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 interspecific. 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 swell 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:

1. An antibody producing lymphocyte cell e.g. spleen cells of mouse.
2. Myeloma (cancer) cells which are capable of multiplying indefinately.

These fused hybrid cells or hybridoma haves 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 rabbit is immunized to a specific antigen for the production of specific antibody.
  
• Tumour is produced in a mouse or rabbit ( It is done by injecting tumour causing antigens).
  
• From the above two types of animals, spleen cells (that produces antibodies) and myeloma cells (that produces tumour) are isolated and cultured seperately. The myeloma cell that is cultured is unusual in two ways, first it has stopped synthes

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 result in each antiserum being a mixture of antibodies with varying affinity, cross reactivities, and effector 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 Milstean 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. Hybridoma or Fused cells 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 to spleen cells (lymphocytes) from an immunized animal. The spleen cells are the source of antibodies. One spleen produce 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 X myeloma hybrids arise and a few of them survive in culture. These hybrids phenotypically resembles 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

Monoclonal antibodies can be used in diagnostic systems to identify serologically similar microorganisms. The merits of monoclonal antibodies as diagnostic reagents is that they provide a standardized product with greatly reduced non-specific reactions. Monoclonal antibodies with markers such as fluorescein, peroxidase-anti peroxidase or radioactive isotopes can be used to identify microbial agents (pathogens) in tissues or cultured cells. Some other clinical applications of monoclonal 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 relationships .
• Treatment of drug overdose.
• Definition of tumor antigens such as human renal antigen.

MOLECULAR BIOLOGY

The term molecular biology was first used in 1945 by William Astbury 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 scientist all over the world, also confirmed that DNA was the genetic material in plants ,animals, and other microorganisms. From all these studies and discoveries has emerged the realization that the basic chemical organization and the metabolic processes of all living things are remarkably similar despite their morphological diversity. In most organisms 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 of DNA. The genetic information for polypeptide synthesis is initially dictated by the disposition of nitrogen bases in DNA molecule and are 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, messenger RNA, and transfer 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. These polypeptide in its turn determines the phenotype of the organism.

Historical Background Of Molecular Biology

The Molecular Biology has come to light recently and thus 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 bacteriophages are 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 are always equal and so are the numbers of guanine and cytosine groups. This is called as Chargaff's Rule.

In 1952 A.D. Harshey and M. Chase demonstrated that only the DNA of T2 bacteriophage enters the host, the bacterium Escherichia coli, where as the proteins remain behind.

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 virus particle.

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

In 1975 E.M. Southern developed method called Southern Blotting Technique for analysing 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 tRNAs.

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

In 1984,86 A. Jaffreys 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. Harshe 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 neucleoprotein. 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 with the help of an experiment using X-rays. Her experiment showed that DNA has a helical structure and which was repeated after a certain distance. 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 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 to 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 DNA molecules contains the right handed helical coiling and has been called B-DNA, which is biologically important form of 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 itself. In the replication process the parent DNA molecule unwinds and unzips. Then each of the old strand serves as the template for the new strands. Each daughter DNA molecule recieves one parental strand and a newly synthesised strand. This type of DNA replication is commonly called as semi-conservative replication, because here each daughter DNA molecule recieves one parental strand.

DNA replication requires following three steps :

1. Unwinding:- The old strand that makes up the parent DNA molecule are unwound and unzipped (weak hydrogen bonds between the pared bases are broken). The hydrogen bonds between the molecules are broken with the help of helicaze 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 polymeraze enzyme for joining the complementary nucleotides. Each daughter molecule contains an old and a new strand.

The replication of DNA strand has an origion 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. The 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.

GENETIC ENGINEERING

During the late 1970s, the science of genetics entered the new era dominated by the use of recombinant DNA technology or genetic engineering to produce outstanding life forms that is not found in the nature. Through this technology, it has been possible to transfer genes from mammals into microorganism like bacteria, causing them to act like tiny factories for making proteins of great economic importance, such as hormones (including growth hormones) and interferon (lymphocyte proteins that prevent replication of a wide variety of viruses). These proteins are produced in such a small amount in humans that the cost of their extraction from tissue has been very expensive.
By genetic engineering it has become possible to produce various blood clotting factors, complement proteins (part of immune system) and other substances for the correction of genetic deficient diseases. Recently, experiments have been conducted in which human cells which are unable to synthesise purines have been obtained from the patient with Lesch-Nyhan Syndrome and grown in culture. These cells have been converted to normal cells by transformation with recombinant DNA.
Thus we can say that genetic engineering could be used as an application for the correction of human defects and providing gene therapy. The therapy given to the patients with the help of genetic materials is called gene therapy. For example, restoring the ability of a diabetic individual to make insulin or correcting immunological deficiencies come under gene therapy.
Recombinant DNA molecules are produced with one of the following three objectives:

1. To obtain a large number of copies of specific DNA fragment
2. To recover large quantities of protein produced by the concerned gene.
3. To integrate the gene in question into the chromosome of the target organism where it expands itself.

Steps Involved In Genetic Engineering

Genetic engineering involves six steps as follows:

1. Isolation of gene or other piece of DNA to be cloned.
2. Insertion of gene into another piece of DNA called a vector that will allow it to be taken up by bacteria and replicate within them as cells grow and replicate.
3. Transfer of recombinant vector into bacterial cells either by transformation or infection through transmission of virus.
4. Selection of those cells which contains desired recombinant vectors.
5. Growth of bacteria that can be continued independently to give as much clone DNA as needed.
6. Expression of gene obtained in the desired product.

Scope And Application Of Genetic Engineering

Genetic Engineering has been applied in several fields some of them are as follows:

• Contribution of Genetic Engineering in the field of medical science in order to get pharmaceuticals like Humulin (human insulin).
• Heamophaelics do not have factor eight which does not allow blood clotting or the blood takes long to clot. Genetic Engineering is used in their treatment to isolate factor eight from another source and introduce in the body of Haemophaelic patients.
• Erythropoetin induces RBC (Red Blood corpuscles) production, and person deficient in this protein/harmone are anaemic. Genetic code for this harmone is transferred into bacteria and reintroduced into the patients suffering from anaemia.
• Tissue Plasminogen Activator (TPA) activates plasmin which dissolves blood clots. Genetically engineered products are used for treating myocardial infection.
• Wounds caused by burns and brittle bones are treated by growth harmone which are produced and introduced through genetic engineering. Thus it can be realised that genetic engineering is also capable of treating person suffering from dwarfism.
• Genetic Engineering is also used for the treatment of cancer patients as it can be used for the production of interferon and interleukins.
• Genetic Engineering is used for vaccination purpose. In vaccination instead of introducing whole organism into the human body only genetically extracted portion of organisms, like bacteria and viruses which can elicit response and help in the production of antibodies against the antigens, is introduced. Vaccination programme has been started for eliminating Polio, DPT, Foot and Mouth disease etc.

for more information you can also refer to http://en.wikipedia.org/wiki/Genetic_engineering

MYCOPLASMA

Mycoplasma are pleuro pneumonia like organism (PPLO) discovered by Nocard and Roux(1989). They were first isolated from bovine sheep suffering from pleuropneumonia. Originally Mycoplasma were included among bacteria and placed in the class Schizomycetes. But now they have been separated from bacteria and placed in separate class having following systematic position.

Class :Mollicutes
Order :Mycoplasmatales
Family :Mycoplasmatacae
Genus :Mycoplasma

Characteristics

1. They are bacteria without cell wall and are immotile.
2. They are called "Joker of the microbial world".
3. They require sterol [cholesterol] for growth in the culture medium.
4. They can be killed only by certain antibiotics like Tetracycline.
5. They are the smallest known cellular organisms with an average 0.1-0.15 micrometer.
6. They are completely insensitive to enzymes.
7. They reproduce by binary fission only.
   In plants they cause "Little leaf of brinjal" disease.
   In animals they cause Pleuropneumonia.In man they cause Human Infertility and abortions due to infection in the urinogenital tract

A well defined cell wall is lacking in Mycoplasma ,so a three layered plasma membrane forms the outer boundary of the cell. They lack organised nucleus , plasmids, lysosomes, endoplasmic reticulam, mitochondria, golgy complex, centriole, flagella. The genetic material is represented by single DNA duplex which is naked because of the absence of the histone association. DNA lies coiled throughout the cytoplasm. Ribosome is 70s type. Enzymes lies both freely in the cytoplasm as well as associated with the plasma membrane.