MOLECULAR BIOLOGY

The term molecular biology was first used in 1945 by William Asbury who was referring to the study of the chemical and physical structure of biological macromolecules.

The roots of molecular biology were established in 1953 when an Englishman, Francis Crick and young American, James Watson working at Medical Research Council Unit, Cavendish Laboratory, Cambridge, proposed a double helical model for the structure of DNA (deoxyribonucleic acid) molecule.

Earlier, discoveries made by Griffith (1928), Avery, Macleod, and McCarthy (1944) had clearly revealed that the DNA was the chemical bearer of genetic information of certain microorganisms (bacteria, bacteriophage, etc).

The discoveries of these scientists brought a great revolution and numerous research works, of many scientists all over the world, also confirmed that DNA was the genetic material in plants, animals, and other microorganisms.

All these studies and discoveries has emerged the realization that the basic chemical organization and the metabolic process of all living things are remarkably similar, despite their morphological diversity.

In most organism the phenotype or the body structure and function ultimately depend on the structural and functional proteins or polypeptides. The synthesis of polypeptides is regulated and governed by self-duplicating genes which are born within molecules of DNA.

The genetic information for polypeptide synthesis is initially dictated by the disposition of nitrogen bases in DNA molecule and is copied down by the process of transcription. In transcription multiple copies of an individual gene is synthesized.

These copies are molecules of RNA (ribonucleic acid) such as ribosomal RNA (r-RNA) , messenger RNA (m-RNA) , and transfer RNA (t-RNA) . These RNA molecules lead to the synthesis of polypeptide chain, and this process is called translation.

In translation the genetic message encoded in messenger RNA molecule is translated into linear sequence of amino acids in a polypeptide. This polypeptide in its turn determines the phenotype of the organism.

Background of Molecular Biology

Molecular Biology has come to light recently, so has a very short history. Some of the discoveries done by scientist all over the world are listed below.

• In 1928 F. Griffith discovered transformation in bacteria.
  

• In 1934 M. Schlesinger demonstrated that bacteriophage is composed of DNA and proteins.

• In 1944 Avery, Macleod, and McCarthy first reported that DNA and not protein is the hereditary chemical.

In 1950 Chargaff demonstrated that in DNA the number of adenine and thymine groups is always equal and so are the numbers of guanine and cytosine groups. This is called as Chargaff's Rule.

• In 1953 J.D. Watson and F.H.C. Crick proposed the helical structure of DNA.

• In 1957 H. Fraenkel-Conart and B. Singer confirmed that RNA is the genetic material of some viruses.

• In 1958 G. Beadle and E. Tatum worked on the biochemical genetics of fungus.

• In 1959 S. Ochoa and A. Kornberg received Nobel Prize for artificial synthesis of nucleic acid.

• In 1964 R.W. Holly gave detailed structure of alanyl tRNA from yeast.

• In 1965 F.H.C. Crick proposed the Wobble Hypothesis for anticodons of tRNA. Another scientist F. Jacob and J. Monad received Nobel Prize for the protein synthesis mechanism in virus.

• In 1968 R.W. Holly, H.G. Khorana and M.W.Neirenberg got Nobel Prize for deciphering the genetic code.

• In 1969 H. Temin and D. Baltimore demonstrated the synthesis of DNA on RNA template tumor viruses. Both were awarded Nobel Prize 1975 for the discovery of enzyme called reverse transcriptase, which is present in the core of virusparticle.

• In 1973 S.H. Kim suggested a three dimensional structure (L-shaped Model) of tRNA.

• In 1975 E.M. Southern developed method called Southern Blotting Technique for analyzing the related genes in a DNA restriction fragment.




• In 1977 P.A. Sharp and R.J. Roberts discovered split genes of adenovirus.

• In 1978 W. Gilbert first of all used the term exon and intron (for split genes).

• In 1979 Khorana reported completion of the total synthesis of a biologically functional gene. Alwini developed Northern Blotting Technique for m-RNA and Towbin et al. developed the Western Blotting Technique for proteins.

• In 1982 A. Klug was awarded Nobel Prize for providing three-dimensional structure of tRNA.

• In 1985 K. Mullis discovered polymerase chain reaction (PCR) which is widely exploited in gene cloning for genetic engineering.

• In 1984, 86 A. Jaffrey discovered the techniques of DNA fingerprinting.

• In 1989 T. Cech and S. Altman were awarded Nobel Prize for showing enzymatic roles of some RNA molecules like ribozymes.

• In 1991 Dr. Lalji Singh developed a new technique of DNA fingerprinting by using BKM-DNA probe (BKM=banded krait minor satellite).

Structure of DNA

In 1952, Alfred D. Harshey and Martha Chase experimentally proved that DNA is the genetic material, which is present in the cells of all plants, animals, prokaryotes including viruses.

In prokaryotes DNA is without any associated protein, but in eukaryotes it is combined with histone protein to form nucleoprotein.

DNA is mainly present in chromosomes and also reported in cytoplasmic organelles like mitochondria and chloroplasts.

Rosalind Franklin first of all studied the structure of the DNA by conducting an experiment using X-rays. Her experiment showed that DNA has a helical structure and which was repeated after a certain interval.

J.D. Watson and F.H.C Crick (1953) proposed a well accepted " Double helical model " of DNA for which they were awarded Nobel Prize. They discovered that a DNA molecule consist of two helically twisted strands, each strand consisting of a phosphate group and deoxyribose (a pentose sugar). The strands are connected together by steps which are made up of a single ring pyrimidine and double ring purine bases.

These bases are connected with the deoxyribose sugar molecules. The two strands intertwined in a clockwise direction and run in opposite directions. The distance between two adjacent base pair is 3.5 Armstrong and each turn is completed after 10 base pairs. Thus the total distance of one turn is 34 Armstrong.

Different forms of DNA

DNA can exist in 5 forms: A, B, C, D, E, and Z. Sugar puckering is the most important feature to distinguishing the DNA forms.

The above described Watson and Crick model of a DNA molecules contains the right handed helical coiling and has been called B-DNA, which is biologically important form of a DNA found in most of the living organisms.

A-form and C-form differ from the B-form in number of monomers per turn or spacing of residues along helical strands.

D-form and E-form of DNA are found in some organisms and they lack in guanine (nitrogenous base).

Z-DNA is a left handed DNA molecule and one complete helix contains 12 base pairs. The angle of rotation in Z-DNA is 60 degree while that of B-DNA is 36 degree.

Replication of DNA

DNA exhibits autocatalytic function according to which the synthesis of DNA (duplication) is under the control of DNA molecule itself.

In the replication process the parent DNA molecule unwinds and unzips. Then each of the old strands serves as the template for the new strands. Each daughter DNA molecule receives one parental strand and a newly synthesized strand. This type of DNA replication is commonly called as semi-conservative replication, because here each daughter DNA molecule receives one parental strand.

DNA replication requires following three steps:

1. Unwinding: - The old strand that makes up the parent DNA molecule is unwound and unzipped (weak hydrogen bonds between the pared bases are broken). The hydrogen bonds between the molecules are broken with the help of helicase enzyme.
   

2. Complementary base pairing: - With the help of enzyme DNA polymerase new complementary nucleotides (that are always present in the nucleus) are positioned adjacent to each other opposite to the parent DNA template.
   

3. Joining:-This step also requires DNA polymerase enzyme for joining the complementary nucleotides. Each daughter molecule contains an old and a new strand.

replication of DNA strand has an origin point at which the replication is initiated. It may also have a terminus point where the replication of DNA is terminated. A ' Y ' shaped structure is formed at the point of replication which is called as "replication fork ".

Replication of DNA may be unidirectional or bidirectional. During DNA replication, one nucleotide is joined with another. Each nucleotide already has a phosphate group at the 5' carbon atom and it is joined to 3' carbon atom of the sugar molecule.

Thus the synthesis of DNA molecule takes place in the 5'->3' direction with the help of DNA polymerase enzyme. But this causes a problem at the replication fork where only one of the new strands run in the 5'->3' direction ( the template for this strand runs in the 3'->5' direction). This strand is called as leading strand.

The template for the other strand runs in the 5'->3' direction, but DNA synthesis could only take place in 5'->3' direction. Thus, this poses a problem and due to this reason synthesis has to begin in the replication fork.

Replication of the 5'->3' parental strand begins as soon as the DNA molecule unwinds and unzips replication of this strand is discontinuous. The replication of this strand results in segment called Okazaki fragments.

Discontinuous replication takes more time than continuous replication therefore the new strand in this case is called the lagging strand.

MONOCLONAL ANTIBODIES

Antibodies are normally obtained by injecting an animal with the antigen for which an immune response is sought. A variety of antibodies appear each specific to a different part of the injected antigen molecule. Blood serum taken from such animals contains the antibody mixture, called polyclonal antibodies.

Most immunogens tend to be rather weak heterogeneous nature of the immune response which results in each antiserum being a mixture of antibodies with varying affinity, cross reactivity, and effecter functions. However, it is possible to enrich antigen-specific lymphocytes.

One of the most important contributions to immunology, that has a strong impact on biotechnology, was made by Kohler and Milstein in 1976. This work has made possible to create pure and uniform antibodies against specific antigens. This technology of producing pure (monoclonal) antibodies is called hybridoma technology.


This technology is greatly helping the development of effective vaccines against diseases of humans, animals, and even plants.The modern approach is to fuse an antibody forming cell (B lymphocyte) with an immortal cell, capable of everlasting proliferation. A mouse is repeatedly immunized with an antigen of choice. As a result, there is proliferation of B lymphocytes making antibodies specific for that antigen. Thereafter, these highly potent B lymphocytes are removed from the mouse and are fused to a mutant Myeloma Cells whose own antibody synthesis has been stopped. The fused product gives us pure and homogeneous bodies regularly.

A Hybridoma or Fused Cell is a hybrid cell that produces monoclonal antibodies in culture. Hybridoma cells are mainly formed by the fusion of a myeloma (cancer) cell with a normal antibody producing lymphocyte. Cultured myeloma (cancer) cells are fused with spleen cells (lymphocytes) obtained from an immunized animal. The spleen cells are the source of antibodies.

One spleen produces only one type of antibody. This means that one hybridoma clone (that is fused product of cancer cells and lymphocytes) will produce only one type of specific antibody. Later, the parental myeloma cells are killed by growing the hybrids in appropriate selective media. The normal spleen cells fail to survive in the culture.

Several spleen-myeloma hybrids arise and a few of them survive in culture. These hybrids phenotypically resemble the myeloma cell parent, and they produce large quantities of the antibodies expressed by the lymphocytes derived from the immunized animal.

Applications of Monoclonal Antibodies

The applications of Monoconal antibodies are as follows:

• Selective elimination of undesired cells, such as tumour cells or activated T lymphocytes in transplantation patients was one of the first possible therapeutic applications of monoclonal antibodies.

• Potential use in radiological scanning for tumor localization

• Counting and distinction of human lymphocyte subsets, using monoclonal antibodies that distinguish human helper and suppressor T cells and thymic lymphocytes at different stages of differentiation.

• Depletion of a particular type of T cell subsets from a mixed population of bone marrow cells to prevent graft verses host reaction.

• Analysis of complex antigen mixtures or of embryological relationship.

• Treatment of drug overdose.

• Definition of tumor antigens such as human renal antigen.

SOMATIC CELL HYBRIDIZATION

It has been known since the 60's that somatic cells from the same or different species in culture could spontaneously fuse to form polyploid cells. The product of fusion was called Homokaryon if the two parental cells come from the same species, and Heterokaryon or Somatic Cell Hybrid if the fusion was site-specific.


The hybrid cells could divide by mitosis and proliferate and thus could be maintained in culture. Cell fusion is followed by nuclear fusion to produce uninucleate hybrid cells or Synkaryons. The rate of cell fusion is very low; about one cell fuses in million of cells.


In 1962 Okada discovered that inactivated Sandai virus could greatly increase the rate of cell fusions. Since then several agents causing cell fusion has been tried among which Polyethylene Glycol (PEG) has some advantages. The exact mechanism of cell fusion is not known. In case of UV-inactivated Sandai virus, it seems that the virus absorbs to the cell surface leading to agglutination of cells. The protein coat of virus forms the connecting bridge between the cells. The membrane of two cells swells into this region and when they come in contact they get dissolved. The cell contents mix up, the nuclei fuse and a Heterokaryon is formed. When cell fusion is mediated by Polyethylene Glycol the two cell membranes directly come in contact.

Basic Hybridoma Technology

The term Hybridoma is applied to fused cells resulting due to fusion of following two types of cells: An antibody producing lymphocyte cell e.g. spleen cells of mouse. Myeloma (cancer) cells which are capable of multiplying indefinitely. These fused hybrid cells or hybridoma have the antibody producing capability inherited from lymphocytes and have the ability to grow continuously like cancer cells. Following steps are involved in the production of monoclonal antibodies (specific antibody for specific antigen).

A mouse is immunized to a specific antigen for the production of specific antibody. Tumor is produced in a mouse (it is done by injecting tumor causing antigens). From the above two types of animals, spleen cells (that produces antibodies) and Myeloma cells (that produces tumor) are isolated and cultured separately.

The Myeloma cell that is cultured is unusual in two ways, first it has stopped synthesizing antibodies and its mutant called HGPRT­- that cannot synthesize the enzyme Hypoxanthine Guanine Phosphoribosyl transferase. Now the fusion of the spleen cells to Myeloma cells is induced by using Polyethylene Glycol to produce hybridoma. The hybrid cells are grown in selective Hypoxanthine aminopterin thymidine medium (HAT).

HAT contains a drug Aminopterin which blocks one pathway for nucleotide synthesis making the cells to depend upon other pathway that needs HGPRT enzyme, which is absent in Myeloma cells. Therefore, Myeloma cells which do not fuse with the B cells will die, since they are HGPRT-. B cells which do not fuse with the Myeloma cells will die, because they lack tumorgenic property of immortal growth. Therefore HAT medium allows selection of Hybridoma cells which inherit HGPRT gene from B cells and tumorgenic property from Myeloma cells.

Now the selection of hybridoma cells is done for cloning and antibody productions. This is facilitated by preparing single cell colonies that produces antibodies. Such antibodies are called Monoclonal antibodies. The hybridoma cells are kept stored at low temperature for future use in the production of monoclonal antibodies.