Textbook - BIOLOGICAL CHEMISTRY - Hubskyi Yu.I. - 2000

Chapter IV. MOLECULAR MECHANISMS OF HEREDITY AND REALIZATION OF GENETIC INFORMATION

CHAPTER 22. REGULATION OF GENE EXPRESSION. GENETIC RECOMBINATIONS

22.4. GENETIC ENGINEERING. RECOMBINANT DNA

The Gene recombinations discussed above are a natural process that occurs in vivo and serves as a biological mechanism aimed at increasing The Diversity of genetic material in the Cells of various organisms.

Introduction/32.html">Genetic Engineering, or Recombinant DNA technology, is an applied scientific field of modern biomedical science. Its methodology is based on isolating individual DNA from cells and purposefully manipulating its molecules—specifically, creating molecular chimeras, which are molecules formed from DNA fragments of different biological species.

Class="center">Biomedical Significance of Genetic engineering Methods

The biotechnological Methods of Genetic Engineering aim to:

1) produce new genotypes (and phenotypes) of organisms through the transplantation of a gene from one Organism into the genotype of another. Specific achievements in this biotechnological field include the creation of chimeric microorganisms containing genes that direct the synthesis of human-useful protein products, particularly medicinal agents (interferons, Hormones such as Insulin, etc.), Enzymes, and immunobiological preparations (such as the hepatitis B vaccine);

2) apply "gene transplantation" for the Treatment of Hereditary diseases in humans and animals—known as Gene Therapy. This area of biotechnological research is in its infancy and promises to introduce fundamentally new medical technologies in the future for treating currently incurable Hereditary diseases, as well as a significant number of common conditions in which hereditary factors play a role in Pathogenesis (atherosclerosis, Diabetes Mellitus, neuropsychiatric disorders, etc.). This approach has already been successfully applied to treat hereditary hypogonadism in mice by implanting the gonadoliberin gene into a fertilized egg Cell.

Gene Transplantation Technology:

1) obtaining a gene with specific properties in a pure form, i.e., as an isolated DNA fragment (encoding the synthesis of a desired enzyme, hormone, etc.);

2) constructing a recombinant (hybrid, chimeric) DNA molecule;

3) introducing the recombinant DNA into a recipient bacterial cell (i.e., a cell where the Replication and cloning of the desired gene will take place);

4) cloning the recombinant DNA.

1. Obtaining the Target Gene.

Obtaining a gene (DNA molecule) intended for replication (cloning) to yield A large number of copies can be accomplished using the following methods:

- chemical gene synthesis (feasible only for short genes consisting of several dozen nitrogenous bases);

- Isolation of the target gene (DNA fragment) from the whole cell genome (a difficult task due to the extreme complexity of Eukaryotic Genomes, which contain numerous non-transcribed introns and repeats—see above);

- construction of complementary DNA (cDNA) based on mRNA (which encodes the Synthesis of the protein desired from the biotechnological Procedure). This method requires Reverse Transcriptase, an enzyme present in certain RNA-containing Viruses that synthesizes DNA on an RNA template According to the reaction equation:

This method is widely used to obtain cDNA and involves isolating the specific mRNA encoding the Translation of a target protein (e.g., interferon, insulin) from the total cellular mRNA, followed by the synthesis of the required cDNA using reverse transcriptase with this mRNA as a template.

2. Construction of Recombinant DNA.

The gene obtained through the procedure described above (cDNA) must be introduced into a bacterial cell in such a way that it integrates into the host genome. To achieve this, a recombinant DNA molecule is formed, consisting of the cDNA and a special DNA molecule that acts as a carrier, or vector, capable of entering the recipient cell.

Viruses or Plasmids are used as vectors for cDNA. Plasmids are small circular DNA molecules located separately from the nucleoid of a bacterial cell (such as E. coli). They contain several genes essential for The Cell's function (e.g., Antibiotic Resistance genes) and can replicate independently of the cell's main genome (DNA). Biologically important and practically useful properties of plasmids for genetic engineering include their ability to transfer from one cell to another via transformation or conjugation, as well as their capacity to integrate into the bacterial chromosome and replicate alongside it.

To construct recombinant DNA, both circular plasmid DNA and linear cDNA are cleaved using highly specific endonucleases targeted to precise nucleotide sequences—the so-called restriction enzymes (or Restriction Endonucleases).

Restriction enzymes (restriction endonucleases) are enzymes present in bacterial cells that "restrict" (hence the name) the replication of certain Bacteriophages by cleaving their phage DNA. The names of restriction enzymes are derived from the bacterial strains from which they are isolated: for example, the restriction enzymes Eco RI and Eco RII originate from the R-strain of E. coli, while Bam HI comes from the H-strain of the microorganism Bacillus amyloliquefaciens.

Restriction enzymes (of which more than 200 types are currently known) cleave double-stranded DNA exclusively at specific nucleotide sequences (typically 4–7 Base Pairs in length, featuring a palindromic Structure). This enables The Use of these endonucleases to "cut" DNA molecules at strictly defined sites. For instance, the restriction enzyme Eco RI hydrolyzes the bonds between G and A within the following sequences:

...GAATTC...

...CTTAAG...

The Cleavage of G-A bonds within these sites of double-stranded DNA by restriction enzymes results in the generation of "overhanging ends" in the molecule, as illustrated in the scheme below.

The overhanging, single-stranded ends of a DNA molecule are also referred to as "sticky ends" because they consist of unpaired NUCLEOTIDES that can readily pair with complementary polynucleotide strands:

Cleavage of plasmid DNA and cDNA (the gene) with specific restriction enzymes yields DNA molecules cut at precise sites with "sticky ends". During in vitro interaction between the cut plasmid and the gene, the polynucleotide strands forming their sticky ends interact through Hydrogen Bonds between complementary bases, as depicted above. The application of DNA ligase, which forms 3'-5' phosphodiester bonds between the terminal nucleotides, "seals" the plasmid and the gene together, completing The formation of the recombinant DNA (Fig. 22.16):

Fig. 22.16. Diagram of recombinant DNA formation from a plasmid and a human DNA fragment.

3. Introduction of recombinant DNA into the recipient cell and cloning of the target gene.

Recombinant DNA molecules, consisting of plasmid DNA and the DNA of the gene to be transplanted (such as a gene encoding a specific human protein), can penetrate into bacterial cells (which serve as natural hosts for the given plasmid) upon interaction.

Inside the host cell, replication (cloning) of the recombinant DNA takes place, yielding many thousands of copies. Subsequently, these cloned DNA molecules are recovered from the bacterial cells, allowing large quantities of the desired gene to be isolated (again using restriction enzymes).

Nowadays, In addition to Bacteria, Yeast, Fungi, plant, and even higher animal cells are utilized for Gene cloning. Using the technology outlined above, genetic engineering has successfully produced human interferon, human insulin, Growth Hormone, Somatostatin, plasminogen activator, protein Diagnostics for AIDS, and other medical products.