MMR) proteins hMLH1 and hMSH2 at time 24 (T24). The mean values obtained from the groups 2, 3, and 4, for hMLH1 and hMSH2 and for test 1 and test 2, were greater than the basal level of group 1 (control). Note the significant increased in the mean values of the MMR proteins in group 4 of test 2, which correlates with the higher deoxyribonucleic acid repair capacity reported. There was also a significant difference between group 1 and group 3 *P < 0.05. The numbers on the figure correspond to each studied subject. C: control HS: heat shock Do: doxorubicin HSʽo: heat shock and doxorubicin


Deoxyribonucleic acid or DNA is a nucleic acid that stores the genetic information of living organisms. Ribonucleic acid or RNA is another nucleic acid which is converted into the amino acid sequence during the protein synthesis. Furthermore, DNA is double stranded while RNA is single stranded. DNA has a long life and is more stable than RNA. DNA has four nitrogenous bases: adenine, guanine, thymine, and cytosine. But RNA does not have a thymine base. It has a uracil base instead.

Moreover, DNA is present in the nucleus and mitochondria while RNA is present in the cytoplasm. DNA content in a cell is fixed. But RNA content has a tendency to vary. RNA is also more resistant to UV compared to DNA.



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