Genetic Engineering

A. GENE MANIPULATION or Genetic Engineering

Genetic engineering is the manipulation of genes by man and refers to artificial synthesis, isolation, modification, combination, addition and repair of the genetic material (DNA) to alter the phenotype to suit human needs.

It is performed by modifying an organism’s own DNA or intruding new DNA to perform desired functions. The discovery of plasmids and the enzymes restriction endonuclease gave the idea of genetic engineering.

B.HISTORY & NEW RESERSERCHES

Genetic engineering was started in 1973 by two scientists of USA : Stanley Cohen and Herbert Boyer, by combining a gene from a bacterium with the plasmid of E. coli. The new technique of genetic engineering is known as recombinant DNA technology.

The technology of genetic engineering came into existence late 1970 when Paul Berg (considered to be father of genetic engineering) was able to introduce a gene of SV−40 virus into bacterium E. coli with the help of lambda phage.

Arber, Smith and Nathans got Nobelprize of 1978 for the discovery of restriction endonuclease.

Restriction enzyme besides its restriction activities also acts as methylases because it modifies DNA through methylation of adenine or cytosine in very specific manner.

C.RECOMBINANT DNA TECHNOLOGY (RDT)

Recombinant DNA technology involves two basic processes, formation of recombinant DNA, and introduction of recombinant in an appropriate host. Recombinant DNA is the DNA formed by combining DNAs from different organisms.

Term 'gene'was given by Johannsen(1909) for any particle to which properties of Mendelian factor or determiner can be given. T.H Morgan (1925) defined gene as ‘any particle on the chromosome which can be separated from other particles by mutation or recombination is called a gene. In general, gene is the basic unit of inheritance.

According to the recent information a gene is a segment of DNA which contains the information for one enzyme or one polypeptide chain coded in the language of nitrogenous bases or the nucleotides. The sequence of nucleotides in a DNA molecule representing one gene determines the sequence of amino acids in the polypeptide chain (the genetic code). The sequence of three nucleotides reads for one amino acid (codon).

(1) Gene action :

Gene act by producing enzymes. Each gene in an organism produces a specific enzyme, which controls a specific metabolic activity. It means each gene synthesizes a particular protein which acts as enzyme and brings about an appropriate change.

(i) One gene one enzyme : This theory was given by Beadle and Tatum (1958), while they were working on red mould or Neurospora(ascomycetes fungus). Which is also called Drosophila of plant kingdom. Wild type Neurospora grows in a minimal medium (containing sucrose, some mineral salts and biotin). The asexual spores i.e. conidia were irradiated with x-rays or UV-rays (mutagenic agent) and these were crossed with wild type. After crossing sexual fruiting body is produced having asci and ascospores. The ascospores produced are of 2 types -

(a) The ascospores, which are able to grow on minimal medium called ‘prototrophs’.

(b) Which do not grow on minimal medium but grow on supplemented medium called ‘auxotrophs’.

(2) Molecular structure of gene :

Gene is chemically DNA but the length of DNA which constitutes a gene, is controversial 3 term i.e. cistron, muton and recon were given by Seymour Benzer to explain the relation between DNA length and gene.

(i) Cistron or functional gene or gene in real sense : Cistron is that particular length of DNA which is capable of producing a protein molecule or polypeptide chain or enzyme molecule.

(ii) Muton or unit of mutation : Muton is that length of DNA which is capable of undergoing mutation. Muton is having one or part of nucleotide.

(iii) Recon : Recon is that length of DNA which is capable of undergoing crossing over or capable of recombination. Recon is having one or two pairs of nucleotides.

(iv) Complon : It is the unit of complementation. It has been used to replace cistron. Certain enzymes are formed of two or more polypeptide chains. Whose active groups are complimentary to each other.

(v) Operon : Operon is the combination of operator gene and sequence of structure genes which act together as a unit. Therefore it is composed of several genes. The effect of operator gene may be additive or suppresive.

(vi) Replicon : It is the unit of replication. Several replicons constitute a chromosome.

(3) Some specific terms

(i) Transposons or Jumping genes : The term ‘transposon’ was first given by Hedges and Jacob(1974) for those DNA segments which can join with other DNA segments completely unrelated and thus causing illegitimate pairing. These DNA segments are transposable and may be present on different place on main DNA. The transposons are thus also called Jumping genes. Hedges and Jacob reported them in bacteria. But actual discovery of these was made by Barbara Mc Clintock (1940) in maize and she named them as controlling elements in maize or mobile genetic elements in maize. For this work, she was awarded nobel prize in (1983).

(ii) Retroposons : The term was given by Rogers (1983) for DNA segments which are formed from RNA or which are formed by reverse transcription under the influence of reverse transcriptase enzyme or RNA dependent DNA polymerase enzyme.

❑ About 10% of DNA of genome in primates and rodents is of this type.

(iii) Split genes or interrupted genes : Certain genes were reported first in mammalian virus and then in eukaryotes by R. Roberts and P. Sharp in (1977) which break up into pieces or which are made of segments called exons and introns. These are called split genes or interrupted genes.

Split gene = Exons + Introns

In mRNA formed from split gene exons are present and not corresponding to introns. So in split genes, exons carry genetic information or informational pieces of split genes are exons.

(iv) Pseudogenes or false genes : DNA sequences presents in multicellular organisms, which are useless to the organism and are considered to be defective copies of functional genes (cistrons) are called pseudogenes or false genes. These have been reported in Drosophila, mouse and human beings.

F. Linkage.

Introduction : "When genes are closely present link together in a group and transmitted as a single unit, the is phenomenon is called linkage".

(1) Theories of linkage

(i) Sutton's hypothesis of linkage (1903) : The number of groups of genes are equivalent to the number of chromosomes.

(ii) Morgan's hypothesis of linkage (1910) : It was given by T. H. Morgan. According to him the genes of homologous parents enter in the same gamete and tend to remain together, which is opposite in heterozygous parents. Linked group are located on the same chromosome and distance between linked group of gene limits the grade of linkage.

(iii) Coupling and repulsion hypothesis : Proposed by Batesonand Punnet (1906) that dominant alleles tend to remain together as well with recessive alleles, called gametic coupling. If dominant and recessive alleles are present in different parents they tend to remain separate and called repulsion. When BBLL and bbll are crossed, the  is BbLl and the test cross of it will show progeny in 7 : 1 : 1 : 7 ratio i.e. BbLl : Bbll : bbLl : bbll (coupling) when BBll is crossed with bbLL the F₁ is BbLl or the test cross progeny will show 1 : 7 : 7 : 1 ratio i.e., BbLl : Bbll : bbLl : bbll (repulsion). Coupled and repulsed genes are known as linked genes. Linkage has coupling phase and repulsion phase. In coupling phase both the linked genes have their dominant alleles in one chromosome and recessive alleles in other chromosomes. The heterozygotes with such constitution is called cis heterozygote. Cis-arrangement is a original arrangement. Which form two types of gametes as (AB) and (ab). In Human X–chromosomes carry 102 genes and Y chromosome carries 10 genes only.

In repulsion phase the normal alleles as well as mutant alleles lie in opposite chromosomes of the homologous pair, such heterozygote is called as trans heterozygote. It is not original arrangement, caused due to crossing over, which form two types of gametes as (Ab) and (aB).

(iv) Chromosomal hypothesis of linkage : It was given by Morgan and Castle. According to them linked genes are bound by chromosomal material and are transmitted as a whole.

(2) Types of linkage : Depending upon the absence or presence of nonparental or new combination of linked genes, linkage has been found to be complete or incomplete.

(i) Complete linkage : Such cases in which linked genes are transmitted together to the offsprings only in their original or parental combination for two or more or several generations exhibit complete linkage. In such cases the linked genes do not separate to form the new or non-parental combinations. This phenomenon is very rare. Some characteristics in males of Drosophila are found to exhibit complete linkage.

H.CLONING

A population of identical molecules (genes), cells or organisms all derived from the same parent by asexual source is known as clone (G. klone = slip, cutting used for propagation). The process of producing genetically similar molecules, cells or organisms from a common precursor by asexual reproduction in vitro or in vivo is termed cloning. Or simply cloning is a biotechnique for copying individual by manipulation at cellular level.

Cloning results from a unique capacity of cell, called totipotency. It is the ability of a nucleated cell to repeatedly divide and differentiate into a complete organism in a suitable nutrient medium. In plants, nearly all the cells are totipotent. In animals, only the zygote and stem cells from early embryos are totipotent.

Pluriopotency is the ability of a cell to develop any type of the cell in the animal body. e.g., kidney cells or heart cells or nerve cells.

Cloning may be studied under the following headings :

I. Genetic Transformation in Plants

❒ This bacterium infects all broad-leaved agricultural crops such as tomato, soyabean, sunflower and cotton etc. It does not infect cereals. It induces formation of cancerous growth called a crown gall tumor. This transforamation of plant cells is due to the effect of Ti plasmid carried by the pathogenic bacterium. Hence, for genetic engineering purposes, Agrobacterium strains are developed in which tumor-forming genes are deleted. These transformed bacteria can still infect plant cells.

❒ The part of Ti plasmid transferred into plant cell DNA, is called the T-DNA. This
T-DNA with desired DNA spliced into it, is inserted into the chromosomes of the host plant where it produces copies of itself, by migrating from one chromosomal position to another at random. But it no longer produces tumors.

Such plant cells are then cultured, induced to multiply and differentiate to form plantlets. These plantlets, when transferred into soil, grow into mature plants, carrying the foreign gene, expressed throughout the new plant.

J. PRACTICAL APPLIcATIONS OF GENETIC ENGiNeERING

Following are the practical applications of genetic engineering :

Diagnosis of disease

Gene therapy (replacement of defective genes responsible for hereditary diseases with the normal gene). e.g. haemophilia, phenylketonuria, alkaptonuria.

Production of genetically modified microorganisms.

Synthesis of pharmaceutically useful chemicals like human insulin interferons, human growth hormone, antibodies, etc. Efficient nitrogen fixation (Rhizobium meliloti used as a biofertilizer).Depollution of environment (Pseudomonas being used).Crop protection (Fungus Trichoderma being employed).Penicillin production.Lignin removal Insect control by Bacillus thuringiensis.

Production of transgenic plants, e.g., tomato, soybean, cotton, corn, brinjal, rice, potato, tobacco, etc. Production of transgenic animals, e.g., sheep, goat, hogs, fish, etc.Help in understanding biological process.Solution of disputed parentage.

K. GENE CLONING

Gene cloning which is done at molecular level refers to the production of a large population of a DNA fragment of interest in pure form.

Gene cloning involves the formation of a recombinant DNA and its introduction into an appropriate host, which may be E. coli or Bacillus subtilis. The bacterium E. coli divides for every 22 minutes under favourable conditions, giving rise to a billion cells in less than 11 hours. This will produce a large clone of recombinant DNA, one copy in each bacterium. With this technique, genes can be isolated, cloned and characterized so that technique has led to significant progress in all fields of molecular biology More recently polymerase chain reaction (PCR) involving a thermostable DNA polymerase has been used to obtain millions of copies of DNA.

Polymerase chain reaction (PCR) involving a thermostable DNA polymerase (Taq polymerase) has been used to obtain millions of copies of a DNA segment of choice. This PCR technique may eventually replace gene cloning in certain areas of research in the field of genetic engineering.

PCR has also been used for developing molecular markers termed ‘Randomly Amplified Polymorphic DNA’s (RAPDs, pronounced as ‘rapids’).