MEDICAL BIOLOGY, ANATOMY, HUMAN PHYSIOLOGY AND PATHOLOGY - Ya.I. Fedonyuk 2010

BIOLOGY

SECTION 1. BIOLOGICAL FOUNDATIONS OF HUMAN VITAL ACTIVITY

1.3. MOLECULAR-GENETIC AND CELLULAR LEVELS OF LIFE ORGANIZATION

1.3.3. Hereditary apparatus of eukaryotic cells and its functioning - molecular level

Introduction/32.html">Genetic Engineering

Genetic (Gene) engineering is a set of experimental Methods used to transfer genes from one Organism to another in order to purposefully endow the latter with new hereditary traits. Genetic engineering knows no taxonomic barriers. It makes it possible to manipulate genetic material from diverse sources and, according to a pre-designed program, construct functionally active recombinant (hybrid, chimeric) DNA molecules in vitro that do not occur in nature. The prefix "re" implies that DNA is not created de novo (anew), but is formed by combining fragments of pre-existing molecules. Recombinant DNA molecules are called chimeric because they can combine seemingly incompatible Genes from different organisms. The theoretical foundation of genetic engineering lies in the universality of METABOLISM/28.html">The Genetic Code.

Genetic engineering encompasses the following stages: 1) obtaining genes through artificial (chemical or template-directed) synthesis or by isolating them from natural sources; 2) inserting the gene into a vector DNA molecule, i.e., creating recombinant DNA molecules; 3) introducing the vector DNA molecule containing the inserted gene into a recipient Cell; 4) establishing conditions for the expression of the transferred gene and its stable inheritance; 5) selecting Cells with the active transferred gene — molecular cloning.

Artificial chemical gene synthesis was first accomplished in 1969 by the Indian scientist H. Khorana and his coworkers. This was the Yeast Alanine tRNA gene consisting of 77 nucleotide pairs. However, the synthesized gene lacked a regulatory region and was therefore functionally inactive. Later, these authors synthesized a functionally active gene — the suppressor Tyrosine tRNA gene of E. coli, which is about 200 nucleotide pairs long. Chemical gene synthesis was greatly advanced by The Development of Methods for determining the Primary Structure of DNA, i.e., The nucleotide sequence in its molecule (sequencing). The method of chemical gene synthesis opened up broad possibilities for the artificial synthesis of human genes. The human Growth Hormone (somatotropin) gene and the human Insulin gene have been obtained via chemical synthesis.

Artificial template-directed gene synthesis is carried out using the enzyme Reverse Transcriptase (revertase). This enzyme is capable of building DNA copies on various RNA templates, including synthetic ones. Virtually any gene can be synthesized on an mRNA template using this method. In this way, researchers synthesized the human interferon gene — a valuable pharmaceutical preparation used to combat viral infections.

The method of isolating a gene from natural DNA is based on the incubation of total DNA with various Restriction Endonucleases (restriction Enzymes). Restriction enzymes act as "molecular scissors" that cut the DNA molecule at specific sites into fragments (restriction). These fragments are separated by Electrophoresis, isolated in pure form, and their nucleotide sequence is determined.

After obtaining genes through synthesis or isolation from natural sources, the next step in genetic engineering is to insert the target gene into a vector DNA molecule. Plasmids, Bacteriophages, certain Viruses, and Mitochondrial DNA serve as vectors. Plasmids are most commonly used as vectors. Plasmids are small, extrachromosomal, ring-shaped DNA molecules capable of autonomous Replication; they are located in the Cytoplasm of a bacterial cell or integrated into its chromosome, in which case they are called episomes. Episomes replicate as part of the chromosome. The ring-shaped vector DNA molecule is cleaved by restriction enzymes into linear fragments. Vector DNA fragments and foreign DNA fragments can join together into a single recombinant (hybrid) molecule via their complementary ("sticky") ends. Phosphodiester bonds between NUCLEOTIDES are formed using ligase enzymes. The transfer of target genes (transgenesis) is achieved through various methods: transformation (if the vector is a plasmid) or Transduction (if the vector is a bacteriophage).

Genetic engineering METHODS have enabled The production of Transgenic Plants and animals (organisms carrying foreign genes). Transgenic Animals are used in biomedicine as models for human diseases (mice carrying a Cancer gene, pigs with human cardiac pathologies, cows producing human IMMUNOGLOBULINS in their Blood). The problem of producing human blood Proteins in the milk of transgenic animals is also being successfully addressed.

Gene banks. Genetic engineering owes much of its success to the creation of gene banks (libraries). A gene bank is a collection of genes derived from recombinant molecules. A geneticist can screen a gene library to isolate the genes needed for research using specially developed genetic, biochemical, radioisotope, or immunological methods. Gene banks have been established for Drosophila, Escherichia coli, and many other organisms, including humans.

Biosafety. Genetic engineering originated in 1972 when American geneticists P. Berg, H. Boyer, and S. Cohen created the first in vitro recombinant DNA molecule, which combined genetic material from three different sources: the complete genome of the simian oncogenic virus SV40, a portion of The Genome of the temperate bacteriophage λ (lambda), and the GENES OF THE galactose Operon of the bacterium Escherichia coli (E. coli). However, this constructed recombinant molecule was not tested for functional activity because the authors feared that genetic engineering methods might lead to the creation of organisms hazardous to human health. Interference with an organism's genotype can lead to unforeseen consequences for humans, plants, animals, and the environment as a whole. For example, the bacterium Escherichia coli, which is harmless under normal conditions, could potentially transfer oncogenic animal viruses into the human intestine. Specially engineered biological agents that target Living organisms are classified as biological weapons. Rules to eliminate the likelihood of harmful consequences from genetic engineering were developed at the International Conference on Recombinant DNA Molecules in Asilomar (USA) in 1975. In 1985, the Biosafety Information Working Group was formed.