Genes: Nature, Concept and Synthesis

The genes are the hereditary units which control characters of living beings. They are small segments of DNA that encode for specific proteins or mRNAs. They control each and every characteristics of living being through the specific proteins. The genes occur in a linear row throughout the length of the DNA. The DNA, in eukaryotic cell, lies in a specific chromosome along with chromosomal proteins. In prokaryotic cell, DNAs occur freely in the protoplasm. The structure of a gene however is the same both in prokaryotic cells and in eukaryotic cells.

Among the genes of an organism, some code for specific enzymes or structural proteins required by the cell. Such genes are known as structural genes. The genes that initiates the expression of the structural gene is called promoter gene. Some other genes code for proteins that induce or stop the expression of the structural genes, and are called regulator genes. A single promoter gene precedes one or a few structural genes. The regulator gene may occur near to the promoter or occur far way from the promoter. If there is a mutation in any one of these genes, the organism fails to produce the particular enzyme or protein. The gene therefore said to be the unit of mutation.

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. It plants, animals and some viruses the genetic material is double stranded (ds) DNA molecule except some viruses such as фX174. In TMV, influenza virus, poliomyelitis virus and bacteriophage the genetic material is single stranded (ss) RNA molecule.

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.

Each deoxyribonucleotide consists of pentose sugar (deoxyribose), a phosphate group and a nitrogenous base (either purine or pyrimidines). Purine bases (adenine and guanine) are heterocyclic and two ringed bases and the pyrimidines (thymine and cytosine) are one ringed bases.

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 positions 1’,2’,3’ and 4’of carbon atoms. 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.

Nitrogenous bases:
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’,7’ and 9’ position.

The pyrimidines 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 two types, thymine (T) and cytosine (C). Uracil (U) is a third pyrimidine. A, G and C are common in both DNA and RNA. U is found only in RNA.

A phosphate group:
In DNA a phosphate group (PO43-) is saturated to the 3’-carbon of deoxyribose sugar and 5’-carbon of another sugar. Therefore, each strand contains 3’ end and 5’ end arranged in an alternate manner. Strong negative chargers of nucleic acid are due to the presence of phosphate group. A nucleotide is a nucleoside phosphate which contains its bond to 3’ and 5’ carbon atoms of pentose sugar that is called phosphodiester.

Nucleosides and nucleotides:
The nitrogenous bases combined with pentose sugar are called nucleosides. A nucleoside linked with phosphate forms a nucleotide.

Nucleoside = pentose sugar + nitrogenous bases
Nucleotide = nucleoside + phosphate

On the basis of different nitrogenous bases the deoxynuclotides are of following types:
1. Adenine (A) = deoxyadenosine-3’/5’-monophosphate (3’/5’-d AMP)
2. Guanine (G) = deoxyguanosine-5’-monophosphate (5’-d GMP)
3. Thymine (T) = deoxythymidine-5’-monophosphate (5’-d TMP)
4. Cytosine (C) = deoxycytidine-5’-monophosphate (5’-d CMP)

In addition to the presence of nucleosides in DNA helix, there are also present in nucleoplasm and cytoplasm in the form of deoxyribonucleotide phosphates e.g. dexoyadenosine triphospahte (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphoshate (dCTP), deoxythymidine triphosphate (dTTP). The advantage of these four dexyribonucleotide 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.

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:
A chemist Erwin Chargaff started using paper chromatography to analyse the bases 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.

The DNA molecule of each species comprises of base composition which is not influenced either by environmental conditions or growth stage 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 isolate from lower plants and animals, and bacteria and viruses was generally rich in G-C and poor in A-T contents. The use of base composition has much significance in establishing relationship between two species and in taxonomy and phylogeny of species.

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. 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 ≡ C).

The two chains are complementary to each other i.e. sequence of nucleotides on one chain is the photocopy sequence of nucleotides on the other chain. The two strands of double helix run in antiparallel direction i.e. they have opposite polarity. In 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 phoshodiester linkage.

The hydrogen bonds between the two strands are such that maintain a distance of 20 Ao. The double helix coils in right hand direction i.e. clockwise direction and completes a turn at every 34 Ao distance. 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 binding of specific protein. The distance between two strands forms a minor groove, one turn of double helix at every 34 Ao. Sugar-phosphate makes the backbone of double helix of DNA molecule.

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 pair of DNA, mutation takes place.
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. Some times one or more or breaks may be present on one or both strands, for example, DNA of phage PM2. Besides some exceptions, the covalently closed circles are twisted into super helix or super coils 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 H1, Fir A, H-NS 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 digree 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 unwinding direction. Such twisted circle appears as 8 i.e. it has one node or crossing over point. If it is twisted at 720 degree before joining, the resulting super helix will contain two nodes.

The enzyme topoisomerases alter the topological form i.e. super coiling of 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. Type II topoisomerases need energy to unwind the DNA molecule resulting in the introduction of super coils. One of the 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.