DNA-THE DEOXYRIBONUCLEIC ACID

THE STRUCTURE AND FUNCTION OF DNA

Biologists in the 1940s had difficulty in conceiving how DNA could be the genetic material because of the apparent simplicity of its chemistry. DNA was known to be a long polymer composed of only four types of subunits, which resemble one another chemically. Early in the 1950s, DNA was examined by x-ray diffraction analysis, a technique for determining the three-dimensional atomic structure of a molecule. The early x-ray diffraction results indicated that DNA was composed of two strands of the polymer wound into a helix. The observation that DNA was double-stranded was of crucial significance and provided one of the major clues that led to the Watson - Crick Model for DNA structure. But only when this model was proposed in 1953 did DNAs potential for replication and information encoding become apparent. In this section we examine the structure of the DNA molecule and explain in general terms how it is able to store hereditary information.

HISTORY OF DNA EVOLUTION

·         In 1868: F. Miescher isolated nucleic acids from white blood cells that were acidic in nature to which he called nuclein.

·         In 1880: Fischer isolated purines and pyrimidines.

·         In 1881: Zacharis identified nuclein with chromatin.

·         In 1899: Altaman replaced the term nuclein with nucleic acid.

·         In 1900s: Kossel identified the presence of histones and protamines with nucleic acids (Nobel laurate).

·         In 1910s: P. A. Levene discovered phosphate and pentose sugars callled deoxyribose molecule.

·         In1928: Frederick Griffith demonstrate the existence of a chemical in bacteria that caries genetic information

·         In 1943: Three American Microbiologist; Ostawald Avery, Colin MacLeod and Maclyn McCarty for the first time presented the evidence that DNA is the genetic material and is made up of genes.

·         In 1944: Oswald Avery showed that degradation of DNA and not protein resulted in loss of genetic information.

·         In 1950s: Rosalind Franklin and her supervisor Maurice Wilkins were working on the X-ray diffraction model for DNA.

Rosalind Franklin (1951):

·         Generated  X-ray crystallography data suggesting a double helix with phosphates on the outside

• Rosalind Franklin who actually proposed the concept of double helix was deprived of Nobel prize due to cruel death in 1958.
• Great revolution in DNA Biology
• In 1953 February: Pauling and R. B. Corey gave a triple helix model of the DNA molecule. However he couldn’t explain the process of DNA replication.
• They were near to present about the double helix model.

• In 1953 April: J.D. Watson (an American Biologist) and F. H. C. Crick (a British Physicist) presented the double helix model of DNA (published in Nature entitled “A structure for deoxyribose nucleic acid’).
• Nobel prize awarded to

o   Watson, Crick and Wilkins

o   in 1962.

DNA STRUCTURE

DNA is composed of nitrogen bases, deoxyribose sugars and phosphate. Adenine and guanine are purine bases while cytosine and thymine are pyrimidine bases. The phosphodiester bond between sugar and phosphate molecules form the backbone of DNA. The glycosidic bond is formed between nitrogen bases and sugar molecules.

Figure 1: Nitrogen bases

Figure 2: Structure of nucleotide showing phosphodiester bond and glycosidic bond

Figure 3: Hydrogen bonding between nitrogen bases

CHARGAFF EQUIVALENT RULE

In 1948, a chemist Erwin Chargaff, on the basis paper chromatography experiment, analyze the base composition of DNA. In 1950, he discovered that: In a DNA molecule of different types of organisms, the total no. of purines is equal to the total no. of pyrimidines. A/T=G/C

Number of Purines (A+G) = Number of Pyrimidines (C+T)

WATSON AND CRICK MODEL FOR DNA

The was proposed by Watson and Crick which was published in Nature in 1953. It is also known as ‘double helix model for DNA’ molecules. However, the photograph for model was taken from X-ray diffraction photograph from Rosalind Franklin.

Figure 4: Double helix structure of DNA

According to the Model:

·         DNA molecule consists of two strands which are connected by H-bonding and they are helically twisted.

·         Each step in one strand consists of nucleotide of purine base which alternately pair with pyrimidine base.

·         DNA is a polymer of four nucleotides (A T G C).

·         Adenine pairs to thymine with 2-H bonding (A=T).

·         Gaunine pairs to cytosine with 3 H-bodings (GºC).

·         Two strands apart 20 A from each other.

·         Helix coils in right hand i.e. clockwise direction and completes at every 34 A distance.

·         Two strands are complementary to each other.

·         One strand runs 5’®3’ while the complementary strand runs 3’®5’.

·         The polarity of DNA is due to direction of phosphodiester linkage.

·         Turning results in deep and wide major groove which is the site of bonding of specific protein.

·         The distance between two strands form a minor groove.

·         One turn of double helix at every 34 A distance includes 10 nucleotides.

·         Each nucleotide is situated at a distance of 3.4 A.

·         Sugar phosphate makes the back bone of double helix of DNA molecules.

·         The DNA model also suggested a copying mechanism of the genetic material which is semi conservative in nature.

·         Experimentally proved by Mathew, Meselson and Frank W. Stahl in 1958.

·         Universally accepted.

DIFFERENT FORMS OF DNA

Three different forms of DNA are found i.e. A form, B form and Z form. The B form (10 bp/turn), which is observed at high humidity, most closely corresponds to the average structure of DNA under physiological conditions. A form (11 bp/turn), which observed under the condition of low humidity, presents in certain DNA/protein complexes. RNA double helix adopts a similar conformation.  Z form (12 bp/turn) more loosely arranged DNA is found during DNA replication.

Figure 5: Different forms of DNA

A DNA Molecule Consists of Two Complementary Chains of Nucleotides:

A deoxyribonucleic acid (DNA) molecule consists of two long polynucleotide chains composed of four types of nucleotide subunits. Each of these chains is known as a DNA chain, or a DNA strand. Hydrogen bonds between the base portions of the nucleotides hold the two chains together. The nucleotides are composed of a five-carbon sugar to which is attached one or more phosphate groups and a nitrogen-containing base. In the case of the nucleotides in DNA, the sugar is deoxyribose attached to a single phosphate group (hence the name deoxyribonucleic acid), and the base maybe either adenine (A), cytosine (C), guanine (G), or thymine (T). The nucleotides are covalently linked together in a chain through the sugars and phosphates, which thus form a "backbone" of alternating sugar-phosphate. Because only the base differs in each of the four types of subunits, each polynucleotide chain in DNA is analogous to a necklace (the backbone) strung with four types of beads (the four bases A, C, G, and T). These same symbols (A, C, G, and T) are also commonly used to denote the four different nucleotides-that is, the bases with their attached sugar and phosphate groups. The way in which the nucleotide subunits are linked together gives a DNA strand a chemical polarity. If we think of each sugar as a block with a protruding knob (the 5'phosphate) on one side and a hole (the 3'hydroxyl) on the other, each completed chain, formed by interlocking knobs with holes, will have all of its subunits lined up in the same orientation. Moreover, the two ends of the chain will be easily distinguishable, as one has a hole (the 3'hydroxyl) and the other a knob (the 5'phosphate) at its terminus. This polarity in a DNA chain is indicated by referring to one end as 3' end and the other as the 5' end. The three-dimensional structure of DNA-the double helix-arises from the chemical and structural features of its two polynucleotide chains. Because these two chains are held together by hydrogen bonding between the bases on the different strands, all the bases are on the inside of the double helix, and the sugar-phosphate backbones are on the outside. In each case, a bulkier two-ring base (a purine) is paired with a single-ring base (a pyrimidine); A always pairs with T and G with C.

DNA TOPOLOGY

In order to fully understand DNA topology, students need to familiarize themselves with three key mathematical concepts: twist (Tw), writhe (Wr), and linking number (Lk). Twist represents the total number of double helical turns in a given segment of DNA. By convention, the right-handed twist of the Watson-Crick structure is assigned a positive value. Writhe is a property of the spatial course of the DNA and is defined as the number of times the double helix crosses itself if the molecule is projected in two dimensions. The helix-helix crossovers (i.e., nodes) are assigned a positive or negative value based on the orientation (i.e., handedness) of the DNA axis. The numerical term that describes the sum of the twist and the writhe is called the linking number, which represents the total linking within a DNA molecule. Mathematically, these properties of DNA can be expressed as:

Lk =  Tw +  Wr

Why is DNA supercoiling important? Duplex DNA is merely the storage form for the genetic information. In order to replicate or express this information, the two strands of DNA must be separated. Since the global underwinding of the genome imparts increased single-stranded character to the double helix, negative supercoiling greatly facilitates this process. As a result, replication origins and gene promoters are more easily opened, and rates of DNA replication and transcription are greatly enhanced.

While negative supercoiling promotes many DNA processes, positive supercoiling inhibits them. When tracking systems, such as replication or transcription complexes travel along the double helix, they do not spiral circumferentially around the DNA. Rather, they move linearly through the DNA and the double helix spins to accommodate this motion. Recall from the earlier discussion that the ends of chromosomal DNA are not free to rotate. As a result, the number of turns of the helix remains invariant unless the nucleic acid chain is broken. Thus, the linear movement of tracking enzymes through DNA does not change the number of turns, but merely compresses them into a shorter segment of the genetic material. Consequently, the double helix becomes increasingly overwound ahead of tracking systems. DNA overwinding, or positive supercoiling, makes it more difficult to open the two strands of the double helix and ultimately blocks essential nucleic acid processes if not alleviated.

Figure 6: DNA supercoiling

Topoisomerases I

Type I topoisomerases are denoted by “odd” numbers (topoisomerase I, III, etc.). These enzymes are monomeric in nature and require no high-energy cofactor. There are two subclasses of type I enzymes, type IA and type IB. Type I topoisomerases act by creating transient single-stranded breaks in the double helix, followed by passage of the opposite intact strand through the break (type IA) or by controlled rotation of the helix around the break (type IB). Type IA enzymes require divalent metal ions for catalytic activity and covalently attach to the 5’-terminal phosphate of the DNA. In contrast, type IB enzymes do not require divalent metal ions and covalently attach to the 3’-terminal phosphate

Topoisomerases II

Type II topoisomerases are denoted by “even” numbers (topoisomerase II, IV, etc.). These enzymes contain multiple polypeptide chains and require ATP for overall catalytic activity. Prokaryotic enzymes have an A₂B₂ structure and eukaryotic enzymes are homodimers in which the bacterial A and B subunits have merged. Based on the structure of the archetypical bacterial type II enzyme, gyrase (see below), the A subunit (or domain) contains the active site tyrosyl residue that links to DNA during the cleavage event and the B subunit (or domain) contains the site of ATP hydrolysis.

Type II topoisomerases modulate DNA topology by generating a transient double-stranded break in the DNA backbone, passing a separate double helix through the opening, and resealing the break. All bacterial and eukaryotic type II enzymes require divalent metal ions for activity and those examined so far appear to utilize a two-metal-ion mechanism similar to that of DNA polymerases and primases. The cleavage reaction of type II topoisomerases generates DNA intermediates with 4-base, 5’-cohesive ends that are covalently attached to the enzyme through their 5’-terminal phosphates 

FOOD BORNE DISEASES

FOOD BORNE DISEASES (INFECTIONS AND INTOXICATION)

Table 1: Bacterial Food Diseases (Infections and Intoxication)

Etiologic Agent	Incubation Period	Clinical Syndrome	Confirmation
Bacillus cereus – Vomiting toxin	1-6 hrs	Vomiting; some patients with diarrhea; fever uncommon	Isolation of organism from stool of two or more ill persons and not from stool of control patients OR Isolation of 105 organisms/g from epidemiologically implicated food, provided specimen is properly handled
Bacillus cereus – Diarrheal toxin	6-24 hrs	Diarrhea, abdominal cramps, and vomiting in some patients; fever uncommon	Isolation of organism from stool of two or more ill persons and not from stool of control patients OR Isolation of 105 organisms/g from epidemiologically implicated food, provided specimen is properly handled
Brucella	Several days to several mos; usually >30 days	Weakness, fever, headache, sweats, chills, arthralgia, weight loss, splenomegaly	Two or more ill persons and isolation of organism in culture of blood or bone marrow; greater than fourfold increase in standard agglutination titer (SAT) over several wks, or single SAT 1:160 in person who has compatible clinical symptoms and history of exposure
Campylobacter jejuni/coli	2-10 days; usually 2-5 days	Diarrhea (often bloody), abdominal pain, fever	Isolation of organism from clinical specimens from two or more ill persons OR Isolation of organism from epidemiologically implicated food
Clostridium botulinum	2 hrs-8 days; usually 12-48 hrs	Illness of variable severity; common symptoms are diplopia, blurred vision, and bulbar weakness; paralysis, which is usually descending and bilateral, might progress rapidly	Detection of botulinum toxin in serum, stool, gastric contents, or implicated food OR Isolation of organism from stool or intestine
Clostridium perfringens	6-24 hrs	Diarrhea, abdominal cramps; vomiting and fever uncommon	Isolation of 106 organisms/g from stool of two or more ill persons, provided specimen is properly handled. OR Demonstration of enterotoxin in the stool of two or more ill persons OR Isolation of 105 organisms/g from epidemiologically implicated food, provided specimen is properly handled
Escherichia coli –Enterohemorrhagic (E. coli O157:H7 and others)	1-10 days; usually 3-4 days	Diarrhea (often bloody), abdominal cramps (often severe), little or no fever	Isolation of E. coli O157:H7 or other Shiga-like toxin-producing E. coli from clinical specimen from two or more ill persons OR Isolation of E. coli O157:H7 or other Shiga-like toxin-producing E. coli from epidemiologically implicated food
Escherichia coli –Enterotoxigenic (ETEC)	6-48 hrs	Diarrhea, abdominal cramps, nausea; vomiting and fever less common	Isolation of organism of same serotype, demonstrated to produce heat-stable (ST) and/or heat-labile (LT) enterotoxin, from stool of two or more ill persons
Escherichia coli –Enteropathogenic (EPEC)	Variable	Diarrhea, fever, abdominal cramps	Isolation of organism of same enteropathogenic serotype from stool of two or more ill persons
Escherichia coli –Enteroinvasive (EIEC)	Variable	Diarrhea (might be bloody), fever, abdominal cramps	Isolation of same enteroinvasive serotype from stool of two or more ill persons
Listeria monocytogenes –Invasive disease	2-6 wks	Meningitis, neonatal sepsis, fever	Isolation of organism from normally sterile site
Listeria monocytogenes –Diarrheal disease	Unknown	Diarrhea, abdominal cramps, fever	Isolation of organism of same serotype from stool of two or more ill persons exposed to food that is epidemiologically implicated or from which organism of same serotype has been isolated
Nontyphoidal Salmonella	6 hrs-10 days; usually 6-48 hrs	Diarrhea, often with fever and abdominal cramps	Isolation of organism of same serotype from clinical specimens from two or more ill persons OR Isolation of organism from epidemiologically implicated food
Salmonella Typhi	3-60 days; usually 7-14 days	Fever, anorexia, malaise, headache, and myalgia; sometimes diarrhea or constipation	Isolation of organism from clinical specimens from two or more ill persons OR Isolation of organism from epidemiologically implicated food
Shigella spp.	12 hrs-6 days; usually 2-4 days	Diarrhea (sometimes bloody), often accompanied by fever and abdominal cramps	Isolation of organism of same species or serotype from clinical specimens from two or more ill persons OR Isolation of organism from epidemiologically implicated food
Staphylococcus aureus	30 min-8 hrs; usually 2-4 hrs	Vomiting, diarrhea	Isolation of organism of same phage type from stool or vomitus of two or more ill persons OR Detection of enterotoxin in epidemiologically implicated food OR Isolation of 105 organisms/g from epidemiologically implicated food, provided specimen is properly handled
Streptococcus, group A	1-4 days	Fever, pharyngitis, scarlet fever, upper respiratory infection	Isolation of organism of same M- or T-type from throats of two or more ill persons OR Isolation of organism of same M- or T-type from epidemiologically implicated food
Vibrio cholerae –O1 or O139	1-5 days	Watery diarrhea, often accompanied by vomiting	Isolation of toxigenic organism from stool or vomitus of two or more ill persons OR Significant rise in vibriocidal, bacterial-agglutinating, or antitoxin antibodies in acute- and early convalescent-phase sera among persons not recently immunized OR Isolation of toxigenic organism from epidemiologically implicated food
Vibrio cholerae –non-O1 and non-O139	1-5 days	Watery diarrhea	Isolation of organism of same serotype from stool of two or more ill persons
Vibrio parahaemolyticus	4-30 hrs	Diarrhea	Isolation of Kanagawa-positive organism from stool of two or more ill persons OR Isolation of 105 Kanagawa-positive organisms/g from epidemiologically implicated food, provided specimen is properly handled
Yersinia enterocolitica	1-10 days; usually 4-6 days	Diarrhea, abdominal pain (often severe)	Isolation of organism from clinical specimen from two or more ill persons OR Isolation of pathogenic strain of organism from epidemiologically implicated food

Table 2: Parasitic Food Diseases (Infections and Intoxication)

Etiologic Agent	Incubation Period	Clinical Syndromes	Confirmation
Cryptosporidium spp.	2-28 days; median: 7 days	Diarrhea, nausea, vomiting; fever	Demonstration of oocysts in stool or in small-bowel biopsy of two or more ill persons OR Demonstration of organism in epidemiologically implicated food
Cyclospora cayetanensis	1-14 days; median: 7 days	Diarrhea, nausea, anorexia, weight loss, cramps, gas, fatigue, low-grade fever; may be relapsing or protracted	Demonstration of the parasite by microscopy or molecular methods in stool or in intestinal aspirate or biopsy specimens from two or more ill persons OR Demonstration of the parasite in epidemiologically implicated food
Giardia intestinalis	3-25 days; median: 7 days	Diarrhea, gas, cramps, nausea, fatigue	Demonstration of the parasite in stool or small-bowel biopsy specimen of two or more ill persons
Trichinella spp.	1-2 days for intestinal phase; 2-4 wks for systemic phase	Fever, myalgia, periorbital edema, high eosinophil count	Two or more ill persons and positive serologic test or demonstration of larvae in muscle biopsy OR Demonstration of larvae in epidemiologically implicated meat

Table 3: Viral Food Diseases (Infections and Intoxication)

Etiologic Agent	Incubation Period	Clinical Syndrome	Confirmation
Hepatitis A	15-50 days; median: 28 days	Jaundice, dark urine, fatigue, anorexia, nausea	Detection of immunoglobulin M antibody to hepatitis A virus (IgM anti-HAV) in serum from two or more persons who consumed epidemiologically implicated food
Norovirus (NoV)	12-48 hrs (median 33 hours)	Diarrhea, vomiting, nausea, abdominal cramps, low-grade fever	Detection of viral RNA in at least two bulk stool or vomitus specimens by real-time or conventional reverse transcriptase-polymerase chain reaction (RT-PCR) OR Visualization of viruses (NoV) with characteristic morphology by electron microscopy in at least two or more bulk stool or vomitus specimens OR Two or more stools positive by commercial enzyme immunoassay (EIA)
Astrovirus	12-48 hrs	Diarrhea, vomiting, nausea, abdominal cramps, low-grade fever	Detection of viral RNA in at least two bulk stool or vomitus specimens by real-time or conventional reverse transcriptase-polymerase chain reaction (RT-PCR) OR Visualization of viruses (NoV) with characteristic morphology by electron microscopy in at least two or more bulk stool or vomitus specimens OR Two or more stools positive by commercial enzyme immunoassay (EIA)

Fungal Intoxication:

·         Aflatoxicosis is caused by aflatoxins produced by the fungi, e.g. Aspergillus flavus.

·         Four types of aflatoxins have been described i.e. aflatoxin B₁, B₂, G₁ and G₂.

·         Animals consuming feeds contaminated with aflatoxin B₁ leads to secretion in the milk of aflatoxin

M₁ and M₂

Table 4: Chemical Food Intoxication

Etiologic Agent	Incubation Period	Clinical Syndrome	Confirmation
Marine toxins – Ciguatoxin	1-48 hrs; usually 2-8 hrs	Usually gastrointestinal symptoms followed by neurologic symptoms (including paresthesia of lips, tongue, throat, or extremities) and reversal of hot and cold sensation	Demonstration of ciguatoxin in epidemiologically implicated fish OR Clinical syndrome among persons who have eaten a type of fish previously associated with ciguatera fish poisoning (e.g., snapper, grouper, or barracuda)
Marine toxins – Scombroid toxin (histamine)	1 min-3 hrs; usually 1 hr	Flushing, dizziness, burning of mouth and throat, headache, gastrointestinal symptoms, urticaria, and generalized pruritis	Demonstration of histamine in epidemiologically implicated fish OR Clinical syndrome among persons who have eaten a type of fish previously associated with histamine fish poisoning (e.g., mahi-mahi or fish of order Scomboidei)
Marine toxins – Paralytic or neurotoxic shellfish poison	30 min-3 hrs	Paresthesia of lips, mouth or face, and extremities; intestinal symptoms or weakness, including respiratory difficulty	Detection of toxin in epidemiologically implicated food OR Detection of large numbers of shellfish-poisoning-associated species of dinoflagellates in water from which epidemiologically implicated mollusks are gathered
Marine toxins – Puffer fish, tetrodotoxin	10 min-3 hrs; usually 10-45 min	Paresthesia of lips, tongue, face, or extremities, often following numbness; loss of proprioception or floating sensations	Demonstration of tetrodotoxin in epidemiologically implicated fish OR Clinical syndrome among persons who have eaten puffer fish
Heavy metals (Antimony, Cadmium, Copper, Iron, Tin, Zinc)	5 min-8 hrs; usually <1 hr="" o:p="">

Source: https://www.cdc.gov/foodsafety/outbreaks/investigating-outbreaks/