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  Section: General Biotechnology / Genes & Genetic Engineering
 
 
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Genes : Nature, Concept and Synthesis

 
     
 

Physical Nature of DNA

Watson and Crick's model of DNA
J.D.Watson and F.H.C. Crick (1953) combined the physical and chemical data generated by earlier workers, and proposed a double helix model for DNA molecule. This model is widely accepted. According to this model, the DNA molecule consists of two strands which are connected together by hydrogen bonds and helically twisted. Each step on one strand consists of a nucleotide of purine base which alternate with that of pyrimidine base. Thus, a strand of DNA molecule is a polymer of four nucleotides i.e. A, G, T, C. The two strands join together to form a double helix. Bases of two nucleotides form hydrogen bonds i.e. A combines with T by two hydrogen bonds (A = T) and G combines with C by three hydrogen bonds (G s C) (Fig.2.3A). However, the sequence of bonding is such that for every A.T.G.C. on one strand there would be T.A.C.G. on the other strand. Therefore, the two chains are complementary to each other i.e. sequence of nucleotides on one chain is the photocopy of sequence of nucleotides on the other chain. The two strands of double helix run in antiparallel direction i.e. they have opposite polarity. In Fig. 2.3A , the left hand strand has 5'3' polarity, whereas the right hand has 3'5' polarity as compared to the first one. The polarity is due to the direction of phosphodiester linkage.


The hydrogen bonds between the two strands are such that maintain a distance of 20A. The double helix coils in right hand direction i.e. clockwise direction and completes a turn at every 34A distance (Fig. 2.3B). The turning of double helix results in the appearance of a deep and wide groove called major groove. The major groove is the site of bonding of specific protein. The distance between two strands forms a minor groove. One turn of double helix at every 34A. Sugar-phosphate (nucleoside) makes the backbone of double helix of DNA molecule (Fig. 2.3B).

 

Content

Chemical nature of DNA

 

Chemical composition

 

Nucleotides, nucleosides

 

Polynucleotides

 

Chargaff's rule of equivalence

Physical nature of DNA

 

Watson and Cricks model of DNA

 

Circular and superhelical DNA

 

Organization of DNA in eukaryotes

Structure of RNA

Gene concept

Units of a gene

 

Cistron

 

Recon

 

Mutan

Split genes (introns)

 

RNA splicing

 

Ribozyme

 

Evolution of split genes

Overlapping gene

Gene organization

Gene expression

Gene regulation

 

Transcription

 

 

The lac operon (structural gene, operator gene, promoter gene and repressor gene)

Artificial synthesis of genes

 

Synthesis of a gene for yeast alanine tRNA

 

Synthesis of a gene for bacterial tyrosine tRNA

 

Synthesis of a human leukocyte interferon gene

Gene synthesis by using mRNA

Gene machine

The PCR

 

Amplification of DNA (melting of target DNA, annealing of primers, primer extension)

 

Application of PCR technology

   

The DNA model also suggested a copying mechanism of the genetic material. DNA replication is the fundamental and unique event underlaying growth and reproduction in all living organisms ranging from the smallest viruses to the most complex of all creatures including man. DNA replicates by semiconservative mechanism which was experimentally proved by Mathew, Meselson and Frank W. Stahlin in 1958. If changes occur in sequence or composition of base pairs of DNA, mutation takes place. Though the presence of adenine, guanine, thymine and cytosine is universal phenomenon, yet unusual bases in DNA molecule also occur. In some bacteriophqges 5-hydroxymethyl- cytosine (HMC) replaces cytosine of the DNA molecule when methylation of adenine, guanine and cytosine occurs. This results in changes of these bases.


Antiparallel orientation of complementary chains (A), and Watson and Crick’s model of DNA double helix (B)   The forms of DNA. A, Nucleoids of E.coli; B, a closed circular bacterial DNA; C, twisted supercoils of double stranded DNA.
Fig. 2.3 Antiparallel orientation of complementary chains (A), and Watson and Crick’s model of DNA double helix (B)
 

Fig. 2.4. The forms of DNA. A, Nucleoids of E.coli; B, a closed circular bacterial DNA; C, twisted supercoils of double stranded DNA.



Circular and Super Helical DNA

Almost in all the prokaryotes and a few viruses, the DNA is organized in the form of closed circle. The two ends of the double helix get covalently sealed to form a closed circle. Thus, a closed circle contains two unbroken complementary strands. Sometimes one or more nicks or breaks may be present on one or both strands, for example, DNA of phage PM2 (Fig 2.4A). Besides some exceptions, the covalently closed circles are twisted into super helix or super coils (Fig. 2.4B) and is associated with basic proteins but not with histones found complexed with all eukaryotic DNA.

These histone like proteins appear to help the organization of bacterial DNA into a coiled chromatin structure with the result of nucleosome like structure, folding and super coiling of DNA, and association or DNA polymerase with nucleoids. These nucleoid-associated proteins include HU proteins, IHF, proteins HI, Fir A, HtNS and Fis. In archaeobacteria (e.g. Archaea) the chromosomal DNA exists in protein associated form. Histone like proteins have been isolated from nucleoprotein complexes in Thermoplasma acidophilum and Halobacterium salinarum.. Thus, the protein associated DNA and nucleosome like structures are defected in a variety of bacteria. If the helix coils clockwise from the axis the coiling is termed as positive or right handed coiling. In contrast, if the path of coiling is anticlockwise, the coil is called left handed or negative coil.

The two ends of a linear DNA helix can be joined to form each strand continuous. However, if one end rotates at 360° with respect to the other to produce some unwinding of the double helix, the ends are joined resulting in formation of a twisted circle in opposite sense i.e. opposite to unwilling direction. Such twisted circle appears as 8 i.e. it has one node or crossing over point. If it is twisted at 720° before joining, the resulting super helix will contain two nodes (Fig. 2.4C).

The enzyme topoisomerases alter the topological form i.e. super coiling of a circular DNA molecule. Type I topoisomerases {e.g. E.coli top A) relax the negatively super coiled DNA by breaking one of the phosphodiester bonds in dsDNA allowing the 3/-OH end to swivel around the 5'-phosphoryl end, and then resealing the nicked phosphodiester backbone (Moat and Foster, 1995). Type II Topoisomerases need energy to unwind the DNA molecules resulting in the introduction of super coils. One of type II isomerases, the DNA gyrase is apparently responsible for the negatively super coiled state of the bacterial chromosome. Super coiling is essential for efficient replication and transcription of prokaryotic DNA. The bacterial chromosomes is believed to contain about 50 negatively super coiled loops or domains. Each domain represents a separate topological unit, the boundaries of which may be defined by the sites on DNA that limit its rotation (Wang,1982; Mout and Foster, 1995).

Organization of DNA in Eukaryotic Cell

In addition to organization of DNA in prokaryotes and lower eukaryotes as discussed earlier, in eukaryotes the DNA helix is highly organized into the well defined DNA-protein complex termed as nucleosomes. Among the proteins the most prominent are the histones. The histones are small and basic proteins rich in amino acids such as lysine and/or arginlne. Almost in all eukaryotic cells there are five types of histones e.g. H1, H2A, H2B, H3 and H4. Eight histone molecules (two each of H2A, H2B, H3 and H4) form an octomer ellipsoidal structure of about 11 nm long and 6.5-7 nm in diameter.
  Internal organization of nucleosomes. A highly super coiled chromatin fiber; B, a single nucleosome.
 

Fig. 2.5. Internal organization of nucleosomes. A highly super coiled chromatin fiber; B, a single nucleosome.

 

DNA coils around the surface of ellipsoidal structure of histones 166 base pairs (about 7/4 turns) before proceeding onto the next and form a complex structure, the nucleosome (Fig. 2.5A-B). Thus a nucleosome is an octomer or four histone proteins complexed with DNA.


The histones play an important role in determining eukaryotic chromosomes by determining the conformation known as chromatin. The nucleosomes are the repeating units of DNA organization which are often termed as beads. The DNA isolated from chromatin looks like string or beads. The 1.46 base pairs of DNA lie in the helical path and the histone-DNA assembly is known as the nucleosome core particle. The stretch of DNA between the nucleosome is known as 'linker' which varies in length from 14 to over 100 base pairs. The H1 is associated with the linker region and helps the folding of DNA into complex structure called chromatin fibers which in turn gets coiled to form chromatin. As a result of maximum folding of DNA, chromatin becomes visible as chromosomes during cell division.

 
     
 
 
     



     
 
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