Introduction:

Gene transfer is the insertion of unrelated genetic information in the form of DNA into cells and Cloning refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms.

Application of gene transfer and cloning: 1.HUMAN PEPTIDE HORMONE GENES

In the human body peptide hormones are secretsd after encoding by peptide hormone genes in the specialized cells like insulin and other Human growth hormones.

a) Insulin
Gilbert abd Villakomaroff(1980) isolated mRNA for insulin from cells of rats pancreas and inserted into Pbr322 plasmid in the middle of a gene normally coding penicillinase, and incorporated it into E.coli cells. E.coli cells produced a hybrid protein (penicillinase+proinsulin) from which the proinsulin was separated by using trypsin . It is estimated that clones of E.coli are capable of producing about one million molecules of insulin per bacterial cell.

b) Somatotropin

Double stranded cDNAs were produced from messenger rna precursor of human growth hormone geneswhich was then incorporated into bacterial cellswhere it expressed in non precursoe form.A recombinant plasmid containing a full length Hgh Cdna (which fails to express) is cleaved with restriction enzymes that release a fregment containing the conplete Hgh coding sequence after codon 24.Several overlapping complementary oligonucleotides are ligated to form one synthetic strand of small DNA fragment which contains the coding sequence for the first 24 amino acids of mature Hgh. The synthetic DNA and Cdna molecules are ligated to yield a new fragment which contains the complete coding sequence of Hgh DNA. It is ligated into a restriction site just down stream of the lac promoter/operatoe region cloned on a plasmid. The Rdna plasmid are allowed to transform E.coli. Synthesis of Hgh is induced by an inducer of lac operon.The Hgh is subsequently purified.

2.GENES FOR VACCINES AND IMMUNOGENIC SUBSTANCES

a)Vaccines for Hepatitis B virus Recombinant vaccine for HBV was produced by cloning HbsAg gene of the virus in yeast cells.The yeast system has its complex membrane and ability of secreting glycosylate protein .This have made it possible to build an autonomously replicating plasmid containing HbsAg gene near the yeast alcohol dehydrogenase I promoter.The HbsAg gene contain 6bp long sequence preceding the AUG that synthesis N-terminal methionone. This is joined to ADH promoter cloned in the yeast vector PMA-56 .The recombinant plasmid is inserted into yeast cells. The transformed yeast cells are multipplied in tryptophan free medium.Transformed cells are selected. Also vaccines for polio virus, Rabies virus, Foot and mouth disease virus ,Smallpox virus, Malaria vaccine, DNA vaccines

3.GENES ASSOCIATED WITH GENETIC DISEASES

For over a decade , genetic scientists have been attempting to map the gene. To produc eth emap they cut human DNA into pieces and grew each piece in a yeast cell for clones. The clones were then cut into fragmentsand fingerprinted to detect overlapping sections. These sections were used as guides to put the pieces back together to get the map. This will help the scientists to discover genetic diseases. a) Phenylketonuria gene: In diseased persons when phenylalanine fails to get converted into tyrosin, disturbances in metabolism result in mental retardedness.It is possible to cure this disease by using recombinant DNA techniques in early period of pregnancy. b) Urokinase gene: Urokinase is involved in dssolution of blood clots . Urokinase has been syntgesized in huge quantity by using genetically engineered bacteria with urokinase gene. c) Thalassaemia genes: It is a condition in which synthsis of and globin chains is reduced and the excess chains precipitate and cause haemolytic anaemia and spleen enlargement. Human globin genes have been identified and sequenced .It has been found that and globin genes are closely related.Himan globin genes has also been developed and cloned. d) Haemophilia genes: Haemophilia is sex linked disease in human whereblood clotting does not take place normally due to deficiency of clotting factor VIII:C. By using gene cloning technique the clotting factor VIII:C gene was cloned which expressed in mammalian cell lines and produced the protein VIII:C responsible for blood clotting.

4.PRODUCTION OF COMMERCIAL CHEMICALS:

There are several chemicals which are produced buy using the recombinant DNA technologies. A few of them are: Vitamins, organic acids ,alcohol, antibiotics etc. Recombinant DNA technology has helped increasse production of antibiotics e.g the rate of penicillin produced at present is about 1,50,000 units/ml against about 10 units/ml in 1950s.

5. ENZYME ENGINEERING:
There is a large number of microorganisms which produce a variety of enzymes. Enzymes differ with respect to substrates. Some of the microorganisms producing enzymes are listed . Bacteria Bacillus cereus B.coagulans B.licheniformis B.megaterium Citrobacter spp. Escherichia coli Klebsiella pneumoniae Actinomycetes Actinoplanes sp. Enzymes Penicillinase -amylase a-amylase, protease Peincillin ,acylase L-asparaginase Penicillin acylase, galactosidase pullulanase Enzymes Glucose isomerase

Fungi Aspergillus flavus A.niger A.oryzae Aureobasidium pullulans Candila lipolytica Mucor micheli and M.pusillus

Enzymes Urate oxidase Amylases, protease, pectinase, glucose oxidase Amylases,lipases, protease Esterase,invertase Lipase rennet

6.PREVENTION, DIAGNOSIS AND CURE OF DISEASES

1. Prevention of disease For preventive measures several immunogenic polypeptides(vaccines), and proteins(antibodies) have been chemically and biologically synthesized. 2. Diagnosis of disease through DNA probe a)Isolate the parasite from the infected tissue of the patient. Extract the DNA from parasite and purify it. b)Break the DNA by using restriction enzymes to get the DNA fragments of varying size. c)Electrophorise the DNA solution of different length by using agarose gel just to get a smear of DNA.

d)Attach the DNA smear to more firmer support by Southern blotting techniques. Thus the filter paper carries the exact replica of the DNA adhered to it e)Hybridize the immobilized DNA on filter paper by incubating it with radiolabelled probe .Probe DNA complementary to certain DNA sequences of parasite DNA sticks to it and forms the hybrid f) Wash the filter paper to remove unhybridized probe .Keep the filter in contact of x-ray film. Dark bands appear where DNA probe had hybridized the specific target sequence. Thus a parasitic disease is diagnosed positive. If dark bands on x-ray film do not appear, the absence of parasite is noted.

7.GENE THERAPY
The ultimate goal of gene therapy is the gene replacement therapy . It permits the physiological regulation of the transgenes and elimination of the possibility of insertional activation of other cellular genes which occur at the time of random integration of foreign gene. Types of gene therapy: Somatic gene therapy Germ line gene therapy Enhancement genetic engineering Eugeniuc Genetic Engineering

8.ABATEMENT OF MICROORGANISM:

POLLUTION

THROUGH

GENETICALLY

ENGINEERED

Genes responsible for degradation of environmental pollutants for eg. Toluene, chlorobenzene acids and other halogenated pesticides and toxic waste have been identified. For every compound one separate plasmid is required. The plasmids are grouped into four categories: 1. OCT plasmid which degrades octane, hexane and decane 2. XYL plasmid which degrades xylene and toluenes 3. CAM plasmid that decompose camphor

4. NAH plasmid which degrades naphthaline