Dominic W. S. Wong

Structures of Nucleic Acids

What is the chemical structure of a deoxyribonucleic acid (DNA) molecule? DNA is a polymer of deoxyribonucleotides. All nucleic acids consist of nucleotides as building units. A nucleotide has three components: sugar, base, and a phosphate group. (The combination of a sugar and a base is a nucleoside.) In the case of DNA, the nucleotide is known as deoxyribonucleotide, because the sugar in this case is deoxyribose. The base is either a purine (adenine or guanine) or a pyrimidine (thymine or cytosine) (Figs. 2.1 and 2.3). Another type of nucleic acid is ribonucleic acid (RNA), a polymer of ribonucleotides also consisting of three components – a sugar, a base and a phosphate. The sugar in this case is a ribose, and that the base thymine is replaced by uracil (Sect. 2.6).

Human Gene Therapy

There are more than 4000 known inherited disorders. The majority of them have minimal effects, but a few causes physical and mental abnormalities that may be life threatening. Genetic diseases that are candidates for gene therapies include severe combined immunodeficiency, thalassaemia, and cystic fibrosis, etc. (Fig. 19.1). Since these genetic diseases are each caused by a single defective gene, one potential treatment is to introduce a normal functional copy of the gene into the cell tissue that is affected. In effect, the normal (therapeutic) gene augments the defective gene in the patient. Gene therapy is not restricted to only treating genetic disorders. The general technology of transferring genetic materials into a patient is also applied to diseases such as cancer, AIDS, and cardiovascular diseases. Many of the approved clinical trials on gene therapy are for the treatment of diseases other than genetic disorders (Table 19.1).

Techniques Used in Cloning

The theoretical and experimental background for cloning techniques is closely tied with the biological processes described in Part 1. Volumes of protocols are available for use in gene cloning. Fortunately, the basic techniques are not difficult to understand.

Enzymes Used in Cloning

The manipulation of DNA utilizes a number of enzymes. These enzymes are naturally occurring in cells involved in transcription, translation, replication, and other biological processes. The reactions catalyzed by these enzymes have become an essential part of gene cloning. Examples of enzyme uses in cloning include cutting and joining DNA, deletion or extension of DNA, generating new DNA fragments, and copying DNA from RNA. These enzymes are available commercially in highly purified forms suitable for cloning work.

Cloning Vectors for Introducing Genes into Host Cells

The introduction of a foreign DNA into a host cell in many cases requires the use of a vector. Vectors are DNA molecules used to transfer a DNA/gene into a host (microbial, plant, animal) cell, and to provide control elements for replication and expression. The vector to be used is determined by the type of host cells and the objectives of the cloning experiment.

Microbial Production of Recombinant Human Insulin

The early success of recombinant DNA technology relies heavily on the elucidation of the biological possesses at the molecular level in microbial systems. The first commercial application is realized in the microbial production of human insulin.

Growth Enhancement in Transgenic Fish

The aquaculture industry produces about 60 million tons of processed fish yearly. Much of the improvement in fish farming has been done by traditional breeding methods. In the past few decades, there has been marked progress in employing recombinant DNA technology to produce transgenic fish with desirable traits, such as increased growth rate and disease resistance.

Transpharmers: Bioreactors for Pharmaceutical Products

The application of transgenic technology to commercially important livestock is expected to generate major effects in agriculture and medicine. Three areas of development have been the focus of intensive investigation: (1) For improved desirable traits, such as increased growth rate, feed conversion, reduction of fat, improved quality of meat and milk. Growth hormone transgenes have been inserted into genomes of pig, sheep, and cow; (2) For improved resistance to diseases – A number of genes contributing to the immune system (such as heavy and light chains of an antibody that binds to a specific antigen) can be introduced to confer in vivo immunization to transgenic animals; (3) To raise transgenic animals for the production of pharmaceutical proteins – The concept of using farm animals as bioreactors has raised the prospect of a revolutionary role of livestock species. The list of proteins includes human lactoferrin, human collagen, α1-antitrypsin, blood coagulation factor, anticlotting agents, and many others.

Transgenic Crops Engineered with Insecticidal Activity

Public concerns over the environmental and health effects of chemical pesticides have intensified the effort to search for alternatives. One attractive option is the use of biopesticides from microorganisms. The role of biopesticides in crop protection is not new. In fact the first such product, based on the insecticidal activity of Bacillus thuringiensis, has been in commercial applications for decades. With the advent of recombinant DNA technology, scientists have produced transgenic crop plants engineered with insecticidal activity.

Structures of Proteins

Proteins are the products of transcription and translation. The structure and hence the functional property of a particular protein are specified by the information encoded in the gene. Some understanding of the molecular architecture of proteins is necessary to make sense of the genetic process.