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

 
     
 

Chemical Nature of DNA

The DNA is found in all plants, animals, prokaryotes and some viruses. In eukaryotes it is present inside the nucleus, chloroplast and mitochondria, whereas in prokaryotes it is dispersed in cytoplasm. In plants, animals and some viruses the genetic material is double stranded (ds) DNA molecule except some viruses such as ΦX174. In TMV, influenza virus poliomyelitis virus and bacteriophages the genetic material is single stranded (ss) RNA molecule (Table 1). The entire genetic message that controls the chemistry of every cell of the body acting in a specific way is actually written in the language of four nitrogenous bases of DNA i.e. purines and pyrimidines. The defined sequence of the four bases constitutes a 'gene' which may be a few or several hundred base pairs long. Genes are the structures of the blue prints called proteins which control the infinite variety of life.


Chemical Composition
Purified DNA isolated from a variety of plants, animals, bacteria and viruses has shown a complex form of polymeric compounds containing four monomers known as deoxyribonucleotide monomers or deoxyribotids (Fig.1). Each deoxyribonucleotide consists of pentose sugar (deoxyribose), a phosphate group and a nitrogenous base (either purine or pyrimidine). Purines bases (adenine and guanine) are heterocyclic and two ringed bases and the pyrimidines (thymine and cytosine) are one ringed bases. The following components of deoxyribonucleotide have been described:

(i) A five carbon ring. Deoxyribose is a pentose sugar consisting of five carbon atoms. Four carbon atoms (1', 2', 3', 4') of this sugar combine with one oxygen atom and form a ring. The fifth atom (5') forms -CH2 group which is present outside this ring. Three -OH groups are attached at position 1', 3' and 5' and the hydrogen atoms combine at position 1', 2', 3' and 4' of carbon atoms (Fig.1). In ribonucleotides, the pentose sugar is ribose which is similar to deoxyribose except that there is an -OH group instead of -H at 2' carbon atom. The absence of -OH group in DNA makes it chemically more stable than the RNA.

 

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

   


(ii)
Nitrogenous base. There are two nitrogenous bases, purines and pyrimidines. The purines are double ring compounds that consists of 5-membered imidazole ring with nitrogen at 1', 3', T and 9' position. The pyrimidmes are single ring compounds, the nitrogen being at position 1'and 3' in 6- membered benzene ring. A single base is attached to 1'-carbon atom of pentose sugar by N-glycosidic bond. Purines are of two types, adenine (A) and guanine (G), and pyrimidines are also of two types, thymine (T) and cytosine (C). Uracil (U) is a third pyrimidine (Fig.2). A, G and C are common in both DNA and RNA. U is found only in RNA.

Table 1. Nature of genetic material.

DNA/RNA

Examples

Double stranded DNA(dsDNA) Higher plants, animals, bacteria, animal viruses (polyoma virus, small pox, herpes virus),  Bacteriophages (T-even)
   
Single stranded DNA(ssDNA) Bacteriophages ($ XI74 and other bacteriophages), Animal viruses (parvovirus)
   
Double stranded RNA(dsRNA) virus Retrovirus, Reovirus, Hepatitis-B virus, animal virus
   
Single stranded RNA(ssRNA) Plant viruses ( tobacco mosaic virus)Animal viruses (influenza virus, poliomyelitis virus)Bacteriophages ( F2, ~ R17)



A typical nucleotide showing its components : base, sugar and phosphate.   Nitrogeneous base of nucleic acids
Fig. 1. A typical nucleotide showing its components : base, sugar and phosphate.
 

Fig.2. Nitrogeneous base of nucleic acids.



Nucleosides and nucleotides

The nitrogenous bases combined with pentose sugar are called nucleosides. A nucleoside linked with phosphate forms a nucleotide (Fig.1).

                      Nucleoside  =   pentose sugar + nitrogenous base

                      Nucleotide  =   nucleoside + phosphate

   
On the basis of different nitrogenous bases the deoxynucleotides are of following types :
   
(i)    Adenine  (A) = deoxyadenosine-3'/5'-monophosphate (3'/5'-d AMP)
   
(ii)  
Guanine  (G) = deoxyguanosine -5'-monophosphate (5'-d GMP)    
(iii) 
Thymine  (T) = deoxythymidine -5'-monophosphate (5'-d TMP)     
(
iv)   Cytosine  (C) = deoxycytidine -5'-monophosphate (5'-d CMP)

In addition to the presence of nucleosides in DNA helix, these are also present in nucleoplasm and cytoplasm in the form of deoxyribonucleotide phosphates e.g. deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP). The advantage of these four deoxyribonucleotide in triphosphate form is that the DNA polymerase acts only on triphosphates of nucleotides during DNA replication.

Similarly, the ribonucleotides contain ribose sugar, nitrogenous bases and phosphate. Except sugar, the other components are similar. However, uracil (U) is found in RNA instead of thymine. Generally, RNA molecule is single stranded besides some exceptions.

Polynucleotide
 The nucleotides undergo the process of polymerization to form a long chain of polynucleotide. The nucleotides are designated by prefixing 'poly' to each repeating unit such as poly A (polyadenylic acid), poly T (polythymidilic acid), poly G (polyguanidylic acid), poly C (polycytidilic acid) and poly U (poly uridylic acid). The polynucleotides that consists of the same repeating unit are called homopolynucleotides such as poly A, poly T, poly G, poly C and poly U.

Chargaff-equivalence Rule
By 1948, a chemist Erwin Chargaff started using paper chromatography to analyze the base composition of DNA from a number of studies. In 1950, Chargaff discovered that in the DNA of different types of organisms the total amount of purines is equal to the total amount of pyrimidines i.e. the total number of A is equal to the total number of T (A-T), and the total number of G is equal to the total number of C (G-C). It means that A/T = G/C i.e. A+T/G+C = 1. In the DNA molecules isolated from several organisms regularity exists in the base composition.

The DNA molecule of each species comprises of base composition which is not influenced either by environmental conditions or growth stages or age. The molar ratio i.e. [A] + [T]/[G]+[C] represents a characteristic composition of DNA of each species. However, in higher plants and animals A-T composition was found generally high and G-C content low, whereas the DNA molecules isolated from lower plants and animals, and bacteria and viruses was generally rich in G-C and poor in A-T contents (Table 2). The two closely related species will have very similar molar % G+C values and vice versa. Thus, the use of base composition has much significance in establishing relationship between two species and in taxonomy and phylogeny of species.

Table 2. Relative amount of nitrogenous bases in DNA isolated from different organisms.

Source

Adenine

Guanine

Thy mine

Cytosine

A+T

G+C

Human sperm

30.9

19.1

31.6

18.4

1.62

Human thymus

30.9

19.9

29.4

19.8

1.52

Sea urchin sperm

32.8

17.7

32.1

18.4

1.85

Wheat germ

26.5

23.5

27.0

23.0

1.19

Yeast

31.3

18.7

32.9

17.1

1.79

Escherichia coli

26.0

24.9

23.9

25.2

1.00

Diplococcus pneumoniae

29.8

20.5

31.6

18.0

1.59

Bacteriophage T2

32.5

18.2

32.6

16.7

1.86

 
     
 
 
     



     
 
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