Gene Expression and Recombinant DNA Technology

A given restriction enzyme cuts DNA segments within a specific nucleotide sequence at restriction site. These recognition sequences are typically four, six, eight, ten, or twelve nucleotides long. Because there are only so many ways to arrange the four nucleotides which compose DNA (Adenine, Thymine, Guanine and Cytosine) into a four-to-twelve nucleotide sequence, recognition sequences tend to occur by chance in any long sequence. Five fragments will be generated if we digest a linear DNA molecule with a restriction enzyme having four recognition sites on the DNA.

Insertional inactivation is a technique of recombinant DNA technology used to select bacteria that carry recombinant plasmids, a fragment of foreign DNA is inserted into a restriction site within a gene for antibiotic resistance, thus causing that gene to become non functional, and hence there is an inactivation of the enzyme.

Adenosine deaminase deficiency also known as ADA deficiency or ADA-SCID  is an autosomal recessive metabolic disorder that causes immunodeficiency. Introduction of gene isolated from bone marrow cells producing ADA into cells at early embryonic stages can be considered as a permanent cure to this disorder.

Antisense RNA  is a single-stranded RNA which is complementary to a messenger RNA strand transcribed within a cell. In tobacco plants resistant to nematodes, the DNA produces both sense and antisense RNA.

RNA interference (RNAi) also known as the post transcriptional gene silencing (PTGS), happens to be  a biological process whereby the RNA molecules leads to the inhibition of gene expression. It occurs in most of the eukaryotes. There is a silencing of a specific mRNA molecule due to a complementary dsRNA molecule. Pest resistant plants are nowadays being produced by this method. 

A gene gun or a biolistic particle delivery system which was actually designed for plant transformation is a device for injecting cells with genetic information. The payload is an elemental particle of a heavy metal coated with plasmid DNA. Microparticles coated with DNA to be bombarded with gene gun are made up of gold or tungsten.

Few isolates of B. thuringiensis produce a group of insecticidal small molecules called beta-exotoxin, commonly known as thuringiensin. The Bt toxin is not toxic to human beings because conversion of pro Bt toxin to Bt toxin takes place only in highly alkaline conditions.

Bacillus thuringiensis form a solid protein crystal, the parasporal body, next to their endospores during spore formation. The parasporal body contains protein toxins that has been found to kill over 100 species of moths by dissolving in the alkaline gut of caterpillars and destroying the epithelium.

A palindromic sequence is a nucleic acid sequence (DNA or RNA) that is the same whether read 5' (five-prime) to 3' (three prime) on one strand or 5' to 3' on the complementary strand with which it forms a double helix.

All the examples are resistant plants, which have been cloned from bacterial Bt toxin gene. This provides resistance to various insects, thus overcoming the necessity for the use of insecticides and subsequent health hazards.

The Bt toxin exists as an inactive protoxin, which needs digestion by an insect to be effective. There are various Bt toxins and each of this is specific to certain target insects. Crop plants have nowadays engineered to contain and express the genes for Bt toxin, which they produce in its active form.

When a person obtains transformants by inserting a recombinant DNA within the coding sequence of â-galactosidase, the recombinant colonies do not produce any colour, whereas non-recombinants form blue coloured colonies. This observation helps in separating out the recombinants from the non-recombinant ones.

Meganucleases are considered to be the most specific naturally occurring restriction enzymes. Meganucleases are "molecular DNA scissors" that can be used to replace, eliminate or modify sequences in a highly targeted way. By modifying their recognition sequence through protein engineering, the targeted sequence can be changed. Meganucleases are used to modify all genome types, whether bacterial, plant or animal. Such modification generates tailor-made plants, which possess all the mentioned characteristics.

Eukaryotic genes do not function properly when cloned into bacterial cell, because of the inability of the bacterial genes to excise introns. Eukaryotic enzymes are often responsible for the destruction of the bacterial restriction enzymes as well.

A restriction endonuclease is an enzyme that cuts the DNA at or near a specific recognition nucleotide sequences known as restriction sites. It breaks the bond between the sugar and phosphate components of nucleic acid molecule.

A plasmid refers to a small DNA molecule that is physically separate from, as well as can replicate independently of, chromosomal DNA within a cell. Plasmids are most frequently found as small circular, double-stranded DNA molecules in bacteria. They are sometimes present in archaea and eukaryotic organisms.

Rosie was the name of the first transgenic cow. Golden rice is an improved variety of genetically engineered rice with high content of Vitamin A. Bt cotton is transgenic cotton created by inserting a crystal protein gene (cry) from the soil bacterium Bacillus thuringiensis into the genome of various cotton cultivars. Radioactive isotopes often behave as DNA probes which are used to identify the various sequences of DNA.