Structural Biochemistry/Eukaryotic Recombination

Overview

Bacterial and viral genes are not the only sequences that can be introduced into a host. Eukaryotic genes can be introduced into bacteria to produce a desired protein product. It is also possible to introduce DNA into higher organisms which has led to gene therapy. Although the manipulation of eukaryotic genes holds many possibilities and advantages, it is also a source of controversy.

Cloning Eukaryotic DNA

Most eukaryotic DNA are interspersed with introns and exons that interrupt genes and cannot be expressed by bacteria. This obstacle is overcome by utilizing DNA that is complementary to mRNA (mRNA splices introns). The key to forming complementary DNA is Reverse Transcription which synthesizes a DNA strand complementary to an RNA template. Complementary DNA for all mRNA that a cell contains can be made, inserted into vectors, and then inserted into bacteria (Such a collection is called a cDNA library).

Complementary DNA molecules can be inserted into vectors that favor their efficient expression in hosts, called expression vectors. In order to maximize transcription, the cDNA is inserted into the vector near a promoter. Clones can be screened on the basis of their capacity to direct the synthesis of a foreign protein in bacteria.

Recombinant DNA in Higher Organisms

Bacteria lack the necessary enzymes to carry out posttranslational modifications. Thus, many eukaryotic genes can be correctly expressed only in eukaryotic host cells. Recombinant DNA molecules can be introduced into animal cells in several ways. First, foreign DNA molecules precipitated by calcium phosphate are taken up by animal cells. Another method involves microinjecting DNA into cells. A third method utilizes viruses, specifically retroviruses since they usually do not kill their hosts and becomes randomly incorporated into host chromosomal DNA, to introduce new genes into animal cells.

Moloney murine leukemia virus accepts inserts as long as 6 kilobase pairs and some genes introduced by this vector are efficiently expressed. Vaccinia virus is a large DNA containing virus that replicates in the cytoplasm of mammalian cells where it shuts down host cell protein synthesis. Baculovirus infects insect cells which can be easily cultured.

Plant Cell Modifications

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Electroporation

What is Electroporation? It is the method used to introduce the foreign DNA into plant cells by helping polar molecules to insert into a host cell through the cell membranes. The process is to initiate a large electric fields to make the plasma membranes permeable to the polar molecules that are allowing the molecules to pass through the cell.

1st: the cellulose wall is removed by adding cellulase to produce protoplast.

2nd: applied electric fulses to disturb the hydrophobic membrane and make its permeable to plasmid DNA to enter.

3rd: The cell wall is then allowed to reform.

Electroporation:Tumor-inducing plasmids (Ti plasmids) carry instructions for the switch to the tumor state. Ti plasmids can deliver foreign genes into some plant cells by integrating into itself into the genome of an infected plant cells (T-DNA). Foreign DNA can be introduced into plant cells by electroporation. The cellulose walls are removed which produces protoplasts. Electric pulses are then applied to a suspension of protoplasts and plasmid DNA. The high electric fields make membranes transiently permeable to large molecules, and the plasmid DNA molecules enter the cells. When the cell wall reforms, the plant cells become viable.

Gene Guns: The most effective means of transforming plant cells fires microprojectiles at the target cells.

Genetically modified organisms create plants with beneficial characteristics such as the ability to grow in poor soils, resistance to climatic variation, resistance to pests, and nutritional fortification. However, their uses are highly controversial because of unknown side effects.