Molecular Biology : DNA DNA: Structure, replication, transcription, control of gene expression, Recombinant DNA technology and Gene therapy. Introduction: What is DNA?DNA is an acronym for Deoxyribonucleic Acid. It is a nucleic acid with two strands of polydeoxyribonucleotide chains twisted around one another and bases mediating between the two chains. It is composed of four deoxyribonucleic acids (i.e) deoxyadenylate(A), deoxyguanylate(G), deoxycytidylate(C) and thymidylate(T).Nucleotides are phosphate esters of nucleosides. Base plus pentose sugar plus phosphoric acid is a nucleotide. The nucleotide are the precursors of nucleic acids(DNA and RNA). A nucleotide is made up of three components namely:(1) A nitrogenous base which could be a purine or a pyrimidine. (2) A ribose or deoxyribose pentose sugar. (3) And a phosphate group esterified to the any of the sugar above. When a base combines with a pentose sugar, a nucleoside is formed. The esterification of the nucleoside to a phosphate group gives a nucleotide or a nucleoside-monophosphate. Hence, the definition given for a nucleotide above. However, if a second and/or a third phosphate group is added, it becomes a nucleotide diphosphate or a nucleoside-triphosphate as the case might be. DNA and RNA are polymers of nucleuoside-monophosphate. There are two types of nitrogenous bases present in nucleic acid nucleic acids which are the purine bases and the pyrimidine bases. Purine bases: There are two of them, namely:=adenine and guanine. Pyrimidine bases: There are three pyrimidine bases; cytosine,thymine and uracil. However, only cytosine and thymine are present in DNA. Uracil,along with cytosine, is present in RNA(thymine is absent).Ribose is a pentose sugar in the fact that it had only five carbon atoms in its structure. It is present in the nucleic acid either as composite ribose or deoxyribise which is ribose deprived of an oxygen atom. Ribose or deoxyrinose combines with bases to form nucleotides in the following fashion. Ribonucleosides. Adenine + Ribose = adenosineGuanine + Ribose = GuanosineUracil + Ribose = UridineCytosine + Ribose = CytidineDeoxyribonucleosides. Adenine + Deoxyribose = Deoxyadenosine(d-adenosine)Guanine + Deoxyribose = d-guanosineCytosine + Deoxyribose = d-cytidineThymine + Deoxyribose = d-thymidine Phosphate groups,when esterified to nucleosides produce nucleotides. When a second phosphate group is added to the nucleotide, it becomes a nucleoside-diphosphate and a nucleotide-triphosphate when a third phosphate group is added. Nucleoside diphosphate contain one high energy bond and nucleoside triphosphate have two high energy bonds. Nucleoside triphosphate are denoted by NTP or d-NTP(if the sugar is deoxyribose) and an example is adenosine triphosphate(ATP) which is the universal energy currency in the body. Brief history on the discovery of DNA. It all began with Gregor Mendel in the year 1843. He was a monk at the Roman Catholic Church and he studied mathematics at the University of Vienna. He performed many experiments amongst which were his works in genetics which involved growing thousands of Lea plants for eight years. He was forced to give up his experiment when he became abbot of the monastery and he died in 1884 but his experiments still form the basis of genetics and gave a fair idea of inheritance.Later on, in 1969, Friedrich Miescher (1844-1895) discovered a substance he called “nuclein” and then he isolated a pure sample of the material now known as DNA from the sperm of salmon, and in 1889 his pupil, Richard Altmann, named it “nucleic acid”. This substance was found to exist only in the chromosomes.Frederick Griffith, a scientist, was working on a project In his 1928 project that formed the basis that DNA was the molecule of inheritance, Frederick Griffith, a scientist performed an experiment which involved mice and two types of pneumonia (one was virulent and the other was non-virulent). He injected the virulent pneumonia into one mouse and the mouse died of pneumonia. Next,he injected the second type of pneumonia which was non-virulent into another mouse and this mouse survived.After this, he heated up the virulent pneumonia to kill it and then injected it into a fresh mouse. This time the animal survived as predicted. Finally,he injected a combination of non-virulent and virulent pneumonia into a mouse. The virulent pneumonia had however been killed by hearing. This time,the mouse died.Griffith speculated that the dead virulent bacteria had passed on a characteristic to the non-virulent one to make it virulent. He believed this characteristic was in the inheritance molecule. This passing on of inheritance molecule was that which he called transformation.A decade later, Oswald Avery continued with Griffith’s experiment to see what the inheritance molecule was. In this experiment he destroyed the lipids, ribonucleic acids, carbohydrates, and proteins of the virulent pneumonia. Transformation still occurred after this. Next he destroyed the deoxyribonucleic acid. Transformation did not occur. He had found the basis of the inheritance which was the DNAIn 1929,Phoebus Levene at the Rockefeller Institute identified the components that make up a DNA Molecule. He showed that the components of DNA were linked in the order phosphate-sugar-base. He said that each of these units is a nucleotide and suggested the DNA molecule consisted of a string of nucleotide units linked together through the phosphate groups. He suggested that these form a ‘backbone’ of the molecule.However, Levene thought the chain was short and that the bases repeated in the same fixed order. It was Torbjorn Caspersson and Einar Hammersten who showed that DNA was a polymer. In the 1940’s another scientist named Erwin Chargaff found the pattern in the amounts of the four bases: adenine, guanine, cytosine, and thymine. He discovered the system of base pairing in the DNA molecule and this is now referred to as chargaff’s rule. Two researchers Rosalind Franklin and Maurice Wilkins tried to make a crystal of the DNA molecule. They wanted to take X ray pictures of the DNA to understand how DNA works. These two scientists were successful and obtained an x-ray pattern. The pattern appeared to contain rungs, like those on a ladder between to strands that are side by side. They found that DNA had a helix shape.In 1953, James Watson and Francis Crick, were trying to put together a model of DNA. They used Franklin and Wilkin’s picture of the X-ray and created their own model.The model of the DNA they created has not been changed much since then. Their model showed a double helix with little rungs connecting the two strands. These rungs were the bases of a nucleotide.They also found that if they paired Thymine with Adenine and Guanine with Cytosine DNA would look uniform. This pairing was also in accordance with Chargaff’s rule.They also found that the two base pairs formed hydrogen bonds. In addition, each side is a complete complement of the other. An English geneticist Alec Jeffreys of the University of Leicester,in 1984 developed DNA profiling and it was first used to convict Colin Pitchfork in 1988 in the Enderby murders case in Leicestershire, England. And from here onward, the study and research of genetics began. Structure of DNA The DNA molecule consisted of four deoxyribonucleotides that have been mentioned above. These deoxyribonucleotides units are linked by 3′ to 5′ phosphodiester bonds to form a long polypeptide chain. The 3′ end of the sugar in one deoxyribonucleotide is attached via a phosphate group to the 5′ end of the another sugar. In the example given here, the adenine is attached to thymidine and thymidine to cytidine.In the DNA, base sequence is important because genetic information is encoded in the specific sequence of bases and if this sequence is altered, the information is also altered. This can lead to abnormalities in the individual that possesses or inherits this altered DNA molecule. The deoxyribose and phosphodiester linkages are the same in all the repeating nucleotides. Therefore, the message will be conveyed even if the base sequences alone are mentioned as shown. 5’P—- Adenine-Thymidine–Cytosine–3’OHOr, 5′———A–T–C—3’This would convey all the salient features of the polynucleotide being described. Watson-Crick Model of DNA Structure. This is the structure of the DNA proposed by James Watson and Francis Crick in 1953. They discovered the structure after viewing the X-ray of the DNA made by Rosalind Franklin and Maurice Wilkins.The salient features of Watson-Crick model of DNA are; • Right Handed Double Helix: The two polydeoxyribonucleic chains that make up DNA are twisted around one another in a right handed double helix similar to a ladder with springs jutting in between. The sugar and phosphate groups comprise the vertical poles of the ladder while the bases jutting inside represent the springs. The bases repeated located perpendicular to the helical axis,whereas the sugars are nearly at right angles to the axis. •Base Pairing: This is based on Erwin chargaff’s base pairing rule and it is often called as Chargaff’s rule. It shows that the two strands of the DNA are always complimentary to each other. So, the thtmine of one strand will pair with adeninine of the opposite strand, while cytosine will pair with guanine.•Hydrogen Bonding: The DNA strands are held together mainly by hydrogen bonds formed among the base pairs. There are two hydrogen bonds between A and T while there are three hydrogen bonds between C and G. The GC bond is therefore stronger than the AT bond. •Antiparallel: The two strands in a DNA molecule run antiparallel to themselves, meaning that on strand runs from the 3′ to 5′ direction while the other is in the 5′ to 3′ direction.Features of the DNA include; Denaturing of DNA strand: DNA can be denatured and it’s double helix separated by heat. This is called melting of DNA and the melted strands can be re-associated at lower temperature through the annealing of DNA. Histones: These are proteins containing unusually higher concentration of basic amino acids. Double stranded DNA is wound around Justine’s to form nucleosomes. Chromatin is DNA in association with Justine’s. Chromatin is then further and further condensed to form chromosomes. Length of DNA : DNA is a very long molecule because the human diploid genome consists of 7 ,000,000,000 base pairs. So, when placed end to end it will be about 2 meters long. If one nucleotide is added per second, it will take 250 years to synthesize the whole DNA of a human cell. The length of a DNA molecule is compressed to 8,000 to 10,000 fold to generate chromosomes. Replication of DNA : During cell division, each daughter cell gets an exact copy of the genetic information of the mother cell. This is called DNA replication. In the daughter cell, one strand is derived from the mother cell; while the other strand us newly synthesize. This is called semi-conservative type of DNA replication. Each strand serves as a template or mold, over which a new contemporary strand is synthesized.The biological importance of DNA are; (1)Coding for protein: DNA hold the codes for synthesis of proteins which are large, complex molecules that play many critical roles in the body. They do most of the work in cells and are required for the structure, function, and regulation of body tissue and organs. (2)Genetic Code and Inheritance: The DNA carries the genetic code of a particular individual and it is passed from parents to offspring. Individuals inherit DNA molecules from both parents. (3)Disease diagnosis and Treatment : The study of DNA has made it possible to identify the causes of various diseases. Most disease states are often associated with change or alterations in an individual’s DNA and some people are also predisposed to certain diseases because of their genetic make up. Knowledge of DNA has also been applied to procure treatment of of most disease state. (4)Forenseics and crime detection: The study of DNA has helped in the justice department to recognise who is guilty or innocent in cases where there is no evidence that a person was present at the scene of a crime. Samples of DNA taken from crime scene can also be used to track down or identify a suspect as either guilty or innocent. (5)Paternity dispute: Paternity dispute cases are very common world wide. Through the assessment of DNA, the paternity of a child can be identified and this leads to children being united with their birth parents and helps prevent or stop family feuds that might have been caused by not knowing whose child a woman is carrying.